Current projects

Our group focuses its resarch on the role of membrane transporters and its various tasks across the placenta and the blood-milk barrier, respectively. These barriers are of fundamental importance in providing healthy and optimal nutrition for the fetus, neonate and infant.

Understanding the membrane transporters is essential in understanding the supply of nutrients to the fetus/infants. Thus, we can better ..... 

There are two main perspectives within our current projects: The «Role of membrane transporters in the physiology and pathophysiology of the human placenta» and the «Role of membrane transporters in mammary gland biology». 

The placenta is an endocrine organ that provides the hormonal equilibrium for the maintenance of pregnancy. It is involved in nutrient exchange/transport and oxygen supply from mother to fetus, and in the removal of metabolites from the fetal to maternal circulation. For additional information on placental structure and transport, please click here. Abnormalities in placenta transport are implicated in the pathogenesis of many pregnancy complications, such as preeclampsia, antiphospholipid syndrome (see article), fetal growth restriction and recurrent pregnancy loss. Fundamental knowledge of the molecular mechanisms of placental transport is crucial for the development of novel therapeutic strategies to combat pregnancy complications.
 
Our research interest is to study the molecular mechanisms of lipid transport across the placenta and its possible implication in placental pathology. The current research projects are focused on the role of the ATP-binding cassette (ABC) transporter family, in particular ABCA1 and ABCG1, in the physiology and pathophysiology of the human placenta.

Figure 1: Syncytiotrophoblast layer, 24 hours in culture; 20x
Figure 1: Syncytiotrophoblast layer, 24 hours in culture; 20x

Methodology and analytical models

  • Isolation and cultivation of primary placenta cells: trophoblast cells from early and term human placentas, amnion epithelial cells, mesenchymal stem cells (see Figure 1 and 2)
  • Identification of subcellular localization of ABC transporters in placental tissue and primary cells using immunohistochemistry and immunocytochemistry techniques (see Figure 3 and 4).
  • Functional assays of cholesterol homeostasis (uptake and efflux) of primary placenta cells
  • Extraction and quantification of lipids in placental tissues
  • mRNA expression analysis using real-time PCR
  • Protein expression analysis ELISA
  • FACS analysis
  • Miscellaneous biochemical assays
     
Figure 2
Figure 2: Amnion epithelial cells, 12 days in culture, 10x (left panel);
human chorion mesenchymal cells, 12 days in culture, 10x (right panel)
Figure 3: Immunohistochemical localization of ABCA1 and ABCG1 in term placenta. ABCA1 staining, 20x (left panel); ABCG1 staining, 20x (right panel)
Figure 3: Immunohistochemical localization of ABCA1 and ABCG1 in term placenta.
ABCA1 staining, 20x (left panel); ABCG1 staining, 20x (right panel)
Figure 4: Immunofluorescence staining of ABCA1 in amnion epithelial cells from term, 20x (left panel); and 1st trimester trophoblast cells, 40x (right panel)
Figure 4: Immunofluorescence staining of ABCA1 in amnion epithelial cells from term,
20x (left panel); and 1st trimester trophoblast cells, 40x (right panel)

The molecular understanding of the movement of nutrients and xenotoxins in the mammary gland is still incomplete. Little is known about the cellular mechanisms by which, for instance, drugs or toxic compounds enter milk or mammary tissue. In terms of the nutritional and health aspects concerning the composition of milk, it is clearly important to better understand the mechanisms by which lipids, ions, organic solutes and vitamins cross the blood-milk barrier.

Current topics in public health and/or economy include:

  1. how to favorably influence the lipid pattern in the milk
  2. to identify precise mechanisms responsible for diseases of the mammary gland and disorders associated with mammary gland function
  3. understanding how the mammary gland adapts to maternal deficiency or excess of nutrients.

To address these questions, precise knowledge of the molecular transport mechanisms and regulatory pathways of the mammary gland is a prerequisite. General principles of secretory routes in mammary epithelial cells are shown in Figure 1.

Figure 1: Secretory routes in mammary epithelial cells
Figure 1: Secretory routes in mammary epithelial cells (MEC). Milk compounds cross the mammary gland plasma membrane through different pathways (I-V). Pathway I: direct movement of monovalent ions, water, glucose, and possibly lipids, drugs and xenobiotics across apical and basal membranes of MEC via transporter proteins. Pathway II: milk fat and lipid secretion with formation of cytoplasmic lipid droplets (CLD) moving to the apical membrane for release as milk fat globules (MFG). Pathways III, IV and V: exocytotic secretion, vesicular transcytosis and transport through the paracellular pathway for plasma components and leukocytes, respectively. Pathway V: open only during pregnancy, involution and in the event of inflammatory states such as mastitis. Abbreviations: BM, basement membrane; BV, blood vessel; DS, desmosome; ER, endoplasmic reticulum; GJ, gap junction; ME, myoepithelial cell; PC, plasma cell; TJ, tight junction. Adapted from McManaman and Neville (2003)

Our research interest is to explore the molecular details of active transmembrane movement of nutritionally important compounds across the blood-milk barrier. In particular, we aim to elucidate the mechanisms implicated in lipid trafficking across the polarized mammary epithelium and in the regulation of lipid homeostasis in mammary epithelial cells at different physiological (developmental) stages of the mammary gland. Currently, we are investigating the involvement of ABC lipid transporters, and of associated regulators in mediating cholesterol exchange between polarized mammary epithelial cells and the external lumen during the pregnancy-lactation cycle. We are studying the potential effects of these transporters on the transfer of cholesterol and therapeutic drugs in the milk in animal models in vivo, as well as in cell culture models in vitro. Of special interest is furthermore the identification of regulatory influences by hormonal and metabolic changes occurring during pregnancy and lactation.

Methodology and analytical models

  • Mammary epithelial cell culture models
  • Identification and sub-cellular localization of ABC transport proteins in mammary tissues and mammary epithelial cells by immunohistochemistry and immunocytochemistry (see Figure 2)
  • Functional assays on cholesterol homeostasis (uptake and efflux) using cultured mammospheres from isolated mammary cells
  • Isolation and characterization of milk fat globules from mature milk (see Figure 3)
  • mRNA expression analysis using real-time PCR
  • Protein expression analysis using ELISA
  • FACS analysis
  • Miscellaneous biochemical assays
Figure 2: Immunofluorescence staining of ABCA1 (left panel) with Alexa Fluor® 405 conjugated antibody and the nuclear counterstain (middle panel) with DAPI in bovine mammary cells (BME-UV1). Overlay of ABCA1 and DAPI staining (right panel). (Unpublished data)
Figure 2: Immunofluorescence staining of ABCA1 (left panel) with Alexa Fluor® 405 conjugated antibody and the nuclear counterstain (middle panel) with DAPI in bovine mammary cells (BME-UV1). Overlay of ABCA1 and DAPI staining (right panel). (Unpublished data)
Figure 3: Characterization of paraformaldehyde fixed milk fat globules (MFG) by confocal laser scanning. Fig A: fluorescent staining of the core triglycerides in MFG by oil red O dye (red). Fig. B: staining of cholesterol in the MFG membrane by filipin (blue). (Unpublished data)
Figure 3: Characterization of paraformaldehyde fixed milk fat globules (MFG) by confocal laser scanning. Fig A: fluorescent staining of the core triglycerides in MFG by oil red O dye (red). Fig. B: staining of cholesterol in the MFG membrane by filipin (blue). (Unpublished data)