S. J. Harper

Heterogeneous vascular endothelial growth factor (VEGF) isoform mRNA and receptor mRNA expression in human glomeruli, and the identification of VEGF148 mRNA, a novel truncated splice variant.

Vascular endothelial growth factor (VEGF) mediates increased vascular permeability and endothelial mitogenesis, and may orchestrate normal glomerular permselectivity and proteinuria. Distinct isoforms result from differential gene splicing. VEGF binds to two cell surface tyrosine-kinase receptors, KDR (kinase domain region) and Flt-1 (fms-like tyrosine kinase-1). The latter also exists in a soluble form (sFlt), which is inhibitory. We have studied patterns of VEGF-isoform and VEGF-receptor expression in isolated single normal human glomeruli. mRNA from 190 glomeruli (from 20 individuals) was harvested on to magnetic beads, and nested reverse transcription-PCR was performed using primers for the VEGF isoforms and VEGF receptors. Simultaneous nested reverse transcription-PCR for CD45 was conducted in order to exclude leucocyte contamination. Unexpected products were isolated, cloned and sequenced. Multiple patterns of glomerular VEGF mRNA isoform expression were identified. Most frequently (58%), all three common forms were expressed. VEGF(189) (i.e. 189-amino-acid form of VEGF) was expressed in 63%, VEGF(165) in 85% and VEGF(121) in 84% of glomeruli. Two unexpected PCR products were also identified: 18% of glomeruli expressed VEGF(145), and 27% of glomeruli expressed a new truncated VEGF splice variant, VEGF(148), lacking exon 6, the terminal part of exon 7 and exon 8. Multiple patterns of VEGF-receptor expression were also identified, the most common being expression of all three isoforms (28%). Overall, KDR was seen in 59% of glomeruli, Flt-1 in 45% and sFlt in 57%. Thus the expression of VEGF within normal glomeruli is complex and variable, with inter- and intra-individual variation. Furthermore, sFlt appears to be the co-dominant form of VEGF receptor expressed within glomeruli, suggesting that, in healthy individuals, a degree of VEGF autoregulation is the norm. The physiological importance of VEGF(148) remains to be confirmed.

ZM323881, a Novel Inhibitor of Vascular Endothelial Growth Factor‐Receptor‐2 Tyrosine Kinase Activity

Objective: Vascular endothelial growth factor (VEGF) increases vascular permeability and angiogenesis in many pathological conditions including cancer, arthritis, and diabetes. VEGF activates VEGF‐Receptor 1(VEGF‐R1) and VEGF‐Receptor 2 (VEGF‐R2), which autophosphorylate to initiate a signaling cascade resulting in angiogenesis and increased microvascular permeability. Here we describe a novel VEGF‐R2 selective inhibitor, ZM323881 (5‐{[7‐(benzyloxy) quinazolin‐4‐yl]amino}‐4‐fluoro‐2‐methylphenol), that is a potent and selective inhibitor of VEGF‐R2 tyrosine kinase in vitro (IC50 < 2 nM), compared with other receptor tyrosine kinases, including VEGF‐R1 (IC50 > 50 µM).

Detection of VEGF-Axxxb Isoforms in Human Tissues

Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A165a, and anti-angiogenic isoforms typified by VEGF-A165b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A165b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A165b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF165b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A165b antibody. These findings support the conclusion that more information on the biology of VEGF-A165b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls.

Overexpression of VEGF165b in Podocytes Reduces Glomerular Permeability

The observation that therapeutic agents targeting vascular endothelial growth factor-A (VEGF-A) associate with renal toxicity suggests that VEGF plays a role in the maintenance of the glomerular filtration barrier. Alternative mRNA splicing produces the VEGF(xxx)b family, which consists of antiangiogenic peptides that reduce permeability and inhibit tumor growth; the contribution of these peptides to normal glomerular function is unknown. Here, we established and characterized heterozygous and homozygous transgenic mice that overexpress VEGF(165)b specifically in podocytes. We confirmed excess production of glomerular VEGF(165)b by reverse transcriptase-PCR, immunohistochemistry, and ELISA in both heterozygous and homozygous animals. Macroscopically, the mice seemed normal up to 18 months of age, unlike the phenotype of transgenic podocyte-specific VEGF(164)-overexpressing mice. Animals overexpressing VEGF(165)b, however, had a significantly reduced normalized glomerular ultrafiltration fraction with accompanying changes in ultrastructure of the glomerular filtration barrier on the vascular side of the glomerular basement membrane. These data highlight the contrasting properties of VEGF splice variants and their impact on glomerular function and phenotype.

Differential Expression of VEGF-Axxx Isoforms Is Critical for Development of Pulmonary Fibrosis

Rationale: Fibrosis after lung injury is related to poor outcome, and idiopathic pulmonary fibrosis (IPF) can be regarded as an exemplar. Vascular endothelial growth factor (VEGF)‐A has been implicated in this context, but there are conflicting reports as to whether it is a contributory or protective factor. Differential splicing of the VEGF‐A gene produces multiple functional isoforms including VEGF‐A165a and VEGF‐A165b, a member of the inhibitory family. To date there is no clear information on the role of VEGF‐A in IPF. Objectives: To establish VEGF‐A isoform expression and functional effects in IPF. Methods: We used tissue sections, plasma, and lung fibroblasts from patients with IPF and control subjects. In a bleomycin‐induced lung fibrosis model we used wild‐type MMTV mice and a triple transgenic mouse SPC‐rtTA+/−TetoCre+/−LoxP‐VEGF‐A+/+ to conditionally induce VEGF‐A isoform deletion specifically in the alveolar type II (ATII) cells of adult mice. Measurements and Main Results: IPF and normal lung fibroblasts differentially expressed and responded to VEGF‐A165a and VEGF‐A165b in terms of proliferation and matrix expression. Increased VEGF‐A165b was detected in plasma of progressing patients with IPF. In a mouse model of pulmonary fibrosis, ATII‐specific deficiency of VEGF‐A or constitutive overexpression of VEGF‐A165b inhibited the development of pulmonary fibrosis, as did treatment with intraperitoneal delivery of VEGF‐A165b to wild‐type mice. Conclusions: These results indicate that changes in the bioavailability of VEGF‐A sourced from ATII cells, namely the ratio of VEGF‐Axxxa to VEGF‐Axxxb, are critical in development of pulmonary fibrosis and may be a paradigm for the regulation of tissue repair.