Christer Betsholtz's projects in developmental genetics

Vascular malformations

Konstantin Gaengel
Vascular malformations are morphological abnormalities of blood vessels that often go hand-in hand with impaired endothelial or mural-cell function. They can affect all vessel types, and arise either sporadically or as a consequence of inherited mutations. They can be present at birth or be acquired later in life and are often asymptomatic. However, certain diseases cause vascular malformations that severely impact the quality of life or even can become life-threatening.

We use a combination of mouse genetics, advanced microscopy techniques and single cell sequencing approaches to study vascular malformations. Our aim is to characterize the cellular and molecular mechanism that lead to formation and progression of those lesions. In doing so we hope to identify new biomarkers that can predict how lesions might progress and find new treatment approaches.

Further reading: A Leica Science Lab article featuring our research:

Microscope image of a retina with vascular malformations, showing blood vessels and endothelial cells
Vascular malformations in a retina. Blood vessels are shown in red and endothelial cells are blue.

PDGF in organ development – Perivascular fibroblasts in brain and lung

Johanna Andrae
The platelet-derived growth factors (PDGFs) are known mediators in several developmental processes. Our main focus is on processes that depend on proper signalling through the tyrosine-kinase receptor PDGFRα and the two ligands PDGF-A and PDGF-C. We ask questions like:

  • What are the characteristics of the cells that express PDGFR?
  • Where are PDGFRα+ cells located in relation to the ligand-expressing cells?
  • What happens in the absence or overactivation of PDGF?

We are interested to know how different cells contribute to a specific tissue organization.

In general, PDGFRα is expressed on mesenchymal cells whereas the ligands are secreted from adjacent epithelial, neuronal or muscle cells. This is true in both the central nervous system and in the lung, which we are specifically interested in.

The role of perivascular brain fibroblasts

Perivascular brain fibroblasts are a group of relatively undescribed cells, located along certain blood vessels in the brain. These PDGFRα+ cells express several fibroblast markers and are situated outside of endothelial cells, but inside of the astrocytic endfeet. Their biology, origin and functional role is still to be described, but our hypothesis is that PDGF signalling is important and that the cells are needed for the vascular system.

Microscope image of blood vessel in the brain, with perivascular fibroblasts and astrocytes
Green perivascular fibroblasts along a blue blood vessel in the brain, surrounded by orange astrocytes.

PDGF signalling in lung alveolarization 

Lung alveolarization is the developmental process where the small, functional units in the lung are formed. In the absence of PDGF-A, the alveolarization process is negatively affected. In the adult lung, there are several reasons to believe that PDGFRα+ cells have other functions.

We hypothesize that PDGF signalling is needed in the stem cell niche if the lung is exposed to an injury. All cell types use specific molecular signals to communicate with each other, and knowing the normal signalling pathways is crucial to understand potential pathological mechanisms.

Molecular studies on blood-brain barrier

Maarja Andaloussi Mäe, Elisa Vázquez Liébanas
The blood-brain barrier (BBB) is viewed mainly as an organotypic state of differentiation of endothelial cells, regulating the passage of blood-borne molecules into the CNS. The endothelial cells joined together by tight and adherens junctions restrict the paracellular passage of ions and biomolecules from the blood to the brain parenchyma. Moreover, trans-cellular passage of blood-borne molecules is restricted through a low rate of vesicular transcytosis across the endothelial cells. Additionally, the brain endothelium expresses influx and efflux transporters that facilitate the specific transport of nutrients and metabolites in and out of the brain.

The function of pericytes

Pericytes play an important role in blood vessel stabilization and BBB formation. They have also been implicated in tissue repair, scar formation, immune cell guidance and blood flow regulation, however due to a challenging molecular and cellular identification of pericytes, some of these functions remain debated. Recently, we provided a molecular definition of pericytes in relation to other vascular cell types in the brain and lung using single cell RNA sequencing.

This study also revealed that pericytes are organotypic, i.e. that they differ between organs. Diabetic retinopathy is associated with pericyte death, basement membrane changes and microvascular dysfunction and leakage. Relatively recent studies have revealed that pericytes are lost also in other human central nervous system diseases, including Alzheimer’s disease, mild dementia, amyotrophic lateral sclerosis and acute stroke.

The importance of pericytes for the blood-brain barrier

Our main goal is to comprehend the complexity of the BBB. To do so, large part of our research aims to explore how pericytes regulate the BBB and how their deficiency affects the function of this crucial barrier. Additionally, we are studying the role of different vascular proteins at the BBB and neurovascular unit using transgenic mouse models and single cell RNA sequencing in combination with in situ methods like immunostainings and RNA in situ hybridization.

Microscope image of brain vasculature lacking pericytes
Pericyte-deficient brain vasculature. A remaining pericyte (in green) is trying to cover a neighboring
capillary by stretching its processes.

Expression profiling of vascular cells in peripheral organs

Riikka Pietilä, Ying Sun
Arteries, veins and capillaries form a continuous route for blood flow in our bodies and the vasculature has adapted remarkably to different demands of our tissues. There are still a lot of unanswered questions regarding endothelial cell (ECs) heterogeneity across organs and factors that determine their transcriptional profile in normal physiology.

To get a comprehensive idea of the types and diversity of vascular cells in different organs we employ single cell RNA sequencing databases. In these we have integrated single cell data for different organs, created using various isolation methods and single-cell sequencing platforms.

Molecular profiling of organs

With the development of sequencing technologies, single-cell RNA sequencing (scRNA-seq) is becoming a robust and broadly used method for transcriptome analysis to dissect the compositions of tissues and dynamics of transcriptional states. Smart-seq2 and 10x Genomics are frequently used scRNA-seq methods. They have their own advantages in terms of amount of genes detection and rare cell type detection.

In order to get a full and in-depth understanding of the molecular composition and profile for individual organs and comparison across the organs/cell types, we leverage different integration methods to combine multiple scRNA-seq datasets obtained from smart-seq2 or 10X Genomics platforms from different labs/collaborators.

Immunostaining and imaging

To validate the single cell data and to map vascular cell types to their anatomical locations we employ various immunostaining techniques against mRNAs and proteins followed by high-resolution imaging. Visualization of mRNAs in tissues using RNAscope for instance allows direct and reliable validation of single cell data in terms of cell types captured, evaluation of expression levels and thus interpretation of results.

With the data, we can answer to questions like:

  • To which extent transcriptional signature in the arteries, veins and capillaries is determined by genetic factors.
  • How much physiological parameters such as hemodynamic forces or oxygen level, which differ from tissue to tissue and within tissue, contribute to the endothelial cell identity.

Analysis of vascular cells and their zonation in different organs helps greatly the study of vascular defects observed in various disease settings.

Microscope image showing gene expression in lung vasculature
Lung vasculature showing vEC specific gene expression (green). Endothelial cells are
labelled with Pecam1 (white). In red, Lgr6 expression in SMCs.