Projects

Contact:

Piotr Kowalski, 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. 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 (6). Using a new generation of liposomes called SAINT-O-Somes we focus on inflamed endothelial cells which have not been extensively investigated as a target for siRNA therapy so far, in contrast to cancer and liver cells. However, unlike in macrophages or in tumor cells, in endothelial cells the processing of liposomes and subsequent release of drug content is inefficient due to the absence of an adequate intracellular processing machinery which limits pharmacological efficiency. Therefore, we designed and patented a system with superior intracellular release characteristics that is suitable for systemic application of siRNA (7). 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, the formation of new blood vessels in inflammatory sites and cancer.

Frequently used techniques :

- cell culturing

- transfection of eukaryotic cells

- preparation of liposomes with siRNA or other compounds

- 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. Bartsch M, Weeke-Klimp AH, Meijer DK, Scherphof GL,Kamps JA, J Liposome Res. 2005;15(1 
2):59-92.

7 . 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

Contact: Niek G.J. Leus, Jan Kamps or Grietje Molema

The endothelium represents an important therapeutic target because of its pivotal role in many diseases such as cancer and chronic inflammation (e.g. Rheumatoid Arthritis). Endothelial cells form the inner lining of blood vessels and are therefore fully accessible for systemically administered drugs including small RNAs. Small interfering RNA (siRNA) is one of the main groups of small RNAs, a class of double-stranded RNA molecules, 21-23 nt in length. siRNA is involved in the RNA interference (RNAi) pathway where it specifically interferes with the expression of the complementary gene of interest. For this reason, the technology based on RNAi has become a powerful tool in basic research and has great potential to become a powerful approach for disease therapy.

Due to their size and charge, siRNAs do not spontaneously enter unperturbed cells. To overcome this problem, our laboratory developed a lipid-based targeting device for siRNA delivery, so-called SAINTargs, which efficiently and specifically deliver siRNA into endothelial cells. Molecular determinants on the surface of activated (diseased) endothelium, like certain cell adhesion molecules and/or receptors associated with disease, are excellent targets for targeted drug delivery to the endothelial surface and intracellular compartments. We conjugated monoclonal anti-E-selectin and anti-VCAM-1 antibodies to these SAINTargs 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 vivoapplications. Within this research we have challenging opportunities for motivated HLO and/or MSc students to do an internship in our laboratory.

Techniques:

- eukaryotic cell culturing

- eukaryotic cell transfections

- lipoplex and liposome technology

- RNA isolation, integrity check (electrophoresis), cDNA synthesis and

(quantitative) PCR

- flow cytometry

- immunohistochemistry

- western blotting

- fluorescence/confocal/laser-dissection microscopy

Contacts: Titia Woudenberg-Vrenken, Grietje Molema or Jan Kamps 

P38 MAPK inhibitors are potent pharmacological entities that have been developed for the treatment of chronic inflammatory diseases and angiogenesis associated with tumor growth. Their toxicity has hampered further clinical development. By formulating them into a new generation of immunoliposomes that once taken up by the endothelial cells are avidly degraded to release the drug, toxicity may be overcome while its potency is not affected. Their physicochemical features are such that formulation in lipid carriers leads to instable formulations that rapidly exchange the drug with biomembranes in vivo. In this project we collaborate with Syncom and Synvolux  to develop chemical derivatives of the inhibitors that exert superior formulation, release and pharmacological features.

Contact:

Peter Heeringa, Grietje Molema or Gopala Krishna Yakala

The aim of our project is to elucidate the molecular effects of different diet (eg:- high-fat, high-cholesterol, high-carbohydrate) on microvascular (dys)function, and unravel both the nature as well as the kinetics of changes. We will 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. As the microvascular endothelium is numerically underrepresented in all organs in the body, whole tissue analysis for -omics profiling masks the specific components that determine the behaviour of the cells. Our recently established research strategy of laser microdissecting the endothelial cell from tissue biopsies before subsequent transcriptome analysis provides an unique opportunity to achieve our aims by generating new insight in the microvasculature in its pathophysiological context.

To examine the molecular pathways involved in diet induced microvascular dysfunction, in vitro experiments will be performed employing relevant human microvascular cells and cell lines available in the laboratory (CiGEnC, HMEC, arteriolar and venular 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.

Contacts:  Grietje Molema, Neng Fisheri Kurniati

Contact: Francis Wulfert (Department of Anesthesiology); Matijs van Meurs, Jan G Zijlstra (Department of C itical Care); Neng F. Kurniati, 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.

Contact:  Gesiena van der Wal, Grietje Molema

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. 

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.