Projects

Current research projects of the EBVDT-group

ENDOTHELIAL TARGETED DRUG DELIVERY

I. Development of targeted liposomal delivery systems for improved siRNA delivery to diseased endothelium.

Contacts: Jan Kamps or Grietje Molema

Gene silencing by siRNA is a powerful technique with a potential for pharmacological application in the clinic. It is capable of knocking down targets in various diseases in vivo, including hypercholesterolaemia (1), liver cirrhosis (2), hepatitis B virus infection (HBV) (3), and cancer (4). Furthermore, siRNA technology enables to generate transient knock out animals to mimic human diseases (5). However, naked siRNA is unable to cross the cellular membrane due to its high molecular weight and negative charge. Therefore, significant effort is put into the development of suitable delivery systems (6). Especially useful for clinical purposes are vesicles that can be applied systemically, that are safe and allow specific targeting. A good example is liposomes which can be tailored on demand to introduce cell specificity and did not show toxicity in clinical studies (7).

We designed and patented a system with superior intracellular release characteristics that is suitable for systemic application of siRNA (8,9). The aim of the project is the further development of SAINT-O-Somes in order to maximize in vitro and in vivo efficacy of siRNA delivery and gene down regulation in inflamed endothelial cells and endothelial cells that engage in angiogenesis. In addition to SAINT-O-Somes, our laboratory developed a lipid-based targeting device for siRNA delivery, so-called SAINTargs, which efficiently and specifically deliver siRNA into endothelial cells (10).

For both delivery systems, we conjugated monoclonal anti-E-selectin and anti-VCAM-1 antibodies to increase carrier-mediated siRNA uptake in diseased endothelium and enhance cell specific gene silencing.

Current experiments focus on further development of SAINTargs for in vivo applications. Within this research we have challenging opportunities for motivated HLO and/or MSc students to do an internship in our laboratory.

Frequently used techniques :

cell culturing; transfection of eukaryotic cells; preparation of SAINT-O-Somes and SAINTargs with siRNA; physicochemical characterization of the delivery systems; RNA isolation, integrity check; cDNA synthesis and quantitative PCR; flow cytometry, Western Blot; immunohistochemistry; fluorescence/confocal microscopy

References :

1. Frank-Kamenetsky M et al., Proc Natl Acad Sci., 2008 Aug 19;105 (33):11915-20; 2. Sato Y Murase K et al., Nature Biotechnology, 2008 Apr;26(4):431-42; 3. Song E Lee et al., Nat Medicine , 2003 Mar;9(3):347-51; 4. Takeshita Fet al., Proc Natl Acad Sci, 2005 Aug 23;102(34):12177-82; 5. Lee WC et al., Organogenesis, 2008 Jul;4(3):176-81; 6. Kowalski PS, Leus NG, Scherphof GL, Ruiters MH, Kamps JA, Molema G, IUBMB Life 2011;63:648-58; 7. Bartsch M, Weeke-Klimp AH, Meijer DK, Scherphof GL,Kamps JA, J Liposome Res. 2005;15(12):59-92; 8. Joanna E. Adrian, Henriëtte W.M. Morselt, Regine Süss, Sabine Barnert, Jan Willem Kok, Sigridur A. Ásgeirsdóttir, Marcel H.J. Ruiters, Grietje Molema, Jan A.A.M. Kamps, J. Control Release, 2010 Jun 15;144(3):341-9; 9. Kowalski PS, Lintermans LL, Morselt HW, Leus NG, Ruiters MH, Molema G, Kamps JA, Mol Pharm 2013;10:3033-44; 10. Leus NG, Talman EG, Ramana P, Kowalski PS, Woudenberg-Vrenken TE, Ruiters MH, Molema G, Kamps JA, Int J Pharm 2013, in press.

ENDOTHELIAL CELL DYSFUNCTION IN DISEASE

II. Dietar y determinants of microvascular endothelial dysfunction

Contacts: Peter Heeringa and Roel van der Heijden

The aim of our project is to elucidate the molecular effects of diets on microvascular (dys)function, and unravel both the nature as well as the kinetics of changes. We focus on the renal microvasculature that becomes affected in time with concomitant renal function loss, thereby posing an enormous burden on health care in the coming decades.

To examine the molecular pathways involved in diet induced microvascular dysfunction, in vitro experiments are performed employing relevant primary human microvascular cells and cell lines available in the laboratory (CiGEnC, HMEC, artery and vein endothelial cells). To study direct effects, these cells will be subjected to dietary components that induce microvascular dysfunction.

The specific research objectives defined are:

1.To characterize the effects of nutritional challenges on the nature and kinetics of inflammatory and vascular (de)stabilization related gene expression in the renal microvasculature in vivo in mice

2.To molecularly identify and define the pathways underlying the microvascular dysfunction and their potential to interfere with by dietary measures in in vitro cell systems.

III. Tumor angiogenesis and pharmacological efficacy of anti-angiogenic drugs

Contacts: Gesiena van der Wal, Grietje Molema

Blocking new blood vessel formation is a promising strategy for the treatment of cancers, as is demonstrated by preclinical animal experiments. However, the first anti-angiogenesis clinical trials have not been as successful as their preclinical counterparts. We hypothesize that lack of clinical success is partly due to heterogenic behaviour of tumor endothelial cells, and partly due to lack of knowledge regarding the true status of angiogenesis in human tumors. To develop effective anti-angiogenic treatment strategies for cancer therapy, understanding of the molecular status of tumor angiogenesis in the patient an essential prerequisite, and subject of study in this research, which is performed in collaboration with Dr. K.P. de Jong, Dept. of Surgery, Division of Hepatopancreatobiliary Surgery and Liver Transplantation, and Prof. Dr. A.S.H. Gouw, Dept. Pathology & Medical Biology, Pathology section, of UMCG.

ENDOTHELIAL CELL DYSFUNCTION IN DISEASE

I V. Pathogenic mechanisms in the induction and progression of ANCA-associated glomerulonephritis

Contacts: Peter Heeringa and Mirjan van Timmeren

Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated small vessel vasculitides are rare, but life-threatening, systemic inflammatory diseases that affect small- to medium-sized blood vessels. Patients suffering from this disease have circulating autoantibodies that are directed against enzymes present in the neutrophils: myeloperoxidase or proteinase 3. Although multiple different organs can be affected, the lungs and kidneys are often involved. Involvement of the kidneys results in crescentic glomerulonephritis (=inflammation of the blood filtering units in the kidney, the glomeruli).

It has been uncertain for a long time if ANCA are pathogenic and cause the underlying vasculitis. But, over the last decades increasing clinical and experimental evidence indicates that ANCA are causally involved in disease pathogenesis. However, the exact mechanism of disease initiation and progression is still unknown (proposed model in figure 1).

Our group combines in vitro techniques with an animal model for anti-MPO IgG-mediated glomerulonephritis to gain insight into the pathogenesis of ANCA vasculitis and to identify key pathways and mediators involved in disease progression.

Research Questions:

1. What are the triggers for induction and re-activation of ANCA-associated vasculitides?

2. Which features of ANCA determine their pathogenicity?

3. Which are key pathways and mediators involved in the initiation and progression of ANCA-associated vasculitides?

4. What are potential new targets for intervention, and evaluate the efficacy of new interventions.

V. Linking Toll-like receptor and B cell activating factor signalling in anti-neutrophil cytoplasmic autoantibody (ANCA) associated vasculitides

Contact: Nikola Lepse

Anti-neutrophil cytoplasmic autoantibody (ANCA) associated vasculitides (AAV) are systemic autoimmune diseases characterized by inflammation of the upper and lower respiratory tracts and necrotizing glomerulonephritis. The pathogenesis of AAV is thought to be related to infections. Toll-like receptors (TLRs) are pattern recognition receptors that are crucial for pathogen detection by the immune system. The expression and activation of TLRs has been associated with various autoimmune conditions but the underlying mechanisms are unclear. TLR mediated activation of innate immune cells induces production of B-cell activating factor (BAFF).Mice overexpressing BAFF develop autoimmune disorders and elevated levels of BAFF have been detected in patients with various autoimmune conditions. BAFF signalling is involved in B-cell maturation, survival and immunoglobulin (Ig) class switch recombination (CSR). A fraction of B-cells undergo CSR from IgM to IgD, giving rise to a subset of IgM-IgD+plasma B-cells.The function of IgM-IgD + cells is unclear, however, antibodies from these cells have been reported to be auto-reactive.

The central hypothesis of this project is that in AAV infections via activation of TLRs on immune cells cause excess BAFF production that in turn (re)activates self-reactive B-cells. Specifically, this project aims to investigate whether in AAV pathogens via TLR activation are involved in (a) B-cell class switching toward the IgM-IgD+ phenotype in the upper airways resulting in IgD induced basophil activation and BAFF production; (b) whether the excess production of BAFF is related to TLR mediated activation of BAFF producing innate immune cells; (c) whether excess of BAFF is related to (re)activation of auto-reactive B-cells, resulting in production of auto-antibodies (ANCA) .

Keywords: BAFF, TLRs, ANCA-associated vasculitis.

VI. SHOCK RESEARCH

Contact: Matijs van Meurs

The research of the shock group focuses on shock related organ dysfunction and the role of the microvascular endothelium in the processes underlying this dysfunction. The ultimate goal is to rationally design therapeutic regimens to counteract Multiple Organ Dysfunction Syndrome (MODS) pathophysiology using combined translational research tools, in septic and hemorrhagic shock (HS).

Building on the hypothesis that organ specific endothelial dysfunction is an essential process that influences shock related organ damage, our research group published several studies on microvascular endothelial cell behaviour in shock. Through a detailed study on endothelial cell reactions in different organs and within different vascular beds during shock initiation and progression, and after resuscitation, we found that there is an early, and organ specific endothelial cell activation during HS. This endothelial activation implies options for early therapeutic intervention at the microvascular level to attenuate MODS. Moreover, the vascular heterogeneity in responsiveness indicates variation in molecular control of endothelial behaviour, which may provide options for molecularly targeted interventions specific for the vascular beds involved.

As endothelial cells lose the majority of their microvascular bed specific behaviour during in vitro culturing, we continue to use in vivo studies for primary observations. Future studies are planned to examine the effects of specific endothelial intracellular signalling systems and their role in microvascular endothelial cell activation in shock and to develop more clinical relevant shock models where human co morbid diseases are mimicked in animal models (adipositas, aging). These studies will enable further characterization of microvascular endothelial responses in shock states and MODS, and the design of rational therapeutic strategies to prevent and treat MODS at the level of the microvascular compartments.

VII. Shock-induced Organ Injury; Understanding the mechanisms triggering vascular inflammation

Contact: Jill Moser (Department of Critical Care; Department of Pathology & Medical Biology, Medical Biology section), Rianne Jongman (Department of Anesthesiology), Matijs van Meurs, Jan G Zijlstra (Department of Critical Care); Peter Heeringa, Grietje Molema (Department of Pathology & Medical Biology, Medical Biology section).

Organ-specific endothelial dysfunction contributes to the development of both acute inflammatory disease as well as chronic inflammatory disorders, highlighting the importance of maintaining endothelial integrity. Current experiments focus on understanding the initial cellular and molecular response to kidney damage and how this leads to shock-induced vascular endothelial cell activation and inflammation. In our diverse shock models (sepsis and hemorrhage), we hope to identify regulators of the endothelial cell inflammatory cascade. Using this approach, we will translate our research findings to human shock models and post-mortem patient kidney biopsies which will hopefully lead to novel intervention strategies in order to treat acute kidney injury.

VIII. Understanding microvascular endothelial cell behavior and pharmacological effects of anti-inflammatory drugs in vivo

Contacts: Grietje Molema, Rebecca Li

To date most studies on endothelial cell behaviour have been carried out in endothelial cell cultures in vitro. In recent years it has become clear that in vitro cultures cannot mimic the in vivo situation of the endothelium. We therefore optimized and further developed the technique of laser microdissecion (LMD) to isolate endothelium from organs. Furthermore, we created experimental conditions in which endothelial cells in vitro are subjected to flow, after which flow changes as occurring in vivo during different shock stages are inflicted. This allows us to study the effects of flow changes per se on endothelial cell behavior and on how drugs interfering with signaling cascades perform.

IX. The endothelial cell in shock

Contact: Francis Wulfert (Department of Anesthesiology); Matijs van Meurs, Jan G Zijlstra (Department of C itical Care); Grietje Molema (Department of Pathology & Medical Biology, Medical Biology section)

In daily care within the Departments of Critical Care and Anesthesiology the development of multiple organ failure (MODS) following hemorrhagic (HS) and septic shock is a problem associated with critically ill patients. MODS contributes significantly to morbidity and mortality of shock patients. The inflammatory response is nowadays considered the leading cause for the development of MODS. In these patients specific therapeutic intervention at different targets has not resulted in significant clinical improvement.

Data from the research initiated in the Molema lab in 2004 indicate that the microvascular endothelial compartment represents a pharmacologically neglected target for therapeutic intervention in inflammatory disease. In our research project, we investigate the hypothesis that organ specific endothelial dysfunction is an essential process that strongly influences shock related organ damage. Through a detailed study on endothelial cell reactions in different organs and different microvascular beds during shock initiation, progression, and after resuscitation, we found that there is an early, and organ specific endothelial cell activation during HS, as presented by induced expression of endothelial associated inflammatory genes. This endothelial activation implies options for early therapeutic intervention at the microvascular level to attenuate MODS.

Our focus in the current research projects is on the molecular basis of this proinflammatory endothelial activation, and the occurrence and nature of microvascular endothelial heterogeneity in the response during different shock states (septic vs hemorrhagic shock). Some more translational projects focus on the translation of knowledge gained in animal models to critically ill shock patients. National and international collaborations with clinical and pre-clinical research groups have led to a vibrant new research network. The knowledge created in these projects will be the basis for studying therapeutic interventions to modify the endothelial pro-inflammatory activation which can be clinically in the future.