VEGF and the ECM

Interactions with HSPG and the ECM

VEGF isoforms have variable affinities for heparan sulfate proteoglycans (HSPGs) on the cell surface and/or within the ECM (Robinson and Stringer, 2001). VEGF121 has lowest affinity, VEGF145 and VEGF165 have similar

Figure 1 Isoforms of Human VEGF-A Generated by Alternative Splicing. Alternative splicing of human VEGF-A has been reported to generate several isoforms. These isoforms differ by the inclusion or exclusion of domains encoded by exons six and seven. The location of a signal peptide and VEGF receptor (VEGFR1 and VEGFR2) binding sites common to all isoforms are indicated on VEGF206. HSPG binding sites common to isoforms with exons six and seven are indicated (*), as is the neuropilin-1 binding site (NP-1). Finally, arrows indicate sites of potential proteolytic processing by either plasmin or uPA. The exons and number of amino acids in each exon is indicated at the bottom of the figure.

Figure 1 Isoforms of Human VEGF-A Generated by Alternative Splicing. Alternative splicing of human VEGF-A has been reported to generate several isoforms. These isoforms differ by the inclusion or exclusion of domains encoded by exons six and seven. The location of a signal peptide and VEGF receptor (VEGFR1 and VEGFR2) binding sites common to all isoforms are indicated on VEGF206. HSPG binding sites common to isoforms with exons six and seven are indicated (*), as is the neuropilin-1 binding site (NP-1). Finally, arrows indicate sites of potential proteolytic processing by either plasmin or uPA. The exons and number of amino acids in each exon is indicated at the bottom of the figure.

Table I VEGF Isoforms from Alternative Splicing in Species Studied to Date.

Species VEGF-A Isoforms (number of amino acids)

Human

121, 145,

165,

183, 189, 206

Mouse

115, 120,

164,

188

Quail

122, 146,

166,

190

Rat

120, 164,

188

Bovine

120, 164

affinities, and VEGF 189 and VEGF206 have the highest affinities for HSPG (and heparin in vitro) (Table II). The high affinity of VEGF189 and VEGF206 is a result of the presence of exons 6 and 7, which mediate binding to HSPGs (Figure 1). Exon 6a in VEGF145 mediates ECM binding independent of HSPGs because of a cell surface retention sequence. The affinity of a VEGF isoform for HSPGs is directly correlated with its diffusibility (Figure 2). As a result, the smallest isoform (VEGF121) is secreted and freely diffusible. Approximately 50 percent of intermediate-sized isoforms (VEGF145 and VEGF165) diffuse from producing cells (based on their presence in conditioned media), whereas the remainder are cell and/or ECM associated. Finally, the largest isoforms (VEGF189 and VEGF206) are secreted from producing cells but completely sequestered within the ECM with low amounts bound to the cell surface

Table II Biochemical Properties of Human VEGF Isoforms.

Isoform

Exons

MW (kDa)

Acidic or basic

Heparin binding

121

1-5, 8

34

Weakly acidic

no

145

1-5, 6a

, 8

41

yes

165

1-5, 7,

8

46

basic

yes

183

1-5, 6a

*, 7, 8

46

yes

189

1-5, 6a

, 7, 8

52

basic

yes

206

1-5, 6a

, 6b, 7, 8

~60

basic

yes

Molecular weight (MW) based on the sum of monomer band sizes. * indicates that exon 6a is truncated.

Molecular weight (MW) based on the sum of monomer band sizes. * indicates that exon 6a is truncated.

(Figure 2). Similar to VEGF 189 and VEGF206, most of VEGF183 remains bound to the cell surface [1], suggesting it has a high affinity for HSPGs. Although most of the iso-forms can bind HSPG and/or the cell surface, they can be released as soluble, biologically active molecules.

Release of VEGF from the ECM

Addition of heparin to cells stably expressing VEGF165 resulted in a two- to fourfold increase in the level of VEGF in conditioned media. Therefore, VEGF165 can be released from the cell surface and/or ECM by heparin. A similar

Figure 2 Diffusibility or Cell-Association of VEGF Isoforms. VEGF is depicted as being produced by an epithelial cell layer and diffusing towards endothelial cells, where it binds to the neu-ropilins (NP-1 or NP-2) or to VEGF receptors (VEGFR1 or VEGFR2). VEGF isoforms are diffusible to different degrees, with VEGF 121 completely diffusible, while VEGF 189 (or VEGF 183) and VEGF206 remain bound to the cell surface and/or within the extracellular matrix. VEGF189 (and 206) can be cleaved by plasmin to generate a diffusible form, VEGF110.

Figure 2 Diffusibility or Cell-Association of VEGF Isoforms. VEGF is depicted as being produced by an epithelial cell layer and diffusing towards endothelial cells, where it binds to the neu-ropilins (NP-1 or NP-2) or to VEGF receptors (VEGFR1 or VEGFR2). VEGF isoforms are diffusible to different degrees, with VEGF 121 completely diffusible, while VEGF 189 (or VEGF 183) and VEGF206 remain bound to the cell surface and/or within the extracellular matrix. VEGF189 (and 206) can be cleaved by plasmin to generate a diffusible form, VEGF110.

effect was observed for heparinase. All VEGF isoforms within the ECM or on the cell surface can be rapidly released by proteolytic cleavage with plasmin, a serine protease (Robinson and Stringer, 2001). Plasmin cleavage produces a 110 amino acid (aa) fragment (Figure 2) that can bind VEGFR2 but not heparin. This 110aa fragment is active as an EC mitogen and vascular permeability factor similar to VEGF121, with activity reduced fiftyfold relative to VEGF165. Plasmin cleavage results in a second smaller, heparin-binding fragment without mitogenic activity because the VEGF receptor-binding domains are absent. Thus, the carboxyl-terminal heparin-binding domains of VEGF are essential for full mitogenic activity, as their deletion by proteolytic cleavage or alternative splicing significantly reduces activity. VEGF189 and perhaps VEGF183 and VEGF206 are also processed by the serine protease urokinase type plasminogen activator (uPA) near the car-boxyl terminus of the domain encoded by exon six. This results in a truncated VEGF (uPA-VEGF189) with activity similar to native VEGF165. These processing steps are required for mitogenic activity and binding toVEGFR2, but not for binding to VEGFR1.

Essentials of Human Physiology

Essentials of Human Physiology

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