Microvasculature
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 microvasculature. 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. Research within this area forms the major part of the work of the Microvascular Research Laboratories.
One of the main areas of research centres on how endothelial cells chronically regulate capillary permeability and how capillaries grow and form new vessels - angiogenesis. New blood vessels appear to have a higher permeability than established vessels and the 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.
The Biology of Vascular Endothelial Growth Factors
Vascular endothelial growth factor (VEGF) consists of a family of gene products (VEGF-A to D and placental growth factor (PlGF)) that are neither specifically targeted at the vascular system or endothelial cells and indeed can either promote or inhibit growth. They are expressed as head-tail dimers and all come in multiple spilce variants. VEGF-A was the first to be described and is thus generally referred to as simply VEGF. While not a particularly potent growth factor, it is a potent vasodilator and a powerful vascular permeabilizing agent (reviewed in Bates 2010), acting via two receptors, VEGFR1 and VEGFR2.
Role of VEGF in angiogenesis
Angiogenesis is the growth of new blood vessels during development, wound healing, tumour formation and many other pathological states. VEGF is strongly upregulated during all these processes and knockout of the gene in mice is lethal Carmeliet et al 1996; Ferrera et al 1996), indicating it's vital role in the development of the vasculature. Multiple splice variants are produced from a single gene sequence, falling into two groups, the pro-angiogenic VEGFxxx and the anti-angiogenic VEGFxxxb families (see Woolard et al 2009). One of the most common isoforms is VEGF165 and it's anti-angiogenic counterpart VEGF165b, which has been a principle focus of work. For example, we have shown that VEGF165b is downregulated in renal cell carcinoma (Bates et al 2002), inhibits VEGF165 directed angiogenesis by acting as a competitive antagonist of the VEGFR2 (Woolard et al 2004) and that it increases hydraulic conductivity, acting via VEGFR1 (Glass et al 2006). We have also shown that expression of VEGF165b inhibits retinal neovascularization (Konopatskaya et al 2006) and also protects retinal epithelial and endothelial cells from ischaemic insult (Magnussen et al 2010). Such effects could play an important therapeutic role in hypoxia-driven angiogenesis in conditions such as diabetic retinopathy.
Role of VEGF in vascular permeability
The 'leakiness' of capillaries is important both in the normal functioning of the vascular system as well as in pathological conditions that affect it. One of the major areas of work is in the role growth factors such as VEGF play in controlling vascular permeability in the capillaries of the glomerulus (Salmon et al 2006). Such work is important as high permeability leads to conditions such as albuminuria, where albumin leaks into the glomerula filtrate. Not only is this toxic to the kidneys themselves, leading to kidney disease and failure, but it also increases the risk of cardiovascular disease to an extent similar to smoking. It is thus a major health problem.
We have shown previously that recombinant VEGF165 increased the ultrafiltration coefficient in isolated glomeruli by increasing endothelial cell permeability to water - an action mediated by VEGFR2 (Salmon et al 2006). In complementary work, we have recently generated a transgenic mouse in which VEGF165b is over expressed specifically in glomerular podocytes (Qui et al 2010). In results that are reminiscent of the angiogenic/anti-angiogenic nature of these two VEGF splice variants, this mouse shows a reduced glomerular permeability to water. Thus the balance of splice variant secretion plays an important role in the local control of permeability.
We are also interested in the role of the endothelial glycocalyx, an extracellular matrix secreted by endothelial cells and made up of polysaccharides, mainly glycosaminoglycans. We have shown recently that angiopoietin-1 reduces glomerular vascular permeability by both increasing the thickness of the glycocalyx and glycosaminoglycan turnover (Salmon et al 2009a). This work adds more layers to the increasingly complex glomerular ultrafiltration system (reviewed in Salmon et al 2009b).
VEGF and tumour growth
Increased vascularisation is an essential element of the growth of any solid tumour. The balance in expression of angiogenic and anti-angiogenic isoforms of VEGF appears to be a primary driver of such vascularisation. Thus we have recently shown that VEGF165 is upregulated in highly tumourigenic neuroblastoma cell lines relative to VEGF165b when compared to less tumorigenic cell lines (Peiris-Pagès et al 2010). Expression of recombinant VEGF165b also reduced the growth and microvascular density of xenografts of such cells. This complements previous work in which we have shown that VEGF165b overexpression slows the growth of multiple tumours types in vivo including malignant prostate tumours, renal cell carcinoma and Ewing's sarcoma (Woolard et al 2004; Rennel et al 2008).
Similar anti-angiogenic and anti-tumour growth properties have been seen with a new splice isoform, VEGF121b (Rennel et al 2009), suggesting that these may be general properties of the VEGFxxxb family, providing a potential additional therapeutic route in cancer treatment.
Ephrins and VEGF function
Recent work has also shown that ephrin-B2, a well known modulator of cell-cell contact inhibition, also controls VEGF-C induced angiogenesis (Wang et al 2010). VEGF-C acts via the VEGFR3 receptor. Expression of recombinant ephrin-B2 resulted in the internalization of VEGFR3, which was also disrupted following knockout of the Efnb2 gene both in vivo and in vitro. Such a knockout also reduced downstream VEGFR3 signalling mechanisms (tyrosine phosphorylation and activation of Rac1). Thus both reverse and forward ephrin-B2-induced signalling mechanisms are important in VEGF-C functioning.The physiological consequence of this disruption of VEGF function is extensive, with over-expression of ephrin-B2 resulting in excessive sprouting of unstable, thin dermal and retinal blood vessels, while knockout results in short stubby vessels with few filopodia (Wang et al 2010). Knockout also reduced chemotaxis towards VEGF, but not other growth actors tested. Similar results have also been found recently for the control of VEGFR2 signalling (Sawamiphak et al 2010). Thus the mechanisms that control cell migration are also fundamental to the generation of new blood vessels.