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Vascular Endothelial Growth Factor Induces Branching Morphogenesis/Tubulogenesis in Renal Epithelial Cells in a Neuropilin-Dependent Fashion

Yale University School of Medicine, New Haven, Connecticut 06510,¹ Department of Nephrology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts²

Received 2 April 2005/ Returned for modification 26 May 2005/ Accepted 6 June 2005

	ABSTRACT

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Vascular endothelial growth factor (VEGF) is well characterized for its role in endothelial cell differentiation and vascular tube formation. Alternate splicing of the VEGF gene in mice results in various VEGF-A isoforms, including VEGF-121 and VEGF-165. VEGF-165 is the most abundant isoform in the kidney and has been implicated in glomerulogenesis. However, its role in the tubular epithelium is not known. We demonstrate that VEGF-165 but not VEGF-121 induces single-cell branching morphogenesis and multicellular tubulogenesis in mouse renal tubular epithelial cells and that these morphogenic effects require activation of the phosphatidylinositol 3-kinase (PI 3-K) and, to a lesser degree, the extracellular signal-regulated kinase and protein kinase C signaling pathways. Further, VEGF-165-stimulated sheet migration is dependent only on PI 3-K signaling. These morphogenic effects of VEGF-165 require activation of both VEGF receptor 2 (VEGFR-2) and neuropilin-1 (Nrp-1), since neutralizing antibodies to either of these receptors or the addition of semaphorin 3A (which blocks VEGF-165 binding to Nrp-1) prevents the morphogenic response and the phosphorylation of VEGFR-2 along with the downstream signaling. We thus conclude that in addition to endothelial vasculogenesis, VEGF can induce renal epithelial cell morphogenesis in a Nrp-1-dependent fashion.

	INTRODUCTION

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Vascular endothelial growth factor A (VEGF-A; classically known as VEGF) is a well-characterized endothelial growth factor that is essential for vasculogenesis and angiogenesis. VEGF belongs to a family of cytokines that include VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor (PlGF). Alternate splicing of the VEGF gene in mice results in isoforms of 120, 144, 164, 182, 188, and 205 amino acids which differ in their receptor specificities, biological activities, and tissue distribution patterns (15). Of these isoforms, VEGF-164 is the most abundant.

In endothelial cells, VEGF-164 can utilize multiple receptors, including Fms-like tyrosine kinase (Flt-1 or VEGF receptor 1 [VEGFR-1]), fetal liver kinase (Flk-1 or VEGFR-2), neuropilin-1 (Nrp-1), and Nrp-2 (5). VEGFR-2 is considered to be the major signaling receptor for VEGF-164 and mediates both the mitogenic and angiogenic responses in endothelial cells. The importance of VEGFR-2 is underscored by the fact that mice lacking this receptor die in utero between days 8.5 and 9.5 (21). In addition, Nrp-1 can act as a coreceptor that increases VEGF-164 activity, possibly by enhancing its binding to VEGFR-2 (23). Nrp-1 was originally identified in neurons as a receptor for semaphorin 3A (SEMA/3A) (15), a soluble member of the semaphorin family that inhibits axon outgrowth by binding to Nrp-1 (6, 25, 26). In contrast to VEGF-164, VEGF-120 interacts with VEGFR-1 and VEGFR-2 but not Nrp-1, whereas PlGF interacts only with VEGFR-1 (15, 16).

In the kidney (human and rodent), VEGF-A mRNA is detected in glomerular podocytes, distal tubules, proximal tubules, and collecting ducts (1, 2, 19, 28) and has been implicated in glomerulogenesis (12, 13, 27, 28). In the adult kidney, VEGF produced by glomerular epithelial cells may be responsible for maintaining the fenestrated phenotype of the nearby endothelial cells, and loss of VEGF signaling in the glomerulus leads to preeclampsia-like pathological lesions (4, 14). Although VEGF is expressed in the renal tubules, little is known about its role in tubular epithelial biology. Recently, VEGF was shown to induce protein synthesis and proliferation in rat proximal tubule cells (30).

The phenomena of endothelial angiogenesis/vasculogenesis and epithelial tubulogenesis exhibit certain similarities. For example, the classical epithelial morphogen, hepatocyte growth factor (HGF), acts as a proangiogenic factor in vivo and in vitro (18, 24). Similarly, endostatin, originally identified as an antiangiogenic factor, negatively regulates ureteric bud (UB) branching in developing kidney (10). Thus, many of the factors involved in endothelial morphogenesis seem to be involved in the epithelial morphogenic program as well. We have thus decided to explore the possibility that VEGF may also evoke a morphogenic response in renal epithelial cells.

In the present study, we demonstrate that renal epithelial cells express VEGFR-1, VEGFR-2, and Nrp-1. Using recombinant human VEGF, we demonstrate that VEGF acts as a morphogen for isolated renal epithelial cells. We further show that this effect is specific for VEGF-165 (the human orthologue of mouse VEGF-164) and requires engagement of VEGFR-2 but not VEGFR-1. Furthermore, we demonstrate that antibodies to Nrp-1 or the addition of SEMA/3A blocks VEGF-165-induced morphogenic effects and blunts VEGFR-2 phosphorylation and downstream signaling. These results highlight a novel role for VEGF in mediating epithelial morphogenesis and suggest that Nrp-1 may play a critical role in VEGF signaling in renal tubular epithelia, in addition to its effects on and role in renal vascular development.

	MATERIALS AND METHODS

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Cell culture. Immortalized inner medullary collecting duct (IMCD) cells were grown in Dulbecco modified Eagle medium (DMEM)-F12 (Gibco BRL) with 10% fetal bovine serum (FBS; Gibco BRL) as described earlier (11). Mouse proximal tubule (MPT) cells, derived from the proximal tubule of the Immortomouse, were a generous gift of John Schwartz (Boston University, Boston, MA) (22) and were maintained at 32°C in DMEM-F12 plus 10% FBS plus gamma interferon (Gibco BRL). These cells were switched to 37°C in the absence of gamma interferon for 5 days (to allow differentiation) before being used for the experiments.

VEGFR expression. Reverse transcription-PCR was performed on RNA prepared from cultured MPT and IMCD cells. Total RNA was prepared using the TRIzol reagent. First-strand cDNA synthesis was performed using the Superscript first-strand synthesis system (Invitrogen). Two micrograms of each RNA sample was reverse transcribed using oligo(dT) priming according to the manufacturer's protocol. A control reaction mixture in which the reverse transcriptase was omitted was prepared for every sample.

PCR was performed using mouse VEGFR-1-, VEGFR-2-, Nrp-1-, and Nrp-2-specific primers. The following oligonucleotides were used as primers: 5'-CGG AAG GAA GAC AGC TCA TC-3' and 5'-TG A ACG CTT GCC TTA TGA-3' (VEGFR-1), 5'-TGA ACG CTT GCC TTA TGA TG-3' and 5'-AGT CGG GCA TCT CCT TTT CT-3' (VEGFR-2), 5'-ACA CCA ACC CCA CAG ATG TT-3' and 5'-CCA GTA GCT CCA TCC TCA GC-3' (Nrp-1), and 5'-GAA GGT TCG ATC CTG TTC CA-3' and 5'-CCT CAC CTG CAA AAG CTG AT-3' (Nrp-2). Primers were used to amplify 497-, 750-, 570-, and 750-bp-long regions corresponding to mouse VEGFR-1, VEGFR-2, Nrp-1, and Nrp-2 cDNA, respectively. PCR amplification was achieved by an initial 94°C soak for 5 min followed by 30 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, with 7 min of extension time at 72°C. The reaction mixture was subjected to electrophoresis on a 1.5% agarose gel, and results were visualized by ethidium bromide staining.

Branching morphogenesis and tubulogenesis. Cells were trypsinized and resuspended in type I collagen plus various doses of human VEGF-165, VEGF-121, or PlGF (R&D Diagnostics) as previously described (11) or VEGF-165 with or without neutralizing antibodies against VEGFR-2 or Nrp-1. For short-term assays, after a 24-h period of incubation at 37°C, 30 single cells were scored for the number of processes per cell. Each well represents an n of 1, and each experiment was repeated three times. Student's t test was used for calculating significance. For long-term tubulogenesis assays, cells were suspended in a 70:30 mixture of collagen and growth factor-reduced Matrigel (BD Biosciences, San Jose, CA) in 3% FBS with or without 50 ng/ml of VEGF-165 and grown for 8 days. Cells were photographed at a magnification of x20 using a Nikon TE200 Hoffman modulation contrast microscope and a SpotRT camera.

Extracellular signal-regulated kinase (ERK), PI 3-K, and PKC inhibitor studies. A branching morphogenesis assay was done using IMCD cells with VEGF-165 (50 ng/ml) with or without the MEK inhibitor UO126 (10 µM; Sigma Aldrich), the phosphatidylinositol 3-kinase (PI 3-K) inhibitor LY294002 (50 µM; Calbiochem), or protein kinase C (PKC) inhibitors Gö6983 and Gö6976 (2 µM; Calbiochem). Numbers of processes per cell were counted at 24 h. Experiments were repeated at least three times. Student's t test was used for calculating significance.

Protein analysis. Serum-starved cells were stimulated with VEGF (50 ng/ml) or vehicle (phosphate-buffered saline) for 0 to 240 min at 37°C. Cell lysates prepared in RIPA buffer (1% Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate, 20 mM Tris, 0.16 M NaCl, 1 mM EGTA, 1 mM EDTA, 15 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, and 0.5 µg/ml pepstatin A) were immunoblotted for VEGFR-1 and VEGFR-2 (1:1000; Santa Cruz, Santa Cruz, CA), Nrp-1 and Nrp-2 (1:1000; both from Zymed, South San Francisco, CA), phospho-VEGFR-2 (Santa Cruz, Santa Cruz, CA), phospho-ERK1/2 (New England Biolabs, Ipsich, MA), and phospho-Akt (Santa Cruz Biotech, Santa Cruz, CA) as described previously (11). Activated PKC was detected using a pan-phospho-PKC antibody (Cell Signaling Technology) that detects endogenous levels of activated PKC , ßI, ßII, , , , , and isoforms when the isoforms are phosphorylated at a residue homologous to threonine 514 of human PKC . To ensure equal loading, membranes were reprobed for total VEGFR-2, ERK, Akt, or PKC (Santa Cruz).

Wound healing assay. A scratch wound was made with a pipette tip in serum-starved confluent IMCD cells, and cells were incubated for 10 h in 95% O₂ and 5% CO₂ at 37°C in serum-free medium alone or serum-free medium with VEGF-121 (50 ng/ml), VEGF-165 (50 ng/ml), or VEGF-165 with or without SEMA/3A (50 ng/ml), UO126 (10 µM), LY294002 (50 µM), Gö6976 (2 µM), or neutralizing antibody against VEGFR-1, VEGFR-2, or Nrp-1 (R&D Diagnostics). In order to calculate the rate of wound healing, pictures of the wound were taken at 0 and 10 h. See Fig. 5A for the means of results of four experiments and Fig. 5B for the means of results of three experiments performed 3 months later. While the basal and stimulated rates of migration were consistent within each group and the percentages of increase were similar between the two groups, the absolute rate of migration was slower in the second group. We are unsure whether this was due to changes in the cells with passaging or subtle differences in the culture conditions. Video images of wound healing were captured every 5 min for 10 h and used to create a time lapse video (available upon request).

View larger version (16K): [in this window] [in a new window]   FIG. 5. VEGF-165 promotes wound healing in IMCD cells. A scratch wound was made with a pipette tip in a confluent IMCD cell monolayer as described in Materials and Methods. (A) The wound healing was monitored in serum-free medium with DMEM-F12 or DMEM-F12 plus VEGF-165 (50 ng/ml), VEGF-121 (50 ng/ml), or VEGF-165 with or without SEMA/3A, LY294002, UO126, Gö8976, or anti-VEGFR-1 (-VEGFR-1) or VEGFR-2 (-VEGFR-2) antibody. The wound was photographed at time point 0 and at 10 h, and the rate of wound healing (nanometers per hour) was calculated. (B) Wound healing was monitored in serum-free medium with DMEM-F12 or DMEM-F12 plus VEGF-165 (50 ng/ml) with or without SEMA/3A or anti-Nrp-1 antibody (-Nrp-1). *, P < 0.001 compared to control cells; **, P < 0.001 compared to the VEGF-165-treated group.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

View larger version (40K): [in this window] [in a new window]   FIG. 1. VEGF receptors are expressed and capable of being activated in IMCD and MPT cells. (A) Total RNA was extracted from IMCD and MPT cells, and reverse transcription-PCR was performed for VEGFR-1, VEGFR-2, Nrp-1, and Nrp-2. (B) Seventy micrograms of protein from IMCD and MPT cells was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted (IB) with anti-VEGFR-1, anti-VEGFR-2, or anti-Nrp-1. (C) IMCD and MPT cells were serum starved overnight and then treated with VEGF-165 (10 min), and lysates were immunoblotted for phosphorylated VEGFR-2, as described in Materials and Methods. Both IMCD and MPT cells show phosphorylation of VEGFR-2. pFlk, phospho-Flk.

View larger version (85K): [in this window] [in a new window]   FIG. 2. VEGF-165 induces branching morphogenesis in renal epithelial cells. IMCD (A and B) and MPT (C and D) cells were grown suspended in collagen type I for 24 h in 0 to 10 ng/ml of VEGF-165, as described in Materials and Methods. Representative fields were photographed at a magnification of x20. (B and D) Quantitation of the average number of processes per cell was performed after 24 h. *, n is 12 and P is <0.001 compared to the control.

(ii) VEGF-stimulated branching morphogenesis requires VEGFR-2 and Nrp-1. As mentioned earlier, VEGF-165 can interact with multiple receptors, including VEGFR-1, VEGFR-2, and Nrp-1. To determine which of these receptors is necessary for epithelial branching morphogenesis, we utilized neutralizing antibodies against VEGFR-1 and VEGFR-2. Incubation of cells in the presence of the neutralizing antibody to VEGFR-2 but not VEGFR-1 resulted in significant inhibition of VEGF-165-stimulated branching morphogenesis (Fig. 3A), demonstrating that activation of VEGFR-2 is required for VEGF responsiveness in these cells. Antibodies alone did not have any effect. In addition, no significant effect on VEGF-165-stimulated process formation was detected with isotype controls for either VEGFR-1 or VEGFR-2 antibody. In agreement with these data, treatment with PlGF (which activates only VEGFR-1) failed to induce branching morphogenesis in these cells (Fig. 3B). Surprisingly, incubation with VEGF-121 (which activates both VEGFR-1 and VEGFR-2) also failed to stimulate branching morphogenesis (Fig. 2C), demonstrating that activation of VEGFR-2, although necessary, is not sufficient for the induction of morphogenesis in renal epithelial cells. This concept is further supported by the observation that renal tubulogenesis is impaired in mice that express VEGF-120 but do not express VEGF-164 (13) and suggests that a second receptor, such as Nrp-1, might be required for normal morphogenic responses.

View larger version (28K): [in this window] [in a new window]   FIG. 3. VEGF-165-dependent branching morphogenesis requires VEGFR-2 and Nrp-1. IMCD cells were suspended in type I collagen for 24 h, and process formation per cell was quantitated as described in Materials and Methods. (A) Cells were treated with 50 ng/ml of VEGF-165 with or without anti-VEGFR-2 antibody (-VEGFR2) or isotype control. -VEGFR1, anti-VEGFR-1 antibody; R1, VEGFR-1; R2, VEGFR-2. (B) Cells were treated with 0 to 100 ng/ml of PlGF. C, control. (C) Cells were treated with 1 to 50 ng/ml of VEGF-121. V-165, VEGF-165. (D) Cells were treated with VEGF-165 with or without SEMA/3A (0.1 to 50 ng/ml). (E) Cells were treated with VEGF-165 with or without anti-Nrp-1 antibody (-Nrp1; 1 to 10 µg/ml). *, P < 0.001 compared to the control group; **, P < 0.001 compared to the VEGF-165-treated group.

Signal transduction: VEGF-165-induced morphogenesis requires PKC, ERK1/2, and PI 3-K activation. Researchers in our lab as well as others have shown that activation of the ERK1/2, PI 3-K, and PKC pathways is required for epithelial tubulogenesis stimulated by growth factors such as HGF and epidermal growth factor (7, 9, 17, 29). In the case of ERK, both transient activation at the membrane (where actin cytoskeletal and cell-matrix interactions appear to be regulated) and sustained activation in the nucleus (where transcriptional events are initiated) are required (8). Recently, VEGF has also been shown to activate ERK1/2 and PI 3-K and induce protein synthesis in MPT cells (20), suggesting that similar signaling events may be utilized in the morphogenic responses to VEGF.

To determine whether VEGF-165-stimulated branching morphogenesis utilizes the PI 3-K and ERK signaling pathways, serum-starved IMCD and MPT cells were stimulated with 50 ng/ml of VEGF-165 for 10 to 240 min, and cell lysates were blotted with antibodies to phospho-ERK1/2 and Akt, a downstream target for the PI 3-K pathway. VEGF-165 induced modest but sustained ERK1/2 and Akt phosphorylation for up to 4 h (Fig. 4A). To examine the importance of these signaling pathways in VEGF-stimulated morphogenesis, we examined VEGF-165-stimulated branching morphogenesis in the presence of the MEK inhibitor, UO126, and the PI 3-K inhibitor, LY294002. Blocking either pathway completely inhibited VEGF-165-stimulated process formation in both IMCD and MPT cells (Fig. 4B), demonstrating that VEGF-165 requires activation of both signaling pathways for induction of cell process extension.

View larger version (16K): [in this window] [in a new window]   FIG. 4. VEGF-165 induces epithelial branching morphogenesis in an ERK-, PI 3-K-, and PKC-dependent fashion. (A) Serum-starved IMCD and MPT cells were stimulated with VEGF-165 (50 ng/ml) for 0 to 240 min, and the cell lysates were Western blotted for phosphorylated ERK1/2 and Akt. IB, immunoblotting; pErk1/2, phospho-ERK1/2; pAkt, phospho-Akt. (B) HGF- and VEGF-165-dependent morphogenesis was blocked by the addition of the inhibitors of ERK (UO126; 10 µM) and PI 3-K (LY294002; 50 µM). Quantitation of the process formation was done at 24 h. *, P < 0.001 compared to thecontrol; **, P < 0.001 compared to the VEGF-165-treated group. (C) Lysates from IMCD cells stimulated with VEGF-165 (50 ng/ml) for 0 to 60 min were Western blotted with pan-phospho-PKC antibody. p-PKC, phospho-PKC. (D) Branching morphogenesis was assessed in IMCD cells in the presence of 50 ng/ml of VEGF-165 with or without PKC inhibitors Gö6983 (10 nM) and Gö6976 (10 nM). PKC inhibitors blocked VEGF-induced process formation. *, P < 0.001 compared to the VEGF-treated group.

Taken together, these data demonstrate that VEGF-165-stimulated branching morphogenesis requires activation of three signaling pathways that are known to be important in HGF responses and further support the hypothesis of conservation of morphogenic signaling pathways between in vitro endothelial vasculogenesis and epithelial tubulogenesis.

Cell migration. (i) VEGF-165 accelerates wound healing. The formation of tubular structures involves not only the initial extension of processes from the cells but also cell proliferation and cell migration. Therefore, we next examined whether VEGF could induce migration of IMCD cells by using a scratch wound healing assay, a model of sheet migration. VEGF-165 but not VEGF-121 significantly accelerated the wound healing process compared to that in control cells (Fig. 5A) (control group, 2,858 ± 489 nm/h; VEGF-165-treated group, 6,270 ± 1,021 nm/h [P < 0.001 compared to control]; VEGF-121-treated group, 3,034 ± 100 nm/h).

In order to determine which signaling pathways might be regulating the VEGF-165-induced migratory response, we used inhibitors of MEK (UO126), PI 3-K (LY294002), and PKC (Gö6976) pathways. Blocking the PI 3-K pathway significantly retarded VEGF-165-mediated wound healing (Fig. 5A). In contrast, blocking either MEK or PKC signaling did not have a significant effect. These results demonstrate that although activation of PI 3-K is required for both VEGF-induced single-cell branching morphogenesis and sheet migration, activation of PKC and ERK1/2 does not appear to be critical in the multicellular migratory response.

To determine which receptor might be necessary for these effects of VEGF-165, we once again utilized neutralizing antibodies against VEGFR-1 and VEGFR-2 (Fig. 5A), as well as SEMA/3A and the blocking antibody against Nrp-1 (Fig. 5B). Similar to the results seen with branching morphogenesis, sheet migration induced by VEGF-165 was significantly blocked by the neutralizing antibody to VEGFR-2 and Nrp-1, as well as by SEMA/3A, whereas anti-VEGFR-1 antibody had no significant effect (Fig. 5A and B; see also time lapse video available upon request). Again, as was seen in the single-cell branching assay, SEMA/3A alone did not affect the basal rate of cell migration. We thus demonstrated that VEGF-165 can enhance epithelial cell sheet migration and that this response requires engagement of VEGFR-2 and Nrp-1 and subsequent activation of the PI 3-K pathway. These data are in concert with a recent observation that VEGF-165-directed endothelial cell migration is dependent on Nrp-1 expression (31).

(ii) VEGF-Nrp-1 interaction is critical for VEGF-mediated signaling. Since the addition of either anti-VEGFR-2 neutralizing antibody or SEMA/3A caused significant inhibition of VEGF-165-mediated branching morphogenesis, we examined the effect of these interventions on VEGF signaling. Addition of either VEGFR-2-neutralizing antibody or SEMA/3A blocked VEGF-165-dependent phosphorylation of VEGFR-2 (Fig. 6A). While the effect of the anti-VEGFR-2 antibody was expected, the marked inhibition by SEMA/3A was surprising. In endothelial cells, Nrp-1 has been shown to potentiate VEGF-mediated signaling. However, our results would suggest an even more critical role for Nrp-1 in VEGF-mediated signaling in epithelial cells. Expectedly, this blockade of the receptor phosphorylation resulted in significant reduction in VEGF-165-mediated ERK1/2 and PI 3-K pathway activation, as revealed by the reduction in phospho-ERK1/2 or phospho-Akt in the presence of VEGFR-2 antibody or SEMA/3A (Fig. 6B and C). The ability of SEMA/3A to effectively block VEGF signaling provides a likely mechanism for the marked inhibition of branching morphogenesis described earlier (Fig. 3D).

View larger version (27K): [in this window] [in a new window]   FIG. 6. Nrp-1 is required for VEGF-mediated signaling in renal tubular epithelial cells. IMCD cells were preincubated with 6 µg of VEGFR-2-neutralizing antibody or 40 ng/ml of SEMA/3A for 30 min and then stimulated with 50 ng/ml of VEGF-165 for 10 min. Lysates were then Western blotted for the phosphorylated form of VEGFR-2 (pVEGFR2) (A), phospho-ERK1/2 (pErk1/2) (B), or phospho-Akt (pAkt) (C). Preincubation with either anti-VEGFR-2 (anti-R2) or SEMA/3A significantly inhibited VEGF-165-mediated VEGFR-2 phosphorylation and downstream signaling. IB, immunoblotting.

In summary, our present data indicate a novel role of VEGF in renal branching morphogenesis and highlight an essential role of Nrp-1 in VEGF-mediated morphogenic effects in epithelial cells. The fact that VEGF induces these morphogenic effects in both kidney epithelial and endothelial cells would suggest that VEGF may be ideally positioned to coordinate the synchronous development of the tubular and vascular architecture in the kidney that is required for the ultimate formation of a functional nephron.

	ACKNOWLEDGMENTS

 
This work was supported by NIH grants to A.K., S.K., and L.G.C.

	FOOTNOTES

 
* Corresponding author. Mailing address: Section of Nephrology, Yale University School of Medicine, New Haven, CT 06510. Phone: (203) 785-7111. Fax: (203) 785-4904. E-mail: Anil.Karihaloo{at}yale.edu.

	REFERENCES

Top Abstract Introduction Materials and Methods Results and Discussion References

 
1. Bacic, M., N. A. Edwards, and M. J. Merrill. 1995. Differential expression of vascular endothelial growth factor (vascular permeability factor) forms in rat tissues. Growth Factors 12:11-15.[Medline]

2. Breier, G., U. Albrecht, S. Sterrer, and W. Risau. 1992. Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation. Development 114:521-532.[Abstract]

3. Cross, M. J., J. Dixelius, T. Matsumoto, and L. Claesson-Welsh. 2003. VEGF-receptor signal transduction. Trends Biochem. Sci. 28:488-494.[CrossRef][Medline]

4. Eremina, V., M. Sood, J. Haigh, A. Nagy, G. Lajoie, N. Ferrara, H. P. Gerber, Y. Kikkawa, J. H. Miner, and S. E. Quaggin. 2003. Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases. J. Clin. Investig. 111:707-716.[CrossRef][Medline]

5. Ferrara, N. 2004. Vascular endothelial growth factor: basic science and clinical progress. Endocr. Rev. 25:581-611.[Abstract/Free Full Text]

6. Goshima, Y., H. Hori, Y. Sasaki, T. Yang, M. Kagoshima-Maezono, C. Li, T. Takenaka, F. Nakamura, T. Takahashi, S. M. Strittmatter, Y. Misu, and T. Kawakami. 1999. Growth cone neuropilin-1 mediates collapsin-1/Sema III facilitation of antero- and retrograde axoplasmic transport. J. Neurobiol. 39:579-589.[CrossRef][Medline]

7. Gual, P., S. Giordano, T. A. Williams, S. Rocchi, E. Van Obberghen, and P. M. Comoglio. 2000. Sustained recruitment of phospholipase C-gamma to Gab1 is required for HGF-induced branching tubulogenesis. Oncogene 19:1509-1518.[CrossRef][Medline]

8. Ishibe, S., D. Joly, X. Zhu, and L. G. Cantley. 2003. Phosphorylation-dependent paxillin-ERK association mediates hepatocyte growth factor-stimulated epithelial morphogenesis. Mol. Cell 12:1275-1285.[CrossRef][Medline]

9. Karihaloo, A., S. Kale, N. D. Rosenblum, and L. G. Cantley. 2004. Hepatocyte growth factor-mediated renal epithelial branching morphogenesis is regulated by glypican-4 expression. Mol. Cell. Biol. 24:8745-8752.[Abstract/Free Full Text]

10. Karihaloo, A., S. A. Karumanchi, J. Barasch, V. Jha, C. H. Nickel, J. Yang, S. Grisaru, K. T. Bush, S. Nigam, N. D. Rosenblum, V. P. Sukhatme, and L. G. Cantley. 2001. Endostatin regulates branching morphogenesis of renal epithelial cells and ureteric bud. Proc. Natl. Acad. Sci. USA 98:12509-12514.[Abstract/Free Full Text]

11. Karihaloo, A., D. A. O'Rourke, C. Nickel, K. Spokes, and L. G. Cantley. 2001. Differential MAPK pathways utilized for HGF- and EGF-dependent renal epithelial morphogenesis. J. Biol. Chem. 276:9166-9173.[Abstract/Free Full Text]

12. Kitamoto, Y., H. Tokunaga, and K. Tomita. 1997. Vascular endothelial growth factor is an essential molecule for mouse kidney development: glomerulogenesis and nephrogenesis. J. Clin. Investig. 99:2351-2357.[Medline]

13. Mattot, V., L. Moons, F. Lupu, D. Chernavvsky, R. A. Gomez, D. Collen, and P. Carmeliet. 2002. Loss of the VEGF(164) and VEGF(188) isoforms impairs postnatal glomerular angiogenesis and renal arteriogenesis in mice. J. Am. Soc. Nephrol. 13:1548-1560.[Abstract/Free Full Text]

14. Maynard, S. E., J. Y. Min, J. Merchan, K. H. Lim, J. Li, S. Mondal, T. A. Libermann, J. P. Morgan, F. W. Sellke, I. E. Stillman, F. H. Epstein, V. P. Sukhatme, and S. A. Karumanchi. 2003. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J. Clin. Investig. 111:649-658.[CrossRef][Medline]

15. Neufeld, G., T. Cohen, S. Gengrinovitch, and Z. Poltorak. 1999. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J. 13:9-22.[Abstract/Free Full Text]

16. Neufeld, G., O. Kessler, and Y. Herzog. 2002. The interaction of neuropilin-1 and neuropilin-2 with tyrosine-kinase receptors for VEGF. Adv. Exp. Med. Biol. 515:81-90.[Medline]

17. Nickel, C., T. Benzing, L. Sellin, P. Gerke, A. Karihaloo, Z. X. Liu, L. G. Cantley, and G. Walz. 2002. The polycystin-1 C-terminal fragment triggers branching morphogenesis and migration of tubular kidney epithelial cells. J. Clin. Investig. 109:481-489.[CrossRef][Medline]

18. Ono, M., Y. Sawa, K. Matsumoto, T. Nakamura, Y. Kaneda, and H. Matsuda. 2002. In vivo gene transfection with hepatocyte growth factor via the pulmonary artery induces angiogenesis in the rat lung. Circulation 106:I264-I269.

19. Peters, K. G., C. De Vries, and L. T. Williams. 1993. Vascular endothelial growth factor receptor expression during embryogenesis and tissue repair suggests a role in endothelial differentiation and blood vessel growth. Proc. Natl. Acad. Sci. USA 90:8915-8919.[Abstract/Free Full Text]

20. Senthil, D., G. G. Choudhury, C. McLaurin, and B. S. Kasinath. 2003. Vascular endothelial growth factor induces protein synthesis in renal epithelial cells: a potential role in diabetic nephropathy. Kidney Int. 64:468-479.[CrossRef][Medline]

21. Shalaby, F., J. Rossant, T. P. Yamaguchi, M. Gertsenstein, X. F. Wu, M. L. Breitman, and A. C. Schuh. 1995. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 376:62-66.[CrossRef][Medline]

22. Sinha, D., Z. Wang, V. R. Price, J. H. Schwartz, and W. Lieberthal. 2003. Chemical anoxia of tubular cells induces activation of c-Src and its translocation to the zonula adherens. Am. J. Physiol. Renal Physiol. 284:F488-F497.[Abstract/Free Full Text]

23. Soker, S., S. Takashima, H. Q. Miao, G. Neufeld, and M. Klagsbrun. 1998. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 92:735-745.[CrossRef][Medline]

24. Strohmeyer, D., F. Strauss, C. Rossing, C. Roberts, O. Kaufmann, G. Bartsch, and P. Effert. 2004. Expression of bFGF, VEGF and c-met and their correlation with microvessel density and progression in prostate carcinoma. Anticancer Res. 24:1797-1804.[Medline]

25. Takahashi, T., A. Fournier, F. Nakamura, L. H. Wang, Y. Murakami, R. G. Kalb, H. Fujisawa, and S. M. Strittmatter. 1999. Plexin-neuropilin-1 complexes form functional semaphorin-3A receptors. Cell 99:59-69.[CrossRef][Medline]

26. Tamagnone, L., S. Artigiani, H. Chen, Z. He, G. I. Ming, H. Song, A. Chedotal, M. L. Winberg, C. S. Goodman, M. Poo, M. Tessier-Lavigne, and P. M. Comoglio. 1999. Plexins are a large family of receptors for transmembrane, secreted, and GPI-anchored semaphorins in vertebrates. Cell 99:71-80.[CrossRef][Medline]

27. Tufro, A. 2000. VEGF spatially directs angiogenesis during metanephric development in vitro. Dev. Biol. 227:558-566.[CrossRef][Medline]

28. Tufro, A., V. F. Norwood, R. M. Carey, and R. A. Gomez. 1999. Vascular endothelial growth factor induces nephrogenesis and vasculogenesis. J. Am. Soc. Nephrol. 10:2125-2134.[Abstract/Free Full Text]

29. Ueland, J. M., J. Gwira, Z. X. Liu, and L. G. Cantley. 2004. The chemokine KC regulates HGF-stimulated epithelial cell morphogenesis. Am. J. Physiol. Renal Physiol. 286:F581-F589.[Abstract/Free Full Text]

30. Villegas, G., B. Lange-Sperandio, and A. Tufro. 2005. Autocrine and paracrine functions of vascular endothelial growth factor (VEGF) in renal tubular epithelial cells. Kidney Int. 67:449-457.[CrossRef][Medline]

31. Wang, L., H. Zeng, P. Wang, S. Soker, and D. Mukhopadhyay. 2003. Neuropilin-1-mediated vascular permeability factor/vascular endothelial growth factor-dependent endothelial cell migration. J. Biol. Chem. 278:48848-48860.[Abstract/Free Full Text]

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