The cardiovascular system is the primary functional organ system to form during ontogenesis in the human embryo .It is mainly comprised of the heart and blood vessels. The heart operates as a muscular pump by conveying blood to all the tissues in the body. The blood vessels along with the endothelial cells develop from the endothelial progenitors, angioblasts and together they constitute the primitive cardiac plexus during early embroyogenesis. These blood vessels resemble pipes that aid in blood and oxygen transportation. Though not directly, but with the help of arteries and veins, they help in circulating blood with oxygen, nutrients and hormonal secretions to the tissues and return the blood to the heart by removing carbon-dioxide and other metabolic wastes from those tissues. Other functions include the regulation of temperature by contracting (vasorestriction) or by relaxing the blood vessels (vasodilation) and the prevention of any infection by the regulation of the immune system.Changes in this blood vessel vasculature and a decreased efficiency in the oxygen and nutrient perfusion to the tissues are the major causes for a number of fatal diseases in humans like Peripheral Arterial Diease (PAD- includes the occlusion of the large arteries in the arms and the legs) and Beurger's disease (characterized by inflammation of the arteries and veins).(Starr & McMillan 2010)
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Blood vessels deteriorate in a number of conditions which raises the need for the formation of new ones. Neovascularisation is an inherent feature of all vascular tissues that involve the formation of new blood vessels. This vessel renewal is induced by a number of factors like inflammation (vasculitis-attack of the immune system on the blood vessels), ischemia (poor blood supply that causes the dysfunction of the affected tissue), tumour metastasis or traumas (Kupatt & Deindl 2007). The commencement of blood vessel formation is dependent on transmission of blood and also on the complex interactions between the localized growth factors. This course of new blood vessel formation is essential for a number of pathological and physiological processes like embryogenesis, fluid shifts, homeostasis and tissue regeneration(Kupatt & Deindl 2007).
Neovascularisation is characterized by three main mechanisms: Angiogenesis, Vasculogenesis and Arteriogenesis. During gastrulation, the angioblasts and the haematopoietic cells differentiate into solid clumps of cells called the blood islands (haemangioblasts). The outer cells of these clusters are converted into endothelial cells while the inner ones turn into the haematopoietic cells. The close proximity between the endothelial cells and the haematopoietic cells right from their inception indicate a common progenitor, haemangioblasts that give rise to these cell lines via an intermediate called the haemogenic endothelium (Yoshimoto & Yoder 2009, Flamme et al. 1997).
Tcell, macrophages, neutrophils, mast cells, dendritic cells and platelets
Fig.1.1. Schematic representation of the stages involved in vessel formation. During normal embryonic development, mesodermal cells give rise to the haemangioblasts that differentiate under the influence of myeloid and stem cells to give rise to the haematopoietic cells and endothelial precursors. These haematopoietic and endothelial progenitors in the presence of Tie-2 and VEGFRs give rise to all blood cell types and endothelial cells. These chemotactic endothelial cells migrate into the blood cells to remodel the tissues, thereby giving rise to the primary vascular labyrinth which in turn gives rise to mature vessels. This entire process that controls the vascularisation of the embryo is termed vasculogenesis with VEGF as the regulator. New vessels and capillaries sprouting from already existing ones are termed angiogenesis and arterigenesis which is modulated by VEGF and Angiopoietins. Adapted from (Testa et al. 2008)
Vasculogenesis is a two-step process that generates blood vessels. This involves the differentiation of endothelial precursor cells from the mesoderm and also the de novo proliferation and networking of the blood vessels to form the primitive blood vascular plexus(Flamme et al. 1997, Semenza 2007).Earlier it was hypothesized that only the embryonic endothelial progenitors mediated vasculogenesis while recent research show involvement of endothelial precursors (ranging from the multipotent progenitors of the bone marrow and pluripotent stem cells to the myeloid cell lineage) in the development of the adult vasculature(Semenza 2007, Drake 2003)
Angiogenesis can be categorized into sprouting and non-sprouting (intussusceptive) angiogenesis. Sprouting angiogenesis is an invasive process that facilitates the growth of new capillaries from already existing ones. This takes place in a cascade of steps starting with the activation of the endothelial cells characterized by the binding of specific growth factors with their receptors. Consequently, there is a disruption of the endothelial basal membrane and the extracellular matrix by activated proteases which allows the endothelial cells to migrate and proliferate through the neighbouring matrix. When these migrating endothelial cells differentiate, a lumen is created forming an immature blood vessel. This immature blood vessel is stabilized by sequestering vascular smooth muscles and pericytes, collectively called the mural cells. It is a very extensive process and has the ability to bridge vascular gaps by endothelial cell proliferation in wound-healing therapies and tissue regeneration (Hillen & Griffioen 2007). A variant of sprouting angiogenesis is Intussusceptive angiogenesis. This type of angiogenesis takes place in a short phase of time without much expense of energy to outline new vascular entities resulting in vascular remodelling. At first, two opposite endothelial cell walls come in contact by a transluminal bridge. This is followed by perforations in the endothelial cell wall that marks the formation of the transcapillary pillar. The newly formed pillar is then enclosed by pericytes and fibroblasts which with their contractile functions widen the pillar, allowing the endothelial cells to segregate into two different vessels (Makanya et al. 2009, Burri et al. 2004).
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Angiogenesis is a complicated process which is regulated by a number of pro- and anti-angiogenic factors, which determines the progression of blood vessel growth(Carmeliet 2005). Angiogenesis is stimulated by a plethora of cytokines like the Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor(FGF), Angiopoietins, integrins, cadherins, Platelet Derived Growth Factor (PDGF),Transforming Growth Factors-Î±,Î², TNF-Î± ,Matrix Metalloproteinase-9 (MMP9) and Stromal cell-derived Factor (SCF)(Carmeliet 2003, Risau 1997).
Fig.1.2. Mechanisms involved in a typical Tumour induced sprouting angiogenesis. Sprouting angiogenesis starts with the degradation of exracellular matrix and the basal membrane with the help of Matrix Metalloproteinases, followed by the proliferation and migration of endothelial cells and ultimately formation of stable vessels. Tumor cells cause the secretion of a variety of factors that facilitate the newly formed blood vessels to end up in the tumor cells. Redrawn from (Nussenbaum & Herman 2010).
Fig.1.3. A three dimensional (a-d) and diagrammatic representation (a'-d') of the steps involved in transluminal bridge formation in Intussusceptive angiogenesis. (a-b, a'-b') represent the opposite walls of the endothelial cells coming into contact. Once in contact, the cells become perforated centrally (c') and this results in the formation of the pillar (d') (Makanya et al. 2009).
Angiogenesis is a crucial process required for several aspects of growth and development but when it goes askew it leads to pathogenesis. When the tissues get depleted in oxygen (hypoxia), there is an induction of cytokine production to maintain the balance in oxygenation. The most important cytokine for vessel growth is VEGF that differentially binds to its characteristic homologous receptor tyrosine kinases, inducing both physiological and pathological angiogenesis and vasculogenesis by a cascade of signal transduction events(Hoeben et al. 2004).
Arteriogenesis is a complex adaptive phenomenon that involves the rapid remodelling of the pre-existing collateral arteries to provide increased perfusion to the endangered ischemic regions(Buschmann & Schaper 1999). Fluid shear stress and inflammation act as the arteriole moulding agents. Mechanical shear stress induces the endothelial cells to activate chemical facilitators like MCP-1 (monocyte chemoattractant protein), MMP (Matrix metalloproteinases), TNF-Î± (tumour necrosis factor) and bFGF (Fibroblast growth factor) which in turn increases the diameter of vessels by promoting endothelial cell proliferation and remodelling(Carmeliet 2000).
1.2. VASCULAR ENDOTHILIAL GROWTH FACTORS:
The Vascular Endothelial Growth Factors are one of the key players of all the processes of Neovascularisation and maintenance of the vascular network in both the embryo and the adult humans. It is the only specific mitogen available for the vascular endothelial cells as it fails to show the same apparent specificity to other cell types. So far seven members of the VEGF family have been identified-Placental growth factor (PIGF), Vascular endothelial growth factors A,B,C,D, the orf virus VEGF-E and the snake venom (VEGF-F) .VEGFs have also found prominent roles in therapeutic vascularisation with the ability to initiate natural reparative mechanisms. Dysfunction in even a single VEGF allele promotes embryonic lethality as a result of a number of acute vascular abnormalities(Ferrara et al. 2003, Neufeld et al. 1999).
1.2.1 Vascular Endothelial Growth Factor -A:
VEGF-A is the master monitor of pathological and physiological neovascularisation. It is present in almost all vascularised tissues especially the perforated and sinusoidal epithelial tissues. It stimulates pronounced angiogenic activities by binding the receptors VEGFR1 and VEGFR2. VEGF-A production is mainly triggered by Hypoxia Inducible Factors (HIFs) and many other cytokines like transforming growth factors, fibroblast growth factors, platelet-derived growth factors and other growth factors(Ferrara et al. 2003, Roy et al. 2006). VEGF-A is otherwise called vascular permeability factor (VPF) for its importance in instigating vascular leakage. This finds a prominent place in inflammation and pathogenesis by increasing the permeability of secluded blood vessels. VEGF is important for stimulating the angioblasts to differentiate into endothelial cells and blood vessels to form the primitive vascular labyrinth in vasculogenesis. Additionally, immature blood vessels formed during vessel sprouting possess VEGF- dependent capillaries which when devoid of mural cell enclosure tend to subsequently regress. Moreover, VEGF is found to be abnormally upregulated during tumour angiogenic conditions than normal circumstances, thereby provoking tumour metastases. This over-expression of VEGF has been credited to the rapidly mutating populations of the neoplastic cells .It is also used for the inhibition of apoptosis in the endothelial proteins. It has significance in the maintenance of homeostasis by vasodilation by stimulating endothelial nitric oxide synthase thereby increasing the in vivo production of nitric oxide. Apart from endothelial cell proliferation, VEGF-A also induces the recruitment of haematopoietic stem cells from the bone marrow. It also acts in colony formation with the aid of mature granulocyte macrophage precursors(Yla-Herttuala et al. 2007, Veikkola & Alitalo 1999).
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The human VEGF-A gene is arranged into eight exons interspersed by seven introns. At present, seven splice variants have been identified VEGF121, VEGF 145, VEGF 148, VEGF165, VEGF 183, VEGF 189 and VEGF 206. Out of these, VEGF165 is the most studied dynamic one for stimulating pro-angiogenic actions as it corresponds to the properties of the native VEGF-A and stays attached to endothelial cell-surface and the extracellular matrix. These splice variants differ in their degrees of availability and occurrence in the endothelial cells. The isoform VEGF121, being an acidic polypeptide, is easily diffusible while the longer basic splice alternatives take part in binding to heparan sulphates with great affinity. VEGF 145 and VEGF 183 are the less frequently observed ones. The isoforms VEGF 189 and VEGF 206 are entirely contained in the extracellular matrix(Robinson & Stringer 2001). The heparan-binding domains containing the proteoglycans establish extracellular matrix binding by an enzyme called plasmin released by plasminogen activators which in turn are secretions of the endothelial cells. The heparan-binding isoforms render stimulatory cues to initiate vascular arborisation (Robinson & Stringer 2001).
Fig.1.4. The family of vascular endothelial growth factors and their associated receptors. Adapted from (Hoeben et al. 2004)
1.2.2. Placental growth factor (PIGF):
As the name suggests, Placental growth factor is expressed mainly in the placenta and also in the adult dormant vasculature and lungs. PIGF binds VEGFR1 for which it exhibits the greatest affinity. Like the VEGF, PIGF also exists as different isoforms hPIGF-1 (PIGF131), hPIGF-2(PIGF152) and mPIGF-1(PIGF203). These have reduced mitogenic activity when compared to the VEGF isoforms, but they can initiate and amplify the expression of VEGF when present in minimal concentrations by forming heterodimers with VEGF that is found upregulated in tumours and hypoxic conditions. PIGF as such is not required for embryonic angiogenesis but PIGF-deficient humans exhibit mitigated responses towards VEGF-A in pathological angiogenesis. They take part in angiogenesis by facilitating interaction between Flt1 and KDR by displacing the VEGF-A bound to FltI, thereby making it available for binding with KDR. But PIGF has not been able to displace VEGF-A from Flt1 entirely, because of the lower affinity of the PIGF towards VEGFR1 when compared to VEGF and due to its limited availability at developmental stages. PIGF can be used for therapeutic myocardial revascularisation in ischemic tissues and also in wound healing therapies. When the heart is hit by ischemia, the myocardium goes into hibernation by temporarily losing its contractile ability which can be restored only in the event of an effective revascularisation triggered by PIGF(Park et al. 1994). PIGF is better than VEGF in recovery of ischemia-affected organs from a functional point of view by its property to effect collateral growth. PIGF can also regenerate haematopoiesis by mobilizing these multipotent haemetopoietic stem cells from the bone marrow in a manner similar to that of VEGF-A. PIGF also plays a key role in the stimulation of arteriogenesis by providing a protective coating of smooth muscle cells in the budding vessels, thereby maintaining the consistency and the resilience of the newly formed capillaries. PIGF is also overexpressed in inflamed cells, one of the trademarks of pathological angiogenesis and collateral growth that tend to provide chemotactic signals for the newly differentiated inflammatory cells to penetrate into the areas of progressing inflammation(Carmeliet et al. 2001, Luttun et al. 2002).
Fig.1.5. Diagrammatic illustration of the roles of VEGF, VEGFR1 and PIGF in embryonic and pathological conditions. In the embryo VEGF induces angiogenesis by contacting VEGFR2. VEGFR1 is initially expressed in its inhibitory soluble form sVEGFR1. By minimal associations with VEGF, VEGFR1 acts as a 'decoy' receptor. In pathological conditions, PIGF is upregulated and binds to VEGFR1 by displacing VEGF from it to VEGFR2. This stimulates angiogenesis in a stronger manner due to the activation of VEGFR1 by PIGF that synergizes with newly coupled VEGF-VEGFR2. Dotted lines represent low expression while strong lines depict high expression. The inhibitory pathway is denoted in red. Figure taken from (Carmeliet et al. 2001).
1.2.3. VEGF-B is encoded by a gene located on chromosome 11 and it binds only to VEGFR1 but not to VEGFR2 or VEGFR3. It has two isoforms VEGF-B167 and VEGF-B187.The former is not glycosylated and hence binds to heparan sulphates while the latter is glycosylated and hence gets secreted. It is predominantly found in the striated muscles and hence find a role in energy metabolism. It also helps in regenerating ischemia-affected collaterals(Roy et al. 2006).
1.2.4. VEGF-C binds to both VEGFR2 and VEGFR3 with equal affinity but not to VEGFR1. Its isoforms are not the products of splicing but formed as a result of proteolytic processing. When bound to VEGFR2, it participates in the migration, mitogenesis and the differentiation of endothelial cells whereas when bound to VEGFR3 it takes part in lymphangiogenesis for the development of lymphatic vasculature that regulates the efficacy of the immune system by trafficking white blood cells(Tammela et al. 2005).
1.2.5. VEGF-D is mainly present adult tissues and is synthesized as a preproprotein like that of the VEGF-C that requires to be processed proteolytically for increased activity. It combines with VEGFR2 and VEGFR3 and mediates angiogenesis as well as lymphangiogenesis. It stimulates lymph vessel maturation and lymphatic metastasis, thereby indicating its survival in certain tumours(Ferrara et al. 2003).
1.2.6.VEGF-E is an Orf- virus encoded VEGF strain that shares ~20% of sequence homology with VEGF-A. They are hypothesized to take part in pathological angiogenesis associated with viral infections (Hoeben et al. 2004).
1.3. VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTORS:
Three VEGF type-III receptor tyrosine kinases have been recognized so far-VEGFR1/Flt1, VEGFR2/KDR and VEGFR3/Flt4.They are a subclass under the Platelet-derived growth factors(PDGF) receptor family, the only difference being PDGFR have 5 IgG domains while these receptors possess 7 IgG domains in their extracellular region(Neufeld et al. 1999, Davis-Smyth et al. 1998).
Human VEGFR1 is otherwise termed fms-like tyrosine kinase receptor-1 (Flt1). VEGFR1 can bind to VEGF-A,B and PIGF ,VEGF-A being the highest affinity. It exhibits a weak mitogenic and tyrosine kinase activity. The receptor gets activated on the formation of a dimer with VEGF resulting in a phosphotyrosine residue which in turn serves as a recruitment site for all signal conducting proteins. It is usually found in endothelial cells, mural cells, osteoblasts , macrophages and also haematopoietic cells and is abnormally upregulated during hypoxic and angiogenic situations.VEGFR1 signalling is important for monocyte mobilization which highly influences the angiogenic functions of VEGFR1(Shibuya 2006).Monoclonal antibodies against VEGFR1 inhibit the vascular growth thereby exerting a negative force during the neovascularisation of tumors and supports pathological angiogenesis. It is for this reason that both PIGF and Flt1 serve as attractive therapeutic agents for the regulation of angiogenesis and inflammation. It is also referred to as a 'decoy' receptor as it averts the binding of the VEGF-A with VEGFR2. A truncated short soluble isoform lacking an IgG domain is obtained by cloning from HUVEC (Human umbilical vein endothelial cells) cDNA library. This sVEGFR1 restrains the VEGF-A activity by sequestering them from other signalling receptors and by the formation of heterodimers with VEGFR2, that have no signalling activity. This spatial organization of VEGF-A mediated by sFlt1 play important roles in endogenous blood vessel formation(Luttun et al. 2002, Shibuya 2006).
Fig.1.6. Role of Anti-Flt1 in the obstruction of inflammation. Antibodies against Flt1 stop the progress of inflammation by affecting the myeloid progenitors mobilization from the bone marrow which in turn affects the differentiation of myeloid cells that produces inactivated macrophages. As a result of this there is a decreased production of the cytokine. Adapted from (Luttun et al. 2002)
This is otherwise called kinase domain region (KDR) which binds to all the members of the VEGF family except PIGF and VEGF-B. VEGFR2 is activated by a process called the dynamic predimerization. Binding with VEGF greatly increases the transcription and expression of this receptor. VEGFR-2 signalling is mitogenic and angiogenic and mediates the microvascular permeability in VEGF-A (Neufeld et al. 1999, Park et al. 1994).
Also named fms-like tyrosine receptor-4 has only six IgG domains instead of seven as the last one is subjected to proteolytic degradation soon after its synthesis. Unlike the other receptors, binds with VEGF-C and VEGF-D playing a prominent role in lymphangiogenesis.VEGFR3 thrives on all adult endothelial cells but is found to have higher expression in nascent blood vessels which gets superfluous as they mature (Ferrara et al. 2003, Veikkola & Alitalo 1999).
1.4. CRYSTAL STRUCTURES AND BINDING INTERFACE WITH VEGFR1:
The three different types of VEGFRs share a common biological structure. Each of the receptors contains seven Immunoglobulin like domains in its ectodomain with a transmembrane area , a conserved tyrosine kinase sequence intercepted by an kinase insert domain and a carboxy terminal end. The human VEGFR1 contains 1338 amino acid residues and possess a greater tendency to bind to VEGF-A than the KDR. The Ig domains 1-4 of Flt1 have been mapped for ligand- specific binding determinants with the second Ig domain holding the most importance. The efficacy of binding to the second IgG domain is unstable without the flanking first and third domains that determine the strength of the ligand-receptor binding. It has been substantiated that the first four domain binding determinants provide the same capacity and affinity as that of an entire VEGFR1 with all the seven IgG domains. The rest of the residues from4 to7 are proposed to initiate signal transduction pathways(Davis-Smyth et al. 1998, Muller et al. 1997). The receptors tend to have a number of potential N-linked glycosylation points and the molecular weights of their related proteins indicate both Flt1 and KDr are heavily glycosylated. This glycosylation accounts for the high affinity binding between the receptor and VEGF. Flt1 and KDR share a ~32% sequence homology which poses difficulty in understanding the specificity between the VEGF system and the receptor units. It has also been observed that out of the 19 FltI residues that are involved in the binding interface only 2 of them in KDR are conserved. This low conservation in the sequence hinders the modelling of a VEGF-VEGFR2 complex with higher accuracy. (Christinger et al. 2004, Wiesmann et al. 1997)
The members of the VEGF family are dimeric glycoproteins. Because of their differential splicing events, both VEGF and PIGF evolve as isoforms that differ in their molecular mass, patterns, of secretion and binding affinities. All the VEGF family members have a homologous domain. The central region is called the cysteine knot motif that has uniformly spaced eight cysteine residues. These invariant residues participate in a three inter- and intra-molecular disulfide interactions (N and C termini) at one end of a centrally conserved two pairs of twisted Î²-parallel sheet in each monomer that form dimers in an anti-parallel fashion(Robinson & Stringer 2001, Muller et al. 1997, Wiesmann et al. 1997).
Fig.1.7. Ribbon representation of VEGF in monomeric (left) and dimeric (right). These forms are produced with the program PyMOL. It shows how the parallel Î²-sheets in the monomer get dimerized in an anti-parallel fashion (Protein Data bank Code- 1VPF).(Research Colloboratory of Structural Bioinformatics, Protein Data Bank.2010)
VEGF-A in humans is a 30-42kDa homodimeric disulfide-bound glycoprotein which mediates its agonistic angiogenicity by differentially binding to both its cognate receptors VEGFR1 and VEGFR2. VEGF-A on binding to its distinct kinase receptors induces autophosphorylation. As mentioned already, a single VEGF-A gene has eight exons that code for atleast seven homodimeric isoforms. Structurally, the VEGF165 resembles PDGF existing as a covalently attached disulfide homodimer with 4 N-linked glycosylation units(Muller et al. 1997, Wiesmann et al. 1997).
Human PIGF possess the same attributes in its structure like the VEGF-A, holding a sequence identity of nearly ~50%.Till date, it has been found to have only three isomers the smallest containing around 131 amino acids. PIGF exclusively binds and induces autophosphorylation in VEGFR1 but not VEGFR2. In a purely dynamic form of the PIGF dimer, the two monomers are covalently attached with the help of disulfide bonds formed between cysteine residues. The interface between the two monomers is filled with the N-termini loops that stabilize the dimeric architecture(Christinger et al. 2004, Iyer et al. 2001). The complete interaction between the VEGFR1-d2 and PIGF is governed by the internal 2-fold symmetry of the ligand dimer and the receptor binds to the opposite ends of the ligand called the hotspots of receptor recognition. Unlike VEGF, receptor binding in the PIGF results in a major conformational change. Considerable conformational movement in PIGF have been perceived in the residues 43-45 and 83-85, those that relate to the receptor recognition spots of VEGF-A to KDR which rules out the structural ability of PIGF to ascertain binding with VEGFR-2(Christinger et al. 2004).
Each receptor monomer extends four distinct segments for contacting five segments in the PIGF homodimer. The residues that are involved in binding fall between 14-107 of VEGF-A, 22-115 of PIGF and 133-224 of VEGFR1-d2. PIGF competes with VEGF-A to bind with the first three domains of Flt1 displaying the fact that both the VEGF members share nearly same contact spots on the receptor. Moreover, the sequence conservation in the binding realm of VEGF-A and PIGF is ~65% which is much higher than the consensus sequence in the receptor entities(Christinger et al. 2004).
Fig.1.8.Ribbon representations of PIGF and VEGF bound to VEGFR1. This is generated using the program Pymol. These structures show the similarity borne between the two growth factors when in conjunction with the receptor. (Protein data bank codes 1RV6 and 1FLT respectively).
It is known that PIGF and VEGF-A have a ~50% sequence homology. These differences in residues are spotted in the hub of the binding interface with respect to VEGFR1 between PIGF and VEGF-A (table is based on Christinger et al. 2004))
The putative hydrophilic associations between PIGF and VEGFR1-d2 and that of VEGFA and VEGFR1d2 based on (Iyer et al. 2001) are as tabulated:
There are a number of other residues that are speculated to participate in weak hydrogen bonding and side chain interactions between the receptor and ligand.
To study the binding interface of PIGF-VEGFR1 and VEGFA-VEGFR1 using their published crystal structures (Christinger et al. 2004, Wiesmann et al. 1997) for the identification of amino acid residues in VEGFR1-ligand binding domain that confer ligand specificity. This will be tested by substituting candidate residues with others using site directed mutagenesis. Using this information I aim to generate a mutant form of VEGFR1 with increased specificity for PlGF. It is expected that data generated in this project will increase understanding of the molecular basis of ligand binding in VEGFR1. To do this, the following experiments are carried out:
Initially VEGFR1: PlGF and VEGFR1: VEGF-A crystal structures will be examined using PYMOL. Analysis of these structures, combined with alignment of primary sequences of VEGFR1 ligands will be used to identify candidate amino acid residues in VEGFR1 that confer ligand specificity. VEGFR-I receptor ectodomain will be cloned into a suitable expression vector.
Site directed mutagenesis will be used to create mutation at defined sites in VEGFR1 ectodomain. This is done using a kit that incorporates the mutations. Wild-type and mutant proteins will be expressed in eukaryotic cells and their expression will be confirmed by immunoblotting.
Binding assays (Competitive ELISA) will be conducted to test the effects of the mutations on binding to PlGF and VEGF-A. These will include ELISA assays in which binding to PlGF is competed with VEGF-A. In addition, relative affinities and off-rates for VEGF-A and PlGF will be determined.
Mutant forms of VEGFR1 ectodomain which exhibit altered ligand binding will be examined in more detail. Specially, the basis for the altered ligand binding will be investigated by examining the known crystal structures of VEGFR1:VEGF-A and VEGFR1:PlGF and the predicted effects of the substituted residues.
If time allows any mutant VEGFR1 forms will be tested in functional assays by examining the ability of specific VEGFR1 mutants to inhibit the functional effects of PlGF and VEGF-A on cells.