University of Bristol

Department of Physiology

Dr David Bates

British Heart Foundation Lecturer in Basic Biomedical Sciences

The Biology of Vascular Endothelial Growth Factors

The primary function of the cardiovascular system is to deliver nutrients to cells, and remove metabolic wastes. Water and nutrients move from the blood to the tissue across the walls of capillaries and venules. The rate at which this happens depends on a number of factors, one of which is the permeability of the capillary wall. The endothelial cells which form the capillary wall control its permeability, in a way which is not very well understood. Our research centres on how endothelial cells chronically regulate capillary permeability and how capillaries grow and form new vessels. My interest in this process - called angiogenesis - stemmed from the observations that new blood vessels appear to have a higher permeability than established vessels, and that a specific growth factor for endothelial cells (VEGF), also causes increased permeability. Development of highly permeable new microvessels is critically important in a number of very common, and potentially lethal diseases. Solid tumours cannot grow more than 0.2mm in diameter without the development of new blood vessels, and yet we do not understand how these vessels are formed, or why they are highly permeable. Other conditions such as diabetes, psoriasis, atherosclerosis and arthritis are also associated with high permeability, and high VEGF165 production.

We are carying out experiments that will help us understand how permeability and angiogenesis are related, and hopefully this will lead to new strategies for drug design,and novel therapies for these conditions, as well as helping us  understand how our bodies function normally. Specifically our research concentrates on the mechanisms by which VEGF causes increased angiogenesis, and transvascular solute and solvent flux in individual capillaries and postcapillary venules. We are now investigating the mechanisms by which the chronic increase in permeability, associated with new vessel growth, is brought about. Some of the areas that we are investigating are:

The role of VEGF induced calcium influx in  increased permeability, and the signal transduction pathways through which VEGF exerts its effects.
How different VEGF isoforms contribute to angiogenesis and permeability
How lymphatic specific VEGFs regulate fluid removal in lymphoedema and skin cancer
The effects of VEGF on the renal glomerulus
The ultrastructural effects brought about by VEGF
The contribution of VEGF to conditions such as diabetes, cancer of the prostate and kidney, and in wound healing.

This work is carried out using whatever methods we feel are most appropriate. Some examples of how we do this are shown below.

Microvascular permeability measurement
Assessment of Angiogenesis
Measurement of glomerular permeability
Calcium measurement in vivo
Immunodetection of lymphatics and lymphatic endothelial cells
Microarray and expression of mRNA in individual vessels
Three dimensional reconstruction of endothelial cell ultrastructure
 
 
 
 
 

Microvascular Permeability.

The movie below shows schematically how we measure permeability of a single capillary. The movie starts with a lower power view of a mesentery with the gut attached.  The gut is the green outer part of the diagram, and the arteries (in red) and veins (in blue) run through the thin connective tissue (the mesentery). Coming off the arteries and  veins are smaller arterioles and venules. These branch down to capillaries which connect to two sides of the arterial system. The movie zooms in on a single capillary.  Capillaries are endothelial lined tubes, usually only one endothelial cell thick, with no smooth muscle surrounding them. The capillary is about 1/50th of a millimetre wide  (20 microns). To measure permeability, we cannulate the capillary with a very fine needle made from glass. The needle (or micropipette) has a bevelled tip, just like a  hypodermic needle, but the tip is only 12-15 microns wide and exceedingly sharp. The micropipette is attached at the back end to a constant, set pressure, so a stream of fluid  comes out of the end of the micropipette. Continued below.

The micropipette is filled with a perfusate, which is a physiological solution containing salts, serum albumin, and a low  concentration of red blood cells. You can see the red blood cells flowing along the capillary. To measure permeability we gently lower a glass rod onto the capillary some  distance down stream from the cannulation site. This blocks the flow out of the end of the capillary and causes the pressure in the capillary to equilibrate with the pressure in  the micropipette. Now the pressure inside the capillary is greater than outside, so fluid is forced across the capillary wall. As the fluid flows out it is replaced by fluid in the  pipette. This means there is a flow of fluid from the pipette into the capillary and across the capillary wall. The red blood cells in the capillary will move with the fluid flow,  seeming to flow along the capillary as the column of fluid in front of them shrinks by filtration. From the speed of the red cells  (dl/dt) and the cross sectional area of the  capillary (calculated from its radius r) we can calculate the rate of fluid flow across the capillary wall (Jv). From this,  the surface area of the vessel (A, calculated from the radius  and the length), and the pressure inside the vessel (which we have set) we can determine the permeability of the wall to water (the hydraulic conductivity). We then remove  the glass rod and allow the perfusate to flow freely. We can add drugs or markers to the vessel either by refilling the pipette while in the vessel, or dripping them on the  outside of the mesentery.
 

Angiogenesis

We are currently intersted to find out how increased vascular permeability is regulated during angiogenesis. To this end we have developed a model for the measureemnt of permeability in rat mesentery during angiogenesis caused by VEGF expression in the surrounding fat cells. This results in increased blood vessel growth of previously characterised vessels, and hence increased permeability. The angiogenesis can be measured by recording the meentery on videotrap and then staining for dividing cells.
 
Figure 1. PCNA staining of vessels in mesentery after exposure to VEGF secretion by cells in the fat pad. Light microscopic veiew of mesentery before (right) and after (left) exposure to VEGF secreting cells. Map of mesenteric microvessels b

 
 

If you have any questions about how this works, please don't hesitate to email me.
You can  also see a video of an actual permeability measurement.
 
 

If you really want to find out more, click herefor a list of my recent publications. Dave's Home Page

Information supplied by: Dave Bates