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【摘要】 Despite production by podocytes of the proangiogenic molecule vascular endothelial growth factor-A (VEGF), the glomeruli are not sites of angiogenesis. We recently described mRNA expression of an inhibitory splice variant of VEGF (VEGF 165 b) in normal kidney (Bates DO, Cui TG, Doughty JM, Winkler M, Sugiono M, Shields JD, Peat D, Gillatt D, and Harper SJ. Cancer Res 62: 4123-4131, 2002). Available anti-VEGF antibodies do not distinguish stimulatory from inhibitory VEGF families. To assess the production of VEGF 165 (stimulatory) and VEGF 165 b (inhibitory) isoforms by human podocytes, we examined both primary cultured and conditionally immortalized human podocytes using family- and isoform-specific RT-PCR. In addition, VEGF protein production was analyzed in podocytes, using isoform-specific double-strand small-interference RNAs (siRNA). RT-PCR demonstrated the production of VEGF 189 mRNA by podocytes of both phenotypes. In contrast, on differentiation there was a splicing change from VEGF 165 to VEGF 165 b mRNA. In addition, VEGF protein in the supernatant of conditionally immortalized, differentiated podocytes was reduced by VEGF 165 b siRNA to 20 ± 11% of the level of mock-transfected cells ( P < 0.01). No reduction was seen with mismatch siRNA. Moreover, there was no reduction in VEGF protein concentration in the supernatant of primary cultured, dedifferentiated human podocytes (109 ± 8% of mismatch siRNA, P 0.1). In conclusion, differentiated but not dedifferentiated human podocytes secrete significant amounts of VEGF 165 b protein. It is possible that this may explain the paradox of high VEGF production in the glomerulus but no angiogenesis. Furthermore, the existence of this splicing switch in relation to podocyte phenotype suggests that alternative splicing of the VEGF pre-RNA is a regulated process that is open to manipulation and therefore could be a target for novel cancer therapies.
【关键词】 angiogenesis smallinterference RNA splicing
VASCULAR ENDOTHELIAL GROWTH factor-A (VEGF) is the most potent and dominant proangiogenic factor in physiological and pathological angiogenesis and, as such, the overexpression of VEGF is believed to be a crucial pathophysiological step in many diseases, including cancer, atherosclerosis, arthritis, and psoriasis ( 8, 11 ). The multiple isoforms of VEGF stimulate endothelial cell proliferation, migration, and increased microvascular permeability by activation of VEGF receptor 1 (flt-1) and VEGFR-2 (KDR/flk1). VEGFR-1 also exists in a soluble form, sVEGFR-1 (sFlt), which is inhibitory when bound to free VEGF ( 12 ). Neuropilin-1 (NP-1) facilitates the binding of VEGF 165 to VEGFR-2 ( 14 ). VEGF receptor signaling is incompletely understood but is rapidly becoming characterized both in vitro and in vivo ( 3, 4 ). Available data suggest that increased permeability is mediated by intracellular calcium ( 3 ) but compliance and mitogenesis via MAPK ( 4 ).
VEGF-A is differentially spliced from eight exons, resulting in different proteins named by their amino acid number: VEGF 121, VEGF 165, (the dominant isoform) VEGF 189, etc. ( Fig. 1 A ). We recently identified an mRNA encoding a novel isoform VEGF 165 b ( 2 ) in normal kidney and other tissues. VEGF 165 b has a 3'-splicing structure that predicts a novel COOH-terminal peptide sequence. Usually, the COOH terminus of VEGF consists of six amino acids encoded by the first 18 nucleotides of exon 8. In VEGF 165 b, that is replaced by six amino acids coded for by an 18-nucleotide open-reading frame formed from a more distal splice site (DSS) selection in exon 8. We initially termed this new open-reading frame "exon 9" ( 2 ). However, there is no true "intron" between the two 18-base sequences and this new open-reading frame should more correctly be termed "exon 8b." Thus DSS selection, "exon 8b," in place of proximal splice site (PSS) selection, "exon 8a," predicts the existence of a family of sister isoforms ( Fig. 1 B ) with a novel COOH terminus. We termed the putative family VEGF xxx b where xxx is the amino acid number. The VEGF xxx b isoforms retain receptor binding and dimerization domains (exons 3&4) ( 2 ) and are the same size as their sister molecules, so are identified as the same product on most published blotting and RT-PCR analysis, which perhaps explains their previous elusiveness.
Fig. 1. COOH-terminal exon structure of stimulatory and inhibitory vascular endothelial growth factor (VEGF) families. PSS and DSS, proximal and distal COOH-terminal splice site selection, respectively.
Glomerular VEGF remains enigmatic. In situ hybridization ( 1 ) and immunohistochemical analyses ( 5 ) define the podocyte as the site of glomerular VEGF production in vivo. Glomerular endothelial cells ( 10 ) and podocytes ( 13, 16 ) express VEGF receptors. A paracrine and perhaps, more intriguingly, an autocrine role would therefore seem possible. However, despite high-level production of VEGF by podocytes and the expression of VEGFR-2 by glomerular endothelial cells, angiogenesis is not a feature of the normal glomerulus. This has previously been explained by two proposals: first, the potential difficulty of podocyte-derived VEGF to target glomerular endothelial cell VEGF receptors, which microanatomically are upstream of the site of production. However, VEGF is clearly able to cross this filtration stream because immunogold electron microscopic studies showed a clear concentration gradient of labeled VEGF particles from glomerulus to endothelial cells, with VEGF being clearly apparent on the endothelial side of the glomerular barrier ( 13 ). Second, the expression of VEGF inhibitors within the normal glomerulus, for example, sFLT ( 27 ) or angiopoietin ( 22 ), prevents angiogenesis. While investigating the apparent paradox, we identified VEGF 165 b. In contrast to all exon 8 proximal splicing variants, VEGF 165 b inhibits VEGF 165 -mediated endothelial cell proliferation and migration in vitro and vasodilatation ex vivo ( 2 ). Furthermore, there is preliminary evidence that VEGF 165 b is not angiogenic in vivo (Woolard JW, unpublished observations).
VEGF 165 b is potentially, therefore, an endogenous inhibitor of VEGF-mediated angiogenesis in the glomerulus. However, there is currently no evidence that VEGF 165 b is endogenously expressed by any human cells. There are no currently available antibodies that can distinguish proximal from distal COOH-terminal splice forms, and previously published work used recombinant VEGF 165 b, overexpressed in a mammalian expression system, so endogenous VEGF 165 b protein expression has not yet been shown in any tissue. Furthermore, we have not shown that the mRNA splice form of VEGF that would result in VEGF 165 b is actually produced in the glomerulus or in any endogenous glomerular cell type. Previous expression work used homogenized renal cortex, and, because isoform-specific in situ hybridization probes have not been successfully developed, the location of expression in the cortex is still uncertain. Although it is not clear whether the renal cortex cell type that expresses VEGF 165 b is the podocyte, the endothelial cells and mesangial cells rarely if ever express VEGF.
Because no antibodies are available that distinguish between the isoforms, we used an interference RNA knockdown technique to determine whether cells endogenously produce VEGF protein. This technique relies on the recently described small-interference RNAs (siRNA), double-strand 19-bp stretches of RNA that use an endogenous RNA degradation mechanism to regulate gene expression. The double-strand RNA is transfected into cells, where it binds to the RNA-induced silencing complex (RISC). RISC uses the siRNA to recognize endogenous mRNA sequences that contain the complementary sequence to the siRNA and degrade the endogenous mRNA ( 19 ). To test the hypothesis that VEGF 165 b protein and mRNA are secreted by human podocytes, we examined isoform-specific mRNA expression by RT-PCR and designed an RNA interference knockdown technique to determine whether VEGF 165 b protein is endogenously produced by human podocytes of both proliferating dedifferentiated and growth-arrested, differentiated phenotypes.
METHODS
Podocytes
Proliferating dedifferentiated podocytes derive from two sources: first, from primary culture from unipolar renal tumor nephrectomy samples collected with local Ethical Committee approval; second, from a conditionally immortalized human podocyte cell line. Podocytes were isolated from the normal pole of specimens by standard sieving techniques ( 17 ). These cells have been previously characterized as cytokeratin and WT-1 positive (immunofluorescence); VEGF, WT-1 and synaptopodin mRNA positive (RT-PCR); and von Willebrand factor, CD45 and smooth muscle myosin negative (RT-PCR), excluding endothelial cell, leucocyte, or mesangial cell contamination, respectively, as we previously described ( 16, 20 ). This phenotype was confirmed by regular sampling.
Differentiated, growth-arrested podocytes were derived from a cell line conditionally transformed from normal human podocytes with a temperature-sensitive mutant of immortalized SV-40 T-antigen as described elsewhere ( 21 ). At the "permissive" temperature of 33°C, the SV-40 T-antigen is active and allows the cells to proliferative rapidly. Thermoswitching the cells to the "nonpermissive" temperature of 37°C inactivates the T-antigen, and the cells become growth arrested and differentiate to express antigens appropriate to in vivo arborized podocytes. Cells were grown for a period of 16 days at 37°C to ensure growth arrest and differentiation.
Molecular Biology
Molecular biology tools were purchased from Invitrogen unless otherwise stated.
RT-PCR
RT-PCR was performed on dedifferentiated proliferating cultured podocytes (PCP), growth-arrested differentiated conditionally immortalized podocytes (DCIP), and sieved human glomeruli. RT-PCR to differentiate between exon 8a- and exon 8b-containing isoforms within the same sample in the same reaction was performed as previously described ( 2 ) using primers specific to exon 7a and 3' untranslated region (UTR). Exon-specific RT-PCR for exon 8a (PSS)-containing angiogenic mRNA isoforms was amplified using primers to exon 4 and exon 8a. Exon 8b (DSS)-containing inhibitory isoforms were detected using exon 4 and exon 8b sequences. Primer sequences are shown in Table 1. mRNA was extracted from 10 sieved glomeruli, primary cultured, or conditionally immortalized podocytes from confluent 75-ml culture flasks using standard techniques ( 6 ). Six percent of the RNA was reverse transcribed using MMLV reverse transcriptase and poly-d(T) primer. One micromolar of each appropriate primer, 1.2 mM MgCl 2, 200 µM dNTPs, and 1 unit of Taq (Abgene) were used. Reactions were cycled 35 times denaturing at 96°C for 30 s, annealing at 55°C for 30 s (exon 8a and 3'UTR primers) or 65°C (exon 8b primers), and extension at 72°C for 60 s. Products were run on 2% agarose gels containing 0.5 µg/ml ethidium bromide and visualized under a UV transluminator.
Table 1. Primer sequences
siRNA Synthesis
Double-strand siRNA was synthesized using in vitro transcription ( Fig. 2 ). Briefly, primers designed to degrade the target sequence were used to make double-strand DNA encoding the T7 RNA polymerase promoter. Single-strand RNA was made from each primer pair, denatured at 96°C, and annealed by cooling to form dsRNA. This was transfected into human embryonic kidney (HEK) cells or podocytes using lipofectamine. Double-strand siRNAs specific for exon 8b-containing mRNA (i.e., across exon 7-8b boundary) were made in this way, but unfortunately, it was not possible to construct a similar siRNA for exon 8a-containing VEGF mRNA species (e.g., VEGF 165 mRNA) because the optimal C-(N) 19 -G sequence across the splice site of exons 7-8a was not present. siRNA was therefore synthesized for an area of the mRNA common to all isoforms, the exon3/exon4 boundary.
Fig. 2. Interference RNA. Primers designed to degrade the target sequence (a) are used to make double-strand DNA (b) encoding the t7RNA polymerase promoter. Single-strand RNA is made from each primer pair and then hybridized to form dsRNA (c). This is transfected (d) into cells where it binds with an RNA-induced silencing complex (RISC), which recognizes the target sequence and enables degradation of the endogenous mRNA.
Primers were synthesized for VEGF 165 b sense (forward and reverse), VEGF 165 b anti-sense (forward and reverse), VEGF 165 b sense with a 2-bp mismatch (reverse only), and VEGF 165 b anti-sense with a 2-bp mismatch (reverse only). Mismatch siRNA acted as a control for nonspecific degradation of secreted protein. For each pair of primers, 1 ) double-strand DNA templates were synthesized using 1 µM primer, 1.2 mM MgCl 2, 200 µM dNTPs, and 1 unit of Taq (Abgene) in 1 x Taq buffer. Reactions were cycled five times denaturing at 94°C for 30 s, annealing at 37°C for 1 min, and extension at 72°C for 3 min. Products were phenol-chloroform extracted and pellets were resuspended in 20 µl diethylpyrocarbonate (DEPC) water. 2 ) Single-strand RNA was synthesized using 1 µM DNA templates, 20 U RNA guard, 200 µM rNTPs, and 80 units of T7 polymerase (Pharmacia) in transcription buffer. The above were incubated for 1 h at 37°C and then digested with 1 unit RNase-free DNAase (Promega) at 37°C to release single-strand RNA. Products were phenol-chloroform extracted and pellets were resuspended in 20 µl DEPC water, and a further round of phenol-chloroform extraction was performed and pellets were resuspended in annealing buffer (10 mM Tris·HCl, 100 mM NaCl). 3 ) Double-strand RNA was produced by mixing equal amounts of the appropriate two products, heating to 95°C for 5 min and cooling to room temperature. Aliquots of each double-strand RNA were run on a 1.5% agarose gel to confirm synthesis.
Transfection Protocol and Assessment of Protein Production
Cells were grown in six-well plates, each well seeded with 3 x 10 5 cells. Initial experiments were conducted on HEK cells transfected with pcDNA3-VEGF 165 b or pcDNA3-VEGF 165 ( 2 ). Stable cell lines were produced using Geneticin selection. Stable or transiently transfected HEK cells and podocytes were transfected with siRNA using lipofectamine. The concentration of VEGF secreted into the media was assayed using a pan-VEGF ELISA (R&D Systems, Duo-set DY293).
RESULTS
RT-PCR
Consistent products were produced on repeated analysis. DCIP produced products consistent with the inhibitory isoform VEGF 165 b and the stimulatory isoform VEGF 189 ( Fig. 3 A ). We were unable to identify exon 8b (inhibitory)-containing VEGF mRNA isoforms in PCP, despite efficient mRNA extraction and reverse transcription as assessed by the presence of 8a products and efficient PCR, as seen in Fig. 3 A. In contrast, the stimulatory VEGF isoform VEGF 165 and VEGF 189 were readily detected ( Fig. 3 B ). RT-PCR for exon 8a- and exon 8b-containing isoforms using exon 7 and 3'UTR primers in DCIP and freshly isolated human glomeruli confirmed the presence of exon 8b-containing isoforms ( Fig. 3 C ). Previous work from this laboratory showed that both exon 8b- and exon 8a-containing isoforms are found in freshly isolated human glomeruli ( 27 ).
Fig. 3. A : exon (Ex)-specific RT-PCR on differentiated, growth-arrested conditionally immortalized podocytes (DCIP). Lanes 1 - 3 : PCR for exon 8b-containing isoforms. Lane 1 : VEGF 165 b cDNA (positive control). Lane 2 : exon 8b product, size compatible with VEGF 165 b. Lane 3 : water control. Lanes 4 and 5 : PCR for exon 8a-containing isoforms. Lane 4 : VEGF 165 cDNA (positive control). Lane 5 : exon 8a product, size compatible with VEGF 189. Lane 6 : molecular weight ladder. B : exon-specific RT-PCR on proliferating primary cultured podocytes (PCP). Lanes 7 and 9 : PCR for exon 8b-containing products. No products were detected. Lanes 8 and 10 : PCR for exon 8a-containing products showed PCR products consistent with VEGF 165 and VEGF 189. C : RT-PCR for composite exon 8a and 8b products in DCIP ( lane 11 ) and freshly harvested human glomeruli ( lane 12 ). No 8a products were identified; however, exon 8b isoforms were identified in both.
VEGF Secretion by Cells in Culture
To determine whether cells in culture could secrete VEGF into the media, conditioned media was tested from a variety of cell lines using the commercially available ELISA. VEGF was not detected in conditioned media from HEK 293Q, Chinese hamster ovary, MCF7 breast cancer cells, fibroblasts, human umbilical vein endothelial cells, or human dermal microvascular endothelial cells. Very low concentrations (50-100 pg/ml) were detected in the conditioned media of A375 cells as previously described ( 23 ). Conditionally immortalized podocytes, on the other hand, expressed significant concentrations of VEGF during proliferation (3.1 ± 0.14 ng/ml), after transfection ( 800 pg/ml), and after differentiation ( 150 pg/ml). We therefore used these cells to determine the effects of siRNA specific for VEGF 165 b on VEGF protein production.
siRNA Studies
HEK cell line. To determine whether siRNA could specifically target VEGF 165 b protein expression, studies were carried out in HEK cells (which do not constitutively express VEGF) stably transfected with VEGF 165 b cDNA. VEGF 165 b siRNA significantly inhibited VEGF 165 b protein expression. VEGF 165 b protein in the supernatant of HEK cells transfected with VEGF 165 b cDNA was significantly lower when cotransfected with VEGF 165 b siRNA (39 ± 0.9 ng/ml) than with mismatch siRNA (123 ± 4 ng/ml, P < 0.007). In contrast, VEGF 165 production was not affected in cells transfected with VEGF 165 cDNA and VEGF 165 b siRNA (76.7 ± 4 ng/ml compared with 80 ± 4 ng/ml with mismatch siRNA; Fig. 4 ). Transient cotransfection of HEK cells with VEGF 165 b and siRNA showed that the inhibition was dose dependent ( Fig. 5 ). The transfected cells produce large amounts of VEGF protein as the production is under the control of the cytomegalovirus promoter. Despite this, the siRNA reduced the production by over 200 ng/ml.
Fig. 4. Small-interference (si)RNA inhibits VEGF 165 b but not VEGF 165 protein expression in transfected human embryonic kidney (HEK) cells, VEGF expression of media of cells cotransfected with siRNA for VEGF 165 b, or a 2-base mismatch RNA and pcDNA 3 -VEGF 165 b ( left ) or pcDNA 3 -VEGF 165 ( right ).
Fig. 5. VEGF 165 b siRNA dose dependently inhibits VEGF 165 b protein expression in transfected cells. VEGF concentration of media taken from HEK293 cells transfected with 2 µg VEGF 165 b cDNA (in expression vector pcDNA3) alone or with increasing amounts of double-strand siRNA directed across the exon 7 exon 8b splice site is shown.
DCIP. DCIP expressed VEGF protein that was blocked by VEGF 165 b-specific siRNA ( Fig. 6 ). Transfection of DCIP with VEGF 165 b siRNA reduced VEGF production to 26 ± 10% compared with before transfection from 151 ± 11 to 38 ± 6.8 pg/ml ( P < 0.001, paired t -test). In contrast, no reduction was seen with mismatch siRNA (from 157 ± 6 to 190 ± 31 pg/ml, P 0.1, paired t -test), demonstrating that this effect was specific for VEGF 165 b and not a nonspecific effect on total protein production.
Fig. 6. DCIP express VEGF protein that is blocked by VEGF 165 b-specific siRNA. Effect of transfecting siRNA for VEGF 165 b or a 2-base mismatch RNA on VEGF expression in the media of DCIP is shown.
Primary cultured podocytes. The VEGF concentration of media from PCP was 3.1 ± 0.14 ng/ml ( n = 15). Transfection of PCP with siRNA reduced the total VEGF production, irrespective of the sequence of the siRNA. However, transfection with pan-VEGF siRNA reduced the VEGF production to a significantly lower level (to 586 ± 17 pg/ml) compared with that with the 2-bp mismatch siRNA (867 ± 41 pg/ml; Fig. 7 ). A pan-VEGF siRNA could therefore significantly reduce the VEGF in primary PCP to 67% of control ( P < 0.01, t -test). This was not seen with VEGF 165 b-specific siRNA. Transfection with VEGF 165 b siRNA reduced VEGF protein production to 928 ± 65 pg/ml, which was not significantly different from that induced by transfection with 2-bp mismatch siRNA 855 ± 69 pg/ml (109% of control). Primary cultured podocytes therefore express VEGF isoforms that do not contain DSS selection, i.e., they express angiogenic, stimulatory forms. The use of VEGF 165 -transfected cells, mismatch sequences, and a pan-VEGF siRNA confirms the specificity of the VEGF 165 b siRNA for sequence and target mRNA.
Fig. 7. Freshly isolated, proliferating, dedifferentiated podocytes express VEGF protein that is not blocked by VEGF 165 b-specific siRNA. Effect of transfecting siRNA for VEGF 165 b, pan-VEGF, or 2-base mismatch RNA (pan or VEGF 165 b, respectively) on VEGF expression in media of proliferating dedifferentiated podocytes is shown.
DISCUSSION
Unipolar renal tumors demonstrate a paradox: one pole contains a highly angiogenic lesion expressing high levels of VEGF mRNA and protein ( 17, 26 ); the opposite, histologically and functionally normal pole also expresses high levels of VEGF mRNA and protein in the absence of new vessel formation. We showed that the VEGF 165 b mRNA ("exon 8b" inhibitory), normally expressed in whole kidney, is downregulated in renal cell carcinoma, in contrast to all other isoforms in all other tumors ( 8, 12 ).
Investigation of exon 8b-containing isoforms in vivo at the protein level presents some difficulties because "exon 8b" distal COOH-terminal splice site-specific antibodies are unavailable. In situ hybridization will not differentiate between exon 8a (PSS) and exon 8b (DSS) mRNA species because the difference between full-length mRNAs is only 18 bases. A single oligonucleotide (for instance, across exon 7-8b splice site) will not produce enough visible signal isotopically or after optimization of hapten-labeling and detection as we previously described ( 15 ). Furthermore, an exon 8b-containing sequence is present in both stimulatory and inhibitory VEGF families, as part of the 3'UTR and as coding sequence, respectively. We therefore approached this study using conventional RT-PCR and siRNA technologies to investigate VEGF expression of human podocytes in culture.
The use of podocytes in vitro attracts two potential criticisms, the issues of purity and differentiation status. We did our utmost to ensure purity of our primary cultures and covered this issue elsewhere ( 16, 20 ). We also studied a conditionally transformed podocyte cell line that can be investigated in both dedifferentiated and differentiated phenotypes and is a pure population ( 18, 21 ).
The data we present are the first evidence that human cells can endogenously express exon 8b-containing isoforms at the protein level and that they can regulate this expression during differentiation, presumably by regulation of splicing. In addition, this study provides new information concerning changes in podocyte VEGF splicing that relate to the level of differentiation. VEGF 189 mRNA is expressed by both podocyte phenotypes. VEGF 189 is avidly heparin bound and in vivo would tend to remain cell and glomerular basement membrane associated. However, to our surprise, a splicing alteration seems to occur in VEGF 165 /VEGF 165 b isoforms in association with podocyte differentiation status. There is a switch from exon 8b-containing inhibitory VEGF 165 b in differentiated podocytes to exon 8a-containing stimulatory VEGF 165 in dedifferentiated podocytes.
The VEGF glomerular literature is conflicting. Floege and colleagues ( 18 ) attempted to inhibit VEGF production in vivo in healthy animals using aptamers, failing to define any glomerular change after 3 wk of administration. However, targeted pan-VEGF (both stimulatory and inhibitory) inhibition and overexpression in podocytes have now shown that a balance of VEGF is required for normal glomerular well-being. A Cre-recombinase knockout of even a single gene copy leads to nephrotic syndrome, uremia, and death 9 wk postpartum, and complete knockout results in death a few hours postpartum ( 9 ). In a transgenic model, VEGF overexpression resulted in death a few days postpartum with renal hemorrhages ( 9 ). In addition, a recent study demonstrated that in vivo inhibition of VEGF with antibody or soluble receptor caused nephrotic syndrome in mature mice ( 24 ).
In glomerular disease, aptamer VEGF inhibition in a model of glomerulonephritis characterized by endothelial cell damage impaired glomerular repair ( 18 ). In contrast, inhibition of VEGF in streptozotocin-induced diabetic animals produced a beneficial reduction in proteinuria ( 7 ).
Some of this data may suggest that VEGF balance is important to glomerular well being and repair. However, overall these contradictory findings cannot be reconciled if VEGF is only considered as a proangiogenic, propermeability vasodilator. However, should the splicing changes we demonstrated in vitro be mirrored in vivo, some of the contradictions are explained.
We hypothesize, therefore, that in healthy glomeruli in vivo there is a balance between COOH-terminal distal and proximal splice site selection. When podocytes dedifferentiate or are injured in glomerular disease, there is a switch from inhibitory exon 8b-containing VEGF isoform expression to stimulatory exon 8a-containing VEGF isoform expression. This would be an appropriate physiological response because exon 8a-containing VEGF isoforms are well-characterized survival factors for endothelial cells. In addition, we recently reported the ability of VEGF 165 to act as an autocrine survival factor for podocytes themselves via a PI3 kinase-dependent pathway ( 13 ). Such a splicing event, therefore, in the context of glomerular injury would be beneficial for both glomerular endothelial cells and podocytes. However, we also hypothesize that this survival response for podocytes and endothelial cells occurs at the expense of proteinuria.
If we accept this hypothesis, some conflicting findings are resolved; for example, the aptamer studies that failed to demonstrate any change in health by aptamer administration but produced a detrimental effect of glomerular repair when administered in a model of glomerulonephritis in which endothelial damage predominated ( 18 ). Aptamers are sequences of nucleic acids that bind to specific areas of proteins because of their three-dimensionsal shape not because of their sequence. They are very specific; indeed the aptamer used in these studies was specific for exon 7 in VEGF 165. Substitution of exon 8a by exon 8b (as in VEGF 165 b) would result in significant conformational change to the terminal part of exon 7 because at least one disulfide bond is lost ( 2 ). This aptamer may well therefore have no effect in healthy glomeruli in which exon 8b isoforms predominate but would have a potent effect on glomerular endothelial cell survival in glomerular disease in which exon 8a isoforms predominate. Furthermore, the detrimental effect of VEGF inhibition in the above animal model contrasts with the beneficial effect of VEGF inhibition in streptozotocin-induced diabetic nephropathy ( 7 ), in which we presume endothelial cell damage is not a major feature but that the proteinuria induced by the splicing change is amenable to manipulation.
The most recent study to shed light on glomerular VEGF biology ( 24 ) contrasted with both the above studies by showing that inhibition of VEGF (a propermeability agent) caused an increase in microvascular permeability and precipitated nephrotic syndrome. This is indeed an unexpected finding. These authors claim that the inhibitory agents affect primarily the circulating VEGF pool. This may be the case but will require further study. What is clear is that many tissues seem to produce both stimulatory and inhibitory VEGF isoforms, at least at the mRNA level, and that within normal tissues a balance may exist ( 2 ). This may also be the case with the circulating VEGF pool, the balance of which may bear no relationship to the balance within a particular microanatomic site. Interpretation of studies based on systemic administration of VEGF inhibitors may always, therefore, be fraught with difficulty because it is impossible to know which pool or pools (circulating and/or tissue, e.g., glomerular) are inhibited and the site at which this inhibition occurs.
The VEGF splicing change we demonstrated is identical to that which tissues undergo during malignant change. We initially demonstrated this in renal cell carcinoma and more recently showed the same change in prostate cancer; indeed in this lesion the splicing change occurs at the prostatic intraepithelial neoplasia (carcinoma in situ) stage ( 25 ). The detailed control of VEGF splicing is one of the few areas of VEGF that have been poorly investigated. A fuller understanding of the VEGF splicing mechanism that podocytes use may therefore bring benefits to glomerular pathology but also to angiogenesis-based disease.
In summary, VEGF 165 b protein, a VEGF isoform that is downregulated in renal and prostate cancer, inhibits VEGF 165 -mediated endothelial cell proliferation and migration, and vasodilatation, is not angiogenic in vivo, and is the dominant form of VEGF expressed by differentiated human podocytes. It is not expressed, however, by PCP.
ACKNOWLEDGMENTS
The authors thank Dr. K. Zavitz for Martini-inspired experimental design.
GRANTS
This work was supported by the Association for International Cancer Research Project Grant 02-053, The Showering Fund SF61, The Luff Cancer Fund, and The Richard Bright VEGF Research Trust. S. J. Harper is supported by Wellcome Trust Grant 057936/Z/99. D. O. Bates is supported by British Heart Foundation Grant BB-2000003.
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作者单位：1 Microvascular Research Laboratories, Department of Physiology, Preclinical Veterinary School, University of Bristol, Bristol BS2 8EJ; 3 Childrens‘ and 4 Academic Renal Unit, University of Bristol, and 5 Bristol Urological Institute, Southmead Hospital, Bristol BS10 5NB, United Kingdom; and 2