AJP - Regu Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 290: R105-R113, 2006; doi:10.1152/ajpregu.00492.2005
0363-6119/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ray, P. E.
Right arrow Articles by Wellstein, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ray, P. E.
Right arrow Articles by Wellstein, A.

CALL FOR PAPERS
Cardiovascular-Kidney Interactions in Health and Disease

Role of fibroblast growth factor-binding protein in the pathogenesis of HIV-associated hemolytic uremic syndrome

Patricio E. Ray,1,2,* Elena Tassi,3,* Xue-Hui Liu,1 and Anton Wellstein3

1Children's Research Institute, Children's National Medical Center, 2George Washington University, Washington, D.C.; and 3Lombardi Comprehensive Cancer Center, Georgetown University, Washington, D.C

Submitted 7 July 2005 ; accepted in final form 27 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
A characteristic finding of childhood HIV-associated hemolytic uremic syndrome (HIV-HUS) is the presence of endothelial injury and microcystic tubular dilation, leading to a rapid progression of the renal disease. We have previously shown that a secreted fibroblast growth factor-binding protein (FGF-BP) is upregulated in kidneys from children affected with HIV-HUS and HIV nephropathy. Here, we sought to determine the potential role of FGF-BP in the pathogenesis of HIV-HUS. By immunohistochemical and in situ hybridization studies, we observed FGF-BP protein and mRNA upregulation in regenerating renal tubular epithelial cells from kidneys of HIV-Tg26 mice with late-stage renal disease, that is, associated with the development of microcystic tubular dilatation and accumulation of FGF-2. Moreover, FGF-BP increased the FGF-2-dependent growth and survival of cultured primary human renal glomerular endothelial cells and enhanced FGF-2-induced MAPK/ERK2 activation, as well as the proliferation of immortalized GM7373 endothelial cells. We propose that HIV-Tg26 mice are a clinically relevant model system to study the role of FGF-BP in the pathogenesis of HIV-associated renal diseases. Furthermore, the upregulation of FGF-BP by regenerating renal tubular epithelial cells may provide a mechanism by which the regenerative and angiogenic activity of FGF-2 in renal capillaries can be modulated in children with HIV-HUS and other renal disease.

renal tubular epithelial and endothelial cells; HIV-nephropathy; HIV-transgenic mice

THE TERM HEMOLYTIC UREMIC SYNDROME (HUS) was first introduced in 1955 by Gasser et al. (17) to describe a heterogeneous group of diseases characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. The classic form of HUS is a well-defined clinical entity in which there is a prodrome of diarrhea, usually bloody, as a result of Shiga-like, toxin-producing bacterial infection (34). In addition, other atypical and familiar forms of HUS, not associated with diarrhea, have been described in association with drugs, inborn errors of metabolism, genetic diseases, and infectious agents (34). HIV-1-infected children can develop an atypical form of HUS characterized by the presence of severe microangiopathic lesions in renal capillaries, in association with microcystic tubular dilation, tubulointerstitial changes, and rapid development of end-stage renal disease (37, 46).

Endothelial damage appears to be a primary pathogenic event in all of the classic and atypical forms of HUS (34). We have demonstrated that FGF-2, an endothelial cell growth factor that can be released by injured endothelial cells, is accumulated in the plasma and urine of children with classic and HIV-HUS, in correlation with the severity of the renal disease (31, 36, 46). In normal conditions, FGF-2 is stored as an inactive form in the extracellular matrix bound to heparan sulfate proteoglycans (HSPG) (18, 22, 40, 52). Interestingly, we have found a significant overexpression of FGF-2 and HSPG in the kidneys of both HIV-transgenic (Tg26) mice (33, 36, 37, 46) and HIV-infected children with renal disease (26). These findings suggest that FGF-2 released by injured endothelial cells can be sequestered from the circulation and stored in the kidneys bound to renal HSPG (26, 31, 33, 36). However, it is crucial to determine how FGF-2 is mobilized from the HSPG milieu to reach and activate its cognate receptor(s) on the target renal endothelial cells.

Fibroblast growth factor-binding protein (FGF-BP) was first characterized as a secreted protein that is able to bind to FGF-2 in a noncovalent, reversible manner and protect it from proteolytic degradation (51). Studies with FGF-BP-negative cell lines showed that expression of FGF-BP increased tumor growth and angiogenesis in a xenograft model (6–8, 50). These and other findings suggest that FGF-BP has a rate-limiting role modulating the angiogenic activity of FGF-2 during the process of tumor growth and embryo development (6, 27, 45). In previous studies, we have also found that FGF-BP is upregulated in regenerating renal tubular epithelial cells in children with HIV-HUS and other renal diseases (26). On the basis of these observations, it is tempting to speculate that the interaction of FGF-BP with FGF-2 may impact on FGF-2-mediated regeneration of renal capillaries adjacent to renal epithelial cells.

In previous studies, we have found that renal epithelial cells isolated from HIV-transgenic (Tg26) mice with renal disease synthesize and release significant levels of FGF-2 into the conditioned media (33). Here, we show for the first time that FGF-BP is upregulated in regenerating renal tubular epithelial cells in the same mouse model. Additionally, we demonstrate the ability of FGF-BP to increase FGF-2-induced survival activity of cultured primary renal glomerular endothelial cells, as well as to enhance FGF-2-dependent MAPK/ERK signaling and mitogenesis in bovine endothelial cells. Overall, when these findings are interpreted in the context of previous studies, they support the notion that FGF-BP, locally released by regenerating tubular epithelial cells, may enhance the angiogenic activity of FGF-2 in children with HIV-HUS and, in this manner, facilitate the healing of renal capillaries and tubules.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
HIV-1 Transgenic Mice

HIV-1 transgenic heterozygous mice (Tg26 line) have been previously described (11, 23). Plasma blood urea nitrogen (BUN) and creatinine levels, as well as urine protein/creatinine ratios in HIV-Tg26 and littermate nontransgenic control mice, were measured following standard techniques as previously described (23, 35).

Patients

This study was approved by the Institutional Review Board from the Children's National Medical Center. Renal sections obtained at autopsy from children with HIV-HUS (n = 2 ), HIV-nephropathy (n = 2), classic HUS (n = 1), and renal sections from HIV-infected children without renal disease (n = 2) were collected to validate the findings obtained in HIV-Tg26 mice. All patients were younger than 16 years of age. The diagnosis of HIV infection, HIV-HUS, HIV nephropathy, and classic HUS was done using standard clinical and pathologic criteria.

Immunohistochemistry

Renal tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Four-micrometer sections were deparaffinized by xylene and rehydrated with decreasing concentration of ethanol and finally H2O. Antigen retrieval was performed by heating the sections twice for 5 min in a microwave oven (2,450 mHz, 850 W) in a 0.01 M sodium citrate solution (pH 6.0). Endogenous peroxidase activity was blocked with 3% H2O2 in 100% methanol for 10 min. Tissues were incubated for 1 h at room temperature with affinity purified IgG fractions (2.5 µg/ml) from a rabbit polyclonal antibody directed against unique peptide sequence of FGF-2 (provided by Dr. Baird, PRIZM Pharmaceuticals, San Diego CA); anti-bovine FGF-2 mouse monoclonal antibody (Upstate Biotechnology, Lake Placid, NY); rabbit polyclonal FGF-BP antibody (1:50 dilution) (8); and biotinylated anti-proliferative cell nuclear antigen (anti-PCNA) monoclonal antibody (clone PC10) (Zymed Laboratories, South San Francisco, CA). Nonimmune mouse or rabbit IgGs were used as negative controls. The specificity of FGF-BP staining in control and diseased renal sections was confirmed by preabsorbing the primary FGF-BP antibody with a 20-fold molar excess of recombinant FGF-BP. Secondary staining was performed with a commercial streptavidin-biotin-peroxidase complex Histostain SP kit (Zymed) and aminoethyl carbazole, according to the manufacturer's instructions. Counterstaining of nuclei was performed by immersion in hematoxylin. Slides were dehydrated with increasing concentrations of ethanol and a final immersion in Xylene 100% and then mounted with Cytoseal 60 low-viscosity mounting medium (Richard-Allan Scientific, Kalamazoo, MI), and coverslips. Immunostaining for von Willebrand factor (vWF) (DAKO, Carpinteria, CA) was carried out as described previously (36, 38).

In Situ Hybridization for FGF-BP mRNA Expression in Tissues

Digoxigenin-labeled mouse and human FGF-BP probes. A 750-bp mouse FGF-BP cDNA (GenBank #U49641) was subcloned into the EcoR I sites of the pRC/CMV vector between T7 and SP6 promoters. The vector was linearized with the restriction enzymes Hind III or Not I and utilized as a template for the synthesis of DIG-labeled antisense or sense riboprobes, respectively. In vitro transcription reactions were performed with SP6 or T7 RNA polymerase in the presence of DIG-UTP by using a DIG RNA labeling Kit (Roche, Indianapolis, IN). The reaction products were purified by phenol extraction and ethanol precipitation and stored at –70°C until use. Labeling efficiency of the riboprobe was estimated by comparison with 10-fold serial dilution of a digoxigenin-labeled control riboprobe and direct detection of the labeled riboprobe with anti-digoxigenin antibodies. Riboprobe concentrations were adjusted to be equivalent on the basis of the labeling efficiency before use in the in situ hybridization studies.

In situ hybridization. Ten percent neutral buffered formalin-fixed and paraffin-embedded renal tissues were cut at 4 µm and floated onto 2% 3-aminopropyltriethoxysilane (Sigma Chemical, St. Louis, MO)-coated slides. Sections were heat-fixed for 30 min at 65°C and deparaffinized. After a 30-min incubation in 5 mmol/l levamisole, the sections were washed in PBS and in diethyl pyrocarbonate-treated water for 5 min, respectively, and then immersed in 0.2 mol/l HCl for 20 min. Then, sections were deproteinized by digestion with 20–40 µg/ml proteinase K (Sigma) for 10 min at 37°C, washed twice in 0.2% glycine for 5 min, once in PBS for 10 min, and postfixed with 1.5% paraformadehyde-1.5% glutaraldehyde for 1 min. After being rinsed twice in PBS, sections were dehydrated through a graded ethanol series and air-dried. The sections were hybridized with the mouse FGF-BP RNA sense or antisense probes (0.1–0.5 ng/µl) in 2x SSC, 10% dextran sulfate, 1x Denhardt's solution, 20 mM vanadyl ribonucleoside complex (Invitrogen, Carlsbad, CA), 0.1 M sodium phosphate under sealed coverslips and incubated overnight in a moist chamber at 42°C. Sections were washed in 0.2x SSC and then blocked with a blocking solution (50 mg/ml skimmed dried milk, 150 mM NaCl, 100 mM Tris·HCl, pH 7.8) at room temperature for 15 min followed by an incubation with alkaline phosphatase-conjugated antidigoxigenin antibody (Roche) (1:750 dilution) for 30 min at 37°C. Colorimetric detection with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Roche) was then performed, and the sections were mounted in 60% glycerol and examined by light microscopy. The negative controls included 1) hybridization with the sense probe, 2) RNase A (100 µg/ml in 10 mM Tris·HCl, pH 8.0, 1 mM EDTA) pretreatment before hybridization, and 3) omission of either the antisense RNA probe or the anti-digoxigenin antibody.

RT-PCR. Total RNA was extracted from control and diseased tissue samples or cells using Trizol reagent (Invitrogen), according to the manufacturer's instructions. RT of 2 µg of total RNA was performed using the Reverse Transcription System (Promega, Madison, WI). The resulting first-strand cDNA was used as a template for a PCR reaction using the Advantage PCR Kit (Clontech, Palo Alto, CA) at an annealing temperature of 58°C for 30 cycles. The following primers were used for amplification: FGF-BP forward: 5'-GCAAGTTTG26TCACCAAAGACC-3', FGF-BP reverse: 5'-CTG26CTTTCTCCTTCCTG26GGC-3'; {beta}-actin forward: 5'-TG26CGTTACACCCTTTCTTG26AC-3', {beta}-actin reverse: 5'-ACTG26GTCTCAAGTCAGTG26TAC-3'.

PCR products were separated on 1.2% agarose gels and stained with ethidium bromide. Amplified cDNA bands were quantified by densitometry image scanning as described before (8). The product yield was expressed relative to {beta}-actin.

Culture of endothelial cells. Primary human glomerular endothelial cells were purified by elutriation from dispase-dissociated normal human cortical glomeruli (Cell Systems, Seattle, WA) and were a generous gift from Dr. Carl Soderland [Applied Cell Biology Research Institute (ACBRI), Kirkland, WA; Ref. (44)]. Cells were used between passages 2 and 4 and were seeded on Attachment Factor-coated Nunclon cell culture flasks (ACBRI). Primary glomerular endothelial cells were maintained in Cell System-Certified endothelial culture medium (ACBRI), containing the following endothelial cells growth supplement (ECGS): recombinant human epidermal growth factor (10 ng/ml), bovine brain extract, heparin (10 µg/ml), amphotericine B (50 ng/ml), gentamycin (50 mg/ml), and 5% (vol/vol) fetal bovine serum. Transformed fetal bovine aortic endothelial GM7373 cells, corresponding to the BFA-1c 1BPT clone (4, 19), were obtained from the National Institute of General Medical Sciences, Human Genetic Mutual Cell Repository, Coriell Institute for Medical Research (Camden, NJ) and were maintained in modified improved MEM (Invitrogen) supplemented with 10% (vol/vol) fetal bovine serum.

Survival assays of human glomerular endothelial cells. Cells were plated in 6-well culture dishes, in the presence of ECGS. At 80% confluence, cells were starved for 12 h; then FGF-BP (30 ng/ml) and FGF-2 (5 and 30 ng/ml) (Invitrogen) were added, and the cells were cultured for 18 h. Cells were then incubated with 1 µCi/ml [3-H]-labeled thymidine for 6–12 h followed by the addition of 1 ml ice-cold 10% TCA for 20 min at 4°C. After the cells were washed, three times with ice-cold water, they were solubilized with 1 ml 0.5 M NaOH/16 mm dish at 37°C for 30 min; 0.5-ml aliquot samples were measured. The results were expressed in counts per minute.

Proliferation assay of immortalized endothelial cells. GM7373 cells (104) were seeded in triplicate in 96-well plates for 8 h in the presence or absence of sodium chlorate (30 mM; Sigma) and serum-starved overnight. Cells were then stimulated with 5 and 10 ng/ml of human recombinant FGF-2 (Invitrogen) in the presence or absence of recombinant human FGF-BP (6 ng/ml) for 48 h. Cell proliferation was assessed using the tetrazolium-based WST-1 reagent (Roche), according to the manufacturer's instructions.

Phosphorylation studies. Forty percent confluent GM7373 cells were plated in complete medium in the presence or absence of 50 mM sodium chlorate for 36 h. Cells were then serum-starved overnight in the presence or absence of sodium chlorate and treated for 5 min with 0.5, 1, and 2 ng/ml of human recombinant FGF-2 (Invitrogen) in the presence or absence of FGF-BP (5 ng/ml) (45). Cells were then washed with cold PBS, pH 7.4, and subsequently lysed at 4°C in a buffer containing 50 mM Tris·HCl, pH 8, 150 mM NaCl, 40 mM b-glycerophosphate, 1 mM EGTA, 0.25% sodium deoxycholate, 1% Nonidet P-40, 50 mM sodium fluoride, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 2 mg/ml leupeptin, 2 mg/ml aprotinin, 1 mg/ml pepstatin, and 100 mg/ml pefabloc. Detection of phosphorylated proteins and phosphorylated ERK1/2 from total cell lysates was carried out using an anti-phosphotyrosine monoclonal antibody (4G10; Upstate Biotechnology, Lake Placid, NY) and an anti phospho-p44/42 MAP Kinase (Thr202/Tyr204) rabbit polyclonal antibody (Cell Signaling, Beverly, MA), respectively, as described previously (45).

Isolation of recombinant FGF-BP protein was done as described previously (45).

Statistical analysis. GraphPad Prism Software was used for statistical analysis. Data are expressed as means ± SE. Differences between two groups were compared by Student's t-test. When more than two means were compared, data were evaluated for statistically significant differences by one-way ANOVA followed by multiple comparisons using the Student-Neuman-Keuls test. The nonparametric Kruskal-Wallis test was used whenever the distribution of the data did not follow a Gaussian distribution. GM7373 proliferation was analyzed by two-way ANOVA with Bonferroni's post hoc test. Statistical significance was defined as P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Indicators of Late-Stage Renal Disease in HIV-Tg26 Mice

The natural history of renal disease in HIV-Tg26 transgenic mouse model has been characterized (23, 33). Interestingly, although born with normal kidneys, HIV-Tg26 mice, at ~40–50 days of life, develop renal disease with high incidence and with many of the pathologic features typical of human immunodeficiency virus associated nephropathy (HIVAN). These renal lesions include hypertrophy and hyperplasia of glomerular epithelial cells, focal and global glomerulosclerosis, varying degrees of tubular distortion and dilation, as well as kidney enlargement (23). These pathologic features are also associated with the development of severe proteinuria and azotemia, as indicated by the following parameters from control vs. HIV-Tg26 mice: urinary protein/creatinine ratios 8.5 ± 0.8 vs. 51 ± 6.6; plasma BUN 23 ± 1.5 vs. 43 ± 4.8 mg/dl; plasma creatinine 0.45 ± 0.0016 vs. 0.6 ± 0.009 mg/dl (mean ± SE, n = 5; P < 0.05 for all parameters compared with controls, respectively).

FGF-BP Protein Upregulation During Late-Stage Renal Disease in HIV-Tg26 Mice

To determine localization and pattern of expression of FGF-BP during disease progression in HIV-Tg26 mouse kidneys, we performed immunohistochemical studies on renal sections from transgenic and wild-type mice. At 20 days of age, HIV-Tg26 mice with no histological evidence of renal disease showed minimal or no expression of FGF-BP in renal tubules (Fig. 1, Bj), with levels comparable to those obtained from 20-day-old control nontransgenic littermates (Fig. 1, Bi). However, 40-day-old HIV-Tg26 mice, showing microcystic dilation, as well as regenerative changes of renal tubules, displayed a significant upregulation of FGF-BP (Fig. 1, Bf). It is noteworthy that no FGF-BP staining was detected in kidney specimens from nontransgenic littermates or from 40-day-old HIV-Tg26 mice stained with nonimmune antiserum (Fig. 1, B, e and g). The expression of FGF-BP was predominantly upregulated in renal tubular epithelial cells, which also stained positive for the proliferating nuclear antigen (PCNA) (Fig. 1, Bh). We detected a significant upregulation (~ 8-fold increase) in the number of proliferating renal tubular cells compared with that of HIV-Tg26 mice without renal disease or control littermates (not shown). These findings correlate with FGF-2 accumulation in the renal interstitium surrounding renal tubular epithelial cells undergoing proliferative changes in HIV-Tg26 mice (33). Moreover, FGF-BP expression in HIV-Tg26 renal tubules coincided with that of PCNA (Fig. 1, Bh), suggesting that FGF-BP synthesis is predominantly restricted to regenerating tubular epithelial cells.



View larger version (83K):
[in this window]
[in a new window]
  		Fig. 1. Fibroblast growth factor-binding protein (FGF-BP) protein detection in the kidneys of HIV-infected children (A; a–d) and HIV-Tg26 mice (B; e–j). Immunostaining of renal tissues without (a, e, i) and with (b, f, j) early and late renal disease (HIV-associated nephropathy, HIVAN). Note the localization of the FGF-BP protein (red stain) in renal tubular epithelial cells (x200 magnification). d, h: Double-staining for FGF-BP (red) and the proliferation marker proliferating cell nuclear antigen (PCNA) (dark blue) in renal tubules (x320 magnification). Negative controls: blockade with an excess of recombinant human FGF-BP protein (c; +FGF-BP) and staining with a control IgG (g).

 
FGF-BP Protein Upregulation in the Kidneys of HIV-Tg26 Mice Mimics the Renal Changes Seen in Children with HIVAN

In a previous study, we had shown upregulation of FGF-BP in the kidneys of children with HUS and other renal disease (26). Renal tubules of children with HIVAN showed a significant upregulation of FGF-BP protein in contrast to renal sections from HIV-infected children without renal disease (Fig. 1, A, a and b). Specificity of the FGF-BP staining was assured by preabsorption of the antibody with recombinant human FGF-BP protein (Fig. 1, Ac). As in the HIV-Tg26 mice, the accumulation of FGF-BP in human HIVAN was predominantly observed in renal tubules undergoing regenerative/proliferative changes, as evidenced by double-staining for PCNA and FGF-BP (Fig. 1, Ad). Thus the transgenic model appears to mimic the human disease.

FGF-BP mRNA Upregulation During the Late-Stage Renal Disease in HIV-Tg26 Mice

The upregulation of FGF-BP protein in the transgenic model was paralleled at the mRNA level. In situ hybridization for FGF-BP mRNA in sections from diseased kidneys from HIV-Tg26 mice showed the same pattern of distribution of FGF-BP mRNA and protein. A significant upregulation of FGF-BP mRNA was detected in regenerating and/or injured renal tubules from 40-day-old HIV-Tg26 mice with renal disease (Fig. 2, Ab). Signal specificity was confirmed by the absence of staining in sections incubated with a sense FGF-BP probe (Fig. 2, Aa). Furthermore, no staining was detected in renal sections from young (20-day-old) or adult (40-day old) HIV-Tg26 without renal disease or control littermates (data not shown). Similarly, RT-PCR of diseased kidneys from 40-day-old HIV-Tg26 mice showed a 2.2-fold increase of FGF-BP mRNA, compared with the levels obtained from control littermate kidneys. Although the differential FGF-BP mRNA expression detected from the RT-PCR analysis appears smaller than that observed from the in situ hybridization, it is likely to be due to the dilution effect of mRNA from other cell types in the tissue. It is noteworthy that no significant differences of FGF-BP mRNA expression were detected between control and HIV-Tg26 mice during the early stages of renal disease (data not shown). We conclude from these data that the renal tubular epithelial cells from HIV-Tg26 mice induce expression of FGF-BP mRNA and do not accumulate the protein from the renal interstitium.



View larger version (49K):
[in this window]
[in a new window]
  		Fig. 2. FGF-BP mRNA detection in kidneys from HIV-Tg26 mice. A: in situ hybridization of a representative section from an HIV-Tg26 mouse with renal disease. Stainings with a murine FGF-BP sense (a) and antisense (b) probe are shown at x320 magnification. B: RT-PCR for FGF-BP mRNA in a HIV-Tg26 kidney with late-stage renal disease compared with a control littermate mouse kidney. FGF-BP (330 bp) and {beta}-actin (550 bp) amplification products are shown. C: densitometric quantitation of FGF-BP normalized for {beta}-actin. Data are representative of at least three independent experiments.

 
Effect of FGF-BP on the Growth and Survival of Primary Glomerular Endothelial Cells

Primary human endothelial cells isolated from renal glomeruli were used to assess the effect of FGF-BP on cell growth. Cytoplasmic expression of von Willebrand factor was used as histological marker to confirm the endothelial characteristics of these cells (Fig. 3A) (36, 38). In the absence of added endothelial cell growth factors, primary glomerular endothelial cells detached from the culture dishes and underwent cell death but were rescued by the addition of FGF-2 (Fig. 3B). The viability of the cultured primary glomerular endothelial cells in the presence of FGF-2 was confirmed by [3H]-labeled thymidine incorporation studies (Fig. 3C). The addition of FGF-BP alone did not affect the growth rate or viability of the cells. However, inclusion of FGF-BP with FGF-2 significantly increased FGF-2-mediated cell survival (P < 0.01). These data, altogether, indicate that the interaction of FGF-BP with FGF-2 is associated with a significant enhancement of FGF-2-induced effects on primary human renal endothelial cells. Therefore, the upregulation of both FGF-2 and FGF-BP detected in HIVAN may thus have a functional impact also on the epithelial/endothelial interactions.



View larger version (53K):
[in this window]
[in a new window]
  		Fig. 3. FGF-BP increases FGF-2-dependent growth and survival of primary human renal glomerular endothelial cells. A: primary human renal glomerular endothelial cells (hematoxylin and eosin stain, left) were characterized by immunostaining for von Willebrand factor (vWF, right). B: representative pictures of cells grown for 36 h in the absence of ECGS or treated with FGF-BP (30 ng/ml) and FGF-2 (5 ng/ml) as indicated (x560 magnification). C: [3H]-labeled thymidine incorporation in the absence of endothelial cells growth supplement (ECGS) or FGF-2 ± FGF-BP to measure viable cells. The graph shows the mean ± SE values corresponding to three different experiments done in duplicate. (**P < 0.01; P < 0.0001 when comparing -FGF-2- and +FGF-2-treated cells).

 
Interaction of FGF-BP, FGF-2 and Heparan Sulfates in Endothelial Cells

FGF-2, like most other FGFs, binds to extracellular HSPGs that serve to store these growth factors and modulate FGF receptor signaling. We had reported earlier that different glycosaminoglycans inhibit FGF-1 and FGF-2 binding to FGF-BP (45), and thus we hypothesized that heparan sulfates would also impact on the FGF-BP interaction with FGF-2 in intact cells. To investigate these interactions with respect to endothelial signaling, we selected an established, immortalized endothelial cell line, GM7373, and used a standard protocol to modulate the proteoglycan sulfate content of the cells by growing the cells in the absence or presence of sodium chlorate. After sodium chlorate pretreatment with 10 to 50 mM, a >70% reduction in proteoglycan sulfation of GM7373 and bovine aortic endothelial cells was reported by different laboratories (3, 30) and this protocol was used by others to evaluate the impact on FGF signaling in different cell types and via different FGF receptor subtypes (9, 10, 20). Here, we stimulated the immortalized endothelial cells with and without sodium chlorate pretreatment and different concentrations of FGF-2 ± FGF-BP and analyzed cells for the induction of protein phosphorylation or followed the growth response of the cells. Anti-phosphotyrosine blots of cells treated with FGF-2 for 5 min showed a distinct induction of p42/p44 at 2 ng/ml of FGF-2. Addition of FGF-BP reduced this threshold concentration of FGF-2 needed to initiate signal transduction to 1 ng/ml (Fig . 4A, top). After growth of the cells in the presence of sodium chlorate, the stimulation by FGF-2 is seen at 1 ng/ml, and the addition of FGF-BP reduced this threshold concentration of FGF-2 to 0.5 ng/ml (Fig. 4A, bottom). The nature of the p42/p44 bands was confirmed by immunoblotting the same whole cell lysates with an anti phospho ERK antibody (Fig. 4B). We conclude from this that less FGF-2 is required for signaling when FGF-BP is present.



View larger version (39K):
[in this window]
[in a new window]
  		Fig. 4. FGF-BP, heparan sulfate, and FGF-2 signaling in immortalized endothelial cells. Western blot analysis for tyrosine phosphorylated proteins (A) and for phospho-ERK (B) in GM7373 endothelial cell lysates treated for 5 min with different concentration of FGF-2 ± FGF-BP (5 ng/ml). Cells were grown in control media or in the presence of sodium chlorate (50 mM) for 36 h to reduce their heparan sulfate content before growth factor stimulation. The arrows indicate pERKs. Data are representative of three independent experiments. C: proliferation rate of GM7373 endothelial cells 48 h after treatment with different concentrations of FGF-2 ± FGF-BP. Cells were grown in control media or with sodium chlorate (30 mM) for 36 h before the growth factor stimulation. Proliferation was measured by WST-1 assay as described under MATERIALS AND METHODS. Data are expressed as means ± SE and are representative of at least three independent experiments. ***P < 0.01.

 
Induction of cell growth was monitored 48 h after growth factor stimulation and required higher concentrations of FGF-2 than the threshold concentrations needed for signaling. Cell proliferation was induced by 2.5-fold with 10 ng/ml of FGF-2. The inclusion of FGF-BP enhanced this FGF-2-induced cell proliferation to 14.7-fold (P < 0.01) (Fig. 4C, left). After chlorate pretreatment of the cells, FGF-2 at 5 and at 10 ng/ml did not stimulate proliferation of the cells. Inclusion of FGF-BP, however, led to a maximum stimulation at both concentrations of FGF-2 (Fig. 4C, right). These data suggest to us that heparan sulfate proteoglycans can modulate the FGF-BP interaction with FGF-2, as well as the resulting cell signaling in endothelial cells. Moreover, FGF-BP appears to be able to supplement heparan sulfate function required for FGF growth stimulation in these cells. A model to explain these interactions is shown in Fig. 5 and discussed below.



View larger version (39K):
[in this window]
[in a new window]
  		Fig. 5. Model describing the function of FGF-BP. FGF-BP (BP) expression and secretion are enhanced in regenerating renal tubular epithelial cells. FGFs (e.g., FGF-2) are produced in renal epithelial cell and stored in the extracellular matrix in the renal interstitum bound to heparan sulfate (HS) proteoglycans. It is proposed that the secreted FGF-BP will release FGF from the HS storage and chaperone the growth factor in an active form to its cognate receptor (FGF-R) on renal capillary endothelial cells. Thus FGF-BP is proposed to enhance endothelial survival and the regeneration of renal capillaries in the context of renal tubular injury.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In recent years, several studies have demonstrated the important role of FGF-2 in the pathogenesis of renal diseases (12, 14, 24, 28, 35, 53, 54). However, to date, it is not clearly understood how FGF-2 trapped in the renal extracellular matrix can induce angiogenic effects in renal capillaries (49). In this study, we describe a potential mechanism by which the renal angiogenic activity of FGF-2 in children with HIV-HUS could be controlled. More specifically, we have found that a secreted FGF-binding protein produced in renal tubular epithelial cells of both HIV-Tg26 mice and HIV-infected children with renal disease (26) enhances the mitogenic activity of FGF-2 and the survival of cultured human renal glomerular endothelial cells. These findings, when interpreted in the context of our previous studies, may be relevant for the pathogenesis of HIV-HUS, HIV-associated nephropathy, and classic HUS (26, 31, 32).

FGF-1 and FGF-2 lack a classic signal peptide for secretion and are not released from cells following a classic secretory pathway (25, 49). Both growth factors are deposited into the extracellular matrix of the cells and tightly bound to HSPGs, and the biological activity of these FGFs is quenched by this binding (2, 22, 29, 40, 43, 47, 49, 52). The mechanisms by which FGF-2 is released and activated in the context of HUS are not clearly understood. We have found high levels of FGF-2 in the plasma and urine of HIV-infected children with classic and HIV-HUS. These patients also show an increased expression of renal heparan sulfate proteoglycans that may facilitate the renal accumulation of FGF-2 (31, 36). In addition, heparanases release by activated peripheral blood mononuclear cells (16, 48), and/or proteolytic enzymes, known to be activated in HUS (i.e, plasmin, thrombin), can digest the protein backbone of the HSPG and release biologically active FGFs from the renal extracellular matrix (1, 13, 22, 47). Here, we report that FGF-BP may represent an alternative mechanism by which the angiogenic activity of FGF-2 can be regulated in the kidney of HIV-infected children with renal disease (26).

Although FGF-PB induced only a modest increase in the FGF-2-induced survival of primary human renal glomerular endothelial cells (see Fig. 3), these results may be biologically relevant in vivo. It should be considered that lethally injured glomerular endothelial cells release FGF-2, and therefore, the ability of FGF-BP to modulate the angiogenic activity of FGF-2 decreases progressively in correlation with the accumulation of FGF-2 in the culture media, which prompted us to also use immortalized endothelial cells for more extensive parallel studies (see Fig. 4). FGF-BP may play a critical role, in particular, during the early stages of renal tubular injury, in which relatively low concentrations of FGF-2 are initially released into the renal interstitum and FGF-2 needs to reach its specific target cells in a biologically active form. The reduction of extracellular heparan sulfates in the immortalized endothelial cells reveals the effect of FGF-BP on FGF-2 growth signaling and supports the notion of a positive modulatory role for FGF-BP (Fig. 4C, right). In addition, in vivo, FGF-BP may further increase the angiogenic activity of FGF-2 by preventing its proteolytic degradation (2, 29, 43). FGF-2 also binds specifically and with high affinity to fibrin and fibrinogen (41, 42), which can potentiate its mitogenic activity. In this manner, fibrin can provide a matrix to support the angiogenic activity of the FGF-2/FGF-BP complex and regulate the process of fibrinolysis and thrombus reorganization, which are essential for the recovery of renal function (34). Alternatively, the FGF-2/FGF-BP complex may also modulate the outcome of HUS by inducing hemodynamic changes through changes in vascular tone (54) contractility (32) and blood pressure (5). Taken together, our data suggest that FGF-BP, acting in synergy with FGF-2, may enhance the regeneration of renal capillaries in children with HIV-HUS by different mechanisms. It is tempting to speculate that, in this manner, FGF-BP may participate in a feedback mechanism to facilitate the regeneration of renal tubules by promoting the survival, regeneration, and proliferation of peritubular endothelial capillaries located in close proximity to the injured tubules (21).

The accumulation of FGF-2 (33) and upregulation of FGF-BP in the kidney of HIV-Tg2626 mice and HIV-infected children with renal disease strongly suggest that both factors may play a relevant role in the pathogenesis of the renal disease. Interestingly, we found that FGF-2 (33) and FGF-BP are upregulated only during the late stages of the renal disease and in association with the development of the tubulointerstitial proliferative lesions in these mice. Since the expression of HIV-1 genes in the kidney is significantly reduced during the late stage of the renal disease (33, 39), our findings suggest that FGF-2 and FGF-BP may play an active role in the pathogenesis of these renal proliferative changes. In support of this notion, we have previously reported that renal tubular epithelial cells harvested from the urine of children with HIV-HUS express high levels of FGF-BP and FGF-2 and that FGF-BP enhances the proliferation of these cells in the presence of FGF-2 (26, 36, 37, 46). Our findings in the HIV-Tg26 model are also clinically relevant, because they mimic the upregulation of FGF-BP seen in renal sections from children with HIV-HUS and HIVAN (26). Thus these mice should provide an excellent small animal model system to determine the role of FGF-BP in the pathogenesis of HIV-associated renal diseases in vivo.

In summary, to the best of our knowledge, we have demonstrated for the first time the presence and upregulation of the secreted FGF-BP in renal tubular epithelial cells of HIV Tg26 mice with renal disease, which mimics the clinical situation seen in HIV-infected children. In addition, we have described a novel mechanism by which the renal angiogenic activity of FGF-2 can be modulated in the context of childhood HIV-HUS. These findings may not be limited to the pathogenesis of HIV-HUS and could be relevant for children with HIV-associated nephropathy (26), classic HUS (31), and renal development (26). Obviously, FGF-BP provides another mechanism of fine-tuning for FGF receptor signaling that is also impacted by extracellular matrix heparan sulfate proteoglycans (15), and we provide a model that would encompass these different players (Fig. 5).


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institutes of Health Grants R-O1-DK-49419 and HL-55605 (P. E. Ray); R-O1-CA71508 (A. Wellstein) and Fundación Argentina para el Desarrollo Infantil (P. E. Ray).


   ACKNOWLEDGMENTS
 
We thank Dr. Carl Soderland from the Applied Cell Biology Research Institute, Seattle, WA, for providing primary human renal glomerular endothelial cells.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Wellstein, Research Bldg. E311, Lombardi Comprehensive Cancer Ctr., Georgetown Univ.; 3970 Reservoir Rd., Washington DC 20057 (e-mail: wellstea{at}georgetown.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* These authors contributed equally to the manuscript. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ashton AW, Dawes J, and Chesterman CN. Acidic and basic fibroblast growth factors have comparable effects on the haemostatic function of vascular endothelium. Growth Factors 12: 111–120, 1995.[Web of Science][Medline]
  2. Bashkin P, Doctrow S, Klagsbrun M, Svahn CM, Folkman J, and Vlodavsky I. Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules. Biochemistry 28: 1737–1743, 1989.[CrossRef][Medline]
  3. Berry D, Shriver Z, Natke B, Kwan CP, Venkataraman G, and Sasisekharan R. Heparan sulphate glycosaminoglycans derived from endothelial cells and smooth muscle cells differentially modulate fibroblast growth factor-2 biological activity through fibroblast growth factor receptor-1. Biochem J 373: 241–249, 2003.[CrossRef][Web of Science][Medline]
  4. Coltrini D, Rusnati M, Zoppetti G, Oreste P, Grazioli G, Naggi A, and Presta M. Different effects of mucosal, bovine lung and chemically modified heparin on selected biological properties of basic fibroblast growth factor. Biochem J 303: 583–590, 1994.[Medline]
  5. Cuevas P, Carceller F, Ortega S, Zazo M, Nieto I, and Gimenez-Gallego G. Hypotensive activity of fibroblast growth factor. Science 254: 1208–1210, 1991.[Abstract/Free Full Text]
  6. Czubayko F, Liaudet-Coopman ED, Aigner A, Tuveson AT, Berchem GJ, and Wellstein A. A secreted FGF-binding protein can serve as the angiogenic switch in human cancer. Nat Med 3: 1137–1140, 1997.[CrossRef][Web of Science][Medline]
  7. Czubayko F, Riegel AT, and Wellstein A. Ribozyme-targeting elucidates a direct role of pleiotrophin in tumor growth. J Biol Chem 269: 21,358–21,363, 1994.[Abstract/Free Full Text]
  8. Czubayko F, Smith RV, Chung HC, and Wellstein A. Tumor growth and angiogenesis induced by a secreted binding protein for fibroblast growth factors. J Biol Chem 269: 28243–28248, 1994.[Abstract/Free Full Text]
  9. Delehedde M, Deudon E, Boilly B, and Hondermarck H. Heparan sulfate proteoglycans play a dual role in regulating fibroblast growth factor-2 mitogenic activity in human breast cancer cells. Exp Cell Res 229: 398–406, 1996.[CrossRef][Web of Science][Medline]
  10. Delehedde M, Seve M, Sergeant N, Wartelle I, Lyon M, Rudland PS, and Fernig DG. Fibroblast growth factor-2 stimulation of p42/44MAPK phosphorylation and I{kappa}B degradation is regulated by heparan sulfate/heparin in rat mammary fibroblasts. J Biol Chem 275: 33905–33910, 2000.[Abstract/Free Full Text]
  11. Dickie P, Felser J, Eckhaus M, Bryant J, Silver J, Marinos N, and Notkins AL. HIV-associated nephropathy in transgenic mice expressing HIV-1 genes. Virology 185: 109–119, 1991.[CrossRef][Web of Science][Medline]
  12. Eguchi J, Nomata K, Kanda S, Igawa T, Taide M, Koga S, Matsuya F, Kanetake H, and Saito Y. Gene expression and immunohistochemical localization of basic fibroblast growth factor in renal cell carcinoma. Biochem Biophys Res Commun 183: 937–944, 1992.[CrossRef][Web of Science][Medline]
  13. Flaumenhaft R, Moscatelli D, Saksela O, and Rifkin DB. Role of extracellular matrix in the action of basic fibroblast growth factor: matrix as a source of growth factor for long-term stimulation of plasminogen activator production and DNA synthesis. J Cell Physiol 140: 75–81, 1989.[CrossRef][Web of Science][Medline]
  14. Floege J, Eng E, Lindner V, Alpers CE, Young BA, Reidy MA, and Johnson RJ. Rat glomerular mesangial cells synthesize basic fibroblast growth factor. Release, upregulated synthesis, and mitogenicity in mesangial proliferative glomerulonephritis. J Clin Invest 90: 2362–2369, 1992.[Web of Science][Medline]
  15. Forsten-Williams K, Chua CC, and Nugent MA. The kinetics of FGF-2 binding to heparan sulfate proteoglycans and MAP kinase signaling. J Theor Biol 233: 483–499, 2005.[CrossRef][Web of Science][Medline]
  16. Fridman R, Lider O, Naparstek Y, Fuks Z, Vlodavsky I, and Cohen IR. Soluble antigen induces T lymphocytes to secrete an endoglycosidase that degrades the heparan sulfate moiety of subendothelial extracellular matrix. J Cell Physiol 130: 85–92, 1987.[CrossRef][Web of Science][Medline]
  17. Gasser C, Gautier E, Steck A, Siebenmann RE, and Oechslin R. [Scientific raisins from 127 years SMW (Swiss Medical Weekly). Hemolytic-uremic syndrome: bilateral kidney cortex necrosis in acute acquired hemolytic anemia 1925]. Schweiz Med Wochenschr 125: 2528–2532, 1995.[Web of Science][Medline]
  18. Gospodarowicz D, Ferrara N, Schweigerer L, and Neufeld G. Structural characterization and biological functions of fibroblast growth factor. Endocr Rev 8: 95–114, 1987.[Abstract/Free Full Text]
  19. Grinspan JB, Mueller SN, and Levine EM. Bovine endothelial cells transformed in vitro by benzo(a)pyrene. J Cell Physiol 114: 328–338, 1983.[CrossRef][Web of Science][Medline]
  20. Kan M, Wu X, Wang F, and McKeehan WL. Specificity for fibroblast growth factors determined by heparan sulfate in a binary complex with the receptor kinase. J Biol Chem 274: 15947–15952, 1999.[Abstract/Free Full Text]
  21. Kanda S, Hisamatsu H, Igawa T, Eguchi J, Taide M, Sakai H, Kanetake H, Saito Y, Yoshitake Y, and Nishikawa K. Peritubular endothelial cell proliferation in mice during compensatory renal growth after unilateral nephrectomy. Am J Physiol Renal Fluid Electrolyte Physiol 265: F712–F716, 1993.[Abstract/Free Full Text]
  22. Klagsbrun M and Baird A. A dual receptor system is required for basic fibroblast growth factor activity. Cell 67: 229–231, 1991.[CrossRef][Web of Science][Medline]
  23. Kopp JB, Klotman ME, Adler SH, Bruggeman LA, Dickie P, Marinos NJ, Eckhaus M, Bryant JL, Notkins AL, and Klotman PE. Progressive glomerulosclerosis and enhanced renal accumulation of basement membrane components in mice transgenic for human immunodeficiency virus type 1 genes. Proc Natl Acad Sci USA 89: 1577–1581, 1992.[Abstract/Free Full Text]
  24. Kriz W, Hahnel B, Rosener S, and Elger M. Long-term treatment of rats with FGF-2 results in focal segmental glomerulosclerosis. Kidney Int 48: 1435–1450, 1995.[Web of Science][Medline]
  25. Ku PT and D'Amore PA. Regulation of basic fibroblast growth factor (bFGF) gene and protein expression following its release from sublethally injured endothelial cells. J Cell Biochem 58: 328–343, 1995.[CrossRef][Web of Science][Medline]
  26. Liu XH, Aigner A, Wellstein A, and Ray PE. Up-regulation of a fibroblast growth factor binding protein in children with renal diseases. Kidney Int 59: 1717–1728, 2001.[CrossRef][Web of Science][Medline]
  27. McDonnell K, Bowden ET, Cabal-Manzano R, Hoxter B, Riegel AT, and Wellstein A. Vascular leakage in chick embryos after expression of a secreted binding protein for fibroblast growth factors. Lab Invest 85: 747–755, 2005.[CrossRef][Web of Science][Medline]
  28. Morita H, Shinzato T, David G, Mizutani A, Habuchi H, Fujita Y, Ito M, Asai J, Maeda K, and Kimata K. Basic fibroblast growth factor-binding domain of heparan sulfate in the human glomerulosclerosis and renal tubulointerstitial fibrosis. Lab Invest 71: 528–535, 1994.[Web of Science][Medline]
  29. Moscatelli D. Basic fibroblast growth factor (bFGF) dissociates rapidly from heparan sulfates but slowly from receptors. Implications for mechanisms of bFGF release from pericellular matrix. J Biol Chem 267: 25803–25809, 1992.[Abstract/Free Full Text]
  30. Qiao D, Meyer K, Mundhenke C, Drew SA, and Friedl A. Heparan sulfate proteoglycans as regulators of fibroblast growth factor-2 signaling in brain endothelial cells. Specific role for glypican-1 in glioma angiogenesis. J Biol Chem 278: 16045–16053, 2003.[Abstract/Free Full Text]
  31. Ray P, Acheson D, Chitrakar R, Cnaan A, Gibbs K, Hirschman GH, Christen E, and Trachtman H. Basic fibroblast growth factor among children with diarrhea-associated hemolytic uremic syndrome. J Am Soc Nephrol 13: 699–707, 2002.[Abstract/Free Full Text]
  32. Ray P, Onorio A, Sgromo S, Maglio S, Marco I, Liu XH, Xu L, and Gallo GE. Novel role of basic fibroblast growth factor in the pathogenesis of classic hemolytic-uremic syndrome. In: Eschericia Coli 0157:H7 and Other Shiga Toxin-Producing E. coli Strains, edited by Kaper JB and O'Brian AD, Washington, DC: American Society for Microbiology Press, 1998, p. 312–322.
  33. Ray PE, Bruggeman LA, Weeks BS, Kopp JB, Bryant JL, Owens JW, Notkins AL, and Klotman PE. bFGF and its low affinity receptors in the pathogenesis of HIV-associated nephropathy in transgenic mice. Kidney Int 46: 759–772, 1994.[Web of Science][Medline]
  34. Ray PE and Liu XH. Pathogenesis of Shiga toxin-induced hemolytic uremic syndrome. Pediatr Nephrol 16: 823–839, 2001.[CrossRef][Web of Science][Medline]
  35. Ray PE, Liu XH, Robinson LR, Reid W, Xu L, Owens JW, Jones OD, Denaro F, Davis HG, and Bryant JL. A novel HIV-1 transgenic rat model of childhood HIV-1-associated nephropathy. Kidney Int 63: 2242–2253, 2003.[CrossRef][Web of Science][Medline]
  36. Ray PE, Liu XH, Xu L, and Rakusan T. Basic fibroblast growth factor in HIV-associated hemolytic uremic syndrome. Pediatr Nephrol 13: 586–593, 1999.[CrossRef][Web of Science][Medline]
  37. Ray PE, Rakusan T, Loechelt BJ, Selby DM, Liu XH, and Chandra RS. Human immunodeficiency virus (HIV)-associated nephropathy in children from the Washington, D.C. area: 12 years' experience. Semin Nephrol 18: 396–405, 1998.[Web of Science][Medline]
  38. Ray PE, Soler-Garcia AA, Xu L, Soderland C, Blumenthal R, and Puri A. Fusion of HIV-1 envelope-expressing cells to human glomerular endothelial cells through an CXCR4-mediated mechanism. Pediatr Nephrol 20: 1401–1409, 2005.[CrossRef][Web of Science][Medline]
  39. Ray PE, Xu L, Rakusan T, and Liu XH. A 20-year history of childhood HIV-associated nephropathy. Pediatr Nephrol 19: 1075–1092, 2004.[Web of Science][Medline]
  40. Ruoslahti E and Yamaguchi Y. Proteoglycans as modulators of growth factor activities. Cell 64: 867–869, 1991.[CrossRef][Web of Science][Medline]
  41. Sahni A, Odrljin T, and Francis CW. Binding of basic fibroblast growth factor to fibrinogen and fibrin. J Biol Chem 273: 7554–7559, 1998.[Abstract/Free Full Text]
  42. Sahni A, Sporn LA, and Francis CW. Potentiation of endothelial cell proliferation by fibrin(ogen)-bound fibroblast growth factor-2. J Biol Chem 274: 14936–14941, 1999.[Abstract/Free Full Text]
  43. Saksela O, Moscatelli D, Sommer A, and Rifkin DB. Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation. J Cell Biol 107: 743–751, 1988.[Abstract/Free Full Text]
  44. Striker GE, Soderland C, Bowen-Pope DF, Gown AM, Schmer G, Johnson A, Luchtel D, Ross R, and Striker LJ. Isolation, characterization, and propagation in vitro of human glomerular endothelial cells. J Exp Med 160: 323–328, 1984.[Abstract/Free Full Text]
  45. Tassi E, Al-Attar A, Aigner A, Swift MR, McDonnell K, Karavanov A, and Wellstein A. Enhancement of fibroblast growth factor (FGF) activity by an FGF-binding protein. J Biol Chem 276: 40247–40253, 2001.[Abstract/Free Full Text]
  46. Turner ME, Kher K, Rakusan T, D'Angelo L, Kapur S, Selby D, and Ray PE. A typical hemolytic uremic syndrome in human immunodeficiency virus-1-infected children. Pediatr Nephrol 11: 161–163, 1997.[CrossRef][Web of Science][Medline]
  47. Vlodavsky I, Bashkin P, Ishai-Michaeli R, Chajek-Shaul T, Bar-Shavit R, Haimovitz-Friedman A, Klagsbrun M, and Fuks Z. Sequestration and release of basic fibroblast growth factor. Ann NY Acad Sci 638: 207–220, 1991.[Web of Science][Medline]
  48. Vlodavsky I, Folkman J, Sullivan R, Fridman R, Ishai-Michaeli R, Sasse J, and Klagsbrun M. Endothelial cell-derived basic fibroblast growth factor: synthesis and deposition into subendothelial extracellular matrix. Proc Natl Acad Sci USA 84: 2292–2296, 1987.[Abstract/Free Full Text]
  49. Wellstein A and Czubayko F. Inhibition of fibroblast growth factors. Breast Cancer Res Treat 38: 109–119, 1996.[CrossRef][Web of Science][Medline]
  50. Wellstein A, Fang WJ, Khatri A, Lu Y, Swain SS, Dickson RB, Sasse J, Riegel AT, and Lippman ME. A heparin-binding growth factor secreted from breast cancer cells homologous to a developmentally regulated cytokine. J Biol Chem 267: 2582–2587, 1992.[Abstract/Free Full Text]
  51. Wu DQ, Kan MK, Sato GH, Okamoto T, and Sato JD. Characterization and molecular cloning of a putative binding protein for heparin-binding growth factors. J Biol Chem 266: 16778–16785, 1991.[Abstract/Free Full Text]
  52. Yayon A, Klagsbrun M, Esko JD, Leder P, and Ornitz DM. Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 64: 841–848, 1991.[CrossRef][Web of Science][Medline]
  53. Zhang G, Ichimura T, Maier JA, Maciag T, and Stevens JL. A role for fibroblast growth factor type-1 in nephrogenic repair. Autocrine expression in rat kidney proximal tubule epithelial cells in vitro and in the regenerating epithelium following nephrotoxic damage by S-(1,1,2,2-tetrafluoroethyl)-L-cysteine in vivo. J Biol Chem 268: 11542–11547, 1993.[Abstract/Free Full Text]
  54. Zhou M, Sutliff RL, Paul RJ, Lorenz JN, Hoying JB, Haudenschild CC, Yin M, Coffin JD, Kong L, Kranias EG, Luo W, Boivin GP, Duffy JJ, Pawlowski SA, and Doetschman T. Fibroblast growth factor 2 control of vascular tone. Nat Med 4: 201–207, 1998.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
W. Zhang, Y. Chen, M. R. Swift, E. Tassi, D. C. Stylianou, K. A. Gibby, A. T. Riegel, and A. Wellstein
Effect of FGF-binding Protein 3 on Vascular Permeability
J. Biol. Chem., October 17, 2008; 283(42): 28329 - 28337.
[Abstract] [Full Text] [PDF]

Home page
 Mayo Clin Proc.Home page
T. I. de Silva, F. A. Post, M. D. Griffin, and D. H. Dockrell
HIV-1 Infection and the Kidney: An Evolving Challenge in HIV Medicine
Mayo Clin. Proc., September 1, 2007; 82(9): 1103 - 1116.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
E. Tassi, S. Walter, A. Aigner, R. H. Cabal-Manzano, R. Ray, P. J. Reier, and A. Wellstein
Effects on neurite outgrowth and cell survival of a secreted fibroblast growth factor binding protein upregulated during spinal cord injury
Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2007; 293(2): R775 - R783.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
C. S. Wilcox
Special feature: cardiovascular-kidney interactions in health and disease
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2006; 290(1): R34 - R36.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ray, P. E.
Right arrow Articles by Wellstein, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ray, P. E.
Right arrow Articles by Wellstein, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.