(Hypertension. 1996;27:897-905.)
© 1996 American Heart Association, Inc.
Articles |
Angiotensin II Is Mitogenic for Cultured Rat Glomerular Endothelial Cells
From the Department of Medicine, Division of Nephrology and Osteology, University of Hamburg (Germany), and the Renal-Electrolyte and Hypertension Division and the Penn Center for Molecular Studies of Kidney Diseases, University of Pennsylvania School of Medicine, Philadelphia, Pa (F.N.Z.).
Correspondence to Gunter Wolf, MD, University of Hamburg, University Hospital Eppendorf, Department of Medicine, Division of Nephrology and Osteology, Pavilion 61, Martinistraße 52, D-20246 Hamburg, FRG.
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Abstract |
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Abstract Angiotensin II (Ang II) has growth-stimulatory properties on different renal cell types. However, possible growth effects of this vasoactive peptide on endothelial cells isolated from the glomerular microvasculature have not formally been investigated. Therefore, we isolated and characterized primary cultures of rat glomerular endothelial cells. We used a simple technique in which collagenase-treated glomeruli were sparsely plated in several 96-well culture plates and microscopically screened for cobblestone-like outgrowth. After two limiting dilutions, homogeneous cultures were obtained. Cells were characterized by positive staining for the endothelial markers factor VIII, CD 31, endothelial leukocyte adhesion molecule-1, and the lectin Bandeiraea simplificifolia. Ang II stimulated the synthesis and release of endothelin-1 in culture supernatants. Moreover, in contrast to syngeneic mesangial cells, glomerular endothelial cells expressed angiotensin-converting enzyme. Ang II stimulated a mild but significant proliferation of quiescent cells, as measured by [3H]thymidine incorporation and direct cell counting. This mitogenesis was transduced by losartan-blockable angiotensin type 1 receptors. Moreover, Ang II mediated phosphorylation of mitogen-activated protein kinase 2 and induction of transcripts for the immediate early gene Egr-1. Our results indicate that Ang II is a moderate mitogen for primary cultures of rat glomerular endothelial cells and activation of these metabolically active cells may play a role in the pathophysiology of several types of glomerulonephritis. Moreover, remodeling of glomerular endothelial cells by Ang II may be important in the progression of structural renal damage during the course of hypertensive injury.
Key Words: angiotensin II • hyperplasia • glomerular endothelial cells • proto-oncogenes • protein kinases
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Introduction |
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Endothelial cells, which line blood vessels throughout the body, are metabolically active and play a pivotal role in modifying the functional state of adjacent or trafficking cells.1 2 3 4 The endothelium is involved in such diverse functions as coagulation, transcapillary transport of solutes and water, inflammation and recruitment of immune competent cells, modulation of vasomotor tone, and presentation of antigens in the context of class II molecules.1 2 3 4 5 6 On the other hand, endothelial cells themselves, being exposed to the bloodstream, are the target of many different cytokines and growth factors that may alter the functional state of these cells.4 Although most previous studies have been performed with cultured endothelial cells from large vessels or the umbilical cord, there is clear evidence that endothelium from capillary beds such as the glomerular tuft behaves differently from endothelium in larger vessels.1 7 8 9 10 GECs from the kidney are in close juxtaposition to other cell types, such as mesangial cells, and may respond to as well as release different cytokines that subsequently influence the function of mesangial cells.2 8 9 Weak proliferation of GECs has been described in various types of glomerulonephritis and in transplant rejection.11 12 In addition, experimentally induced glomerulonephritis in the rabbit produced by injection of antiendothelial antibodies has been characterized by a slight proliferation of endothelial cells and the formation of subepithelial immune deposits, suggesting that endothelial cells may be an immune target in specific types of glomerulonephritis.13 Vascular remodeling with the formation of a neointima is a common feature of atherosclerosis and hypertension.14 15 Although mainly vascular smooth muscle cells contribute to the observed hyperplasia seen in these conditions, there is also evidence that the endothelium contributes to the overall proliferation.16 Thus, proliferation of GECs may contribute to structural damage of glomeruli, as seen in malignant hypertension.17
In contrast to the easily obtainable endothelial cells from larger vessels,2 in vitro studies of potential growth factors for GECs have been hampered by the difficulties in isolating and propagating these cells in culture.18 More recently, however, successful cultures of bovine, rat, human, and baboon GECs have been described.9 18 19 20
We have been interested in Ang II as a potential growth factor for renal cells.21 Our interest stems from the observation that systemic or intrarenal production of Ang II may influence diverse renal parameters, such as glomerular hemodynamics, tubular transport, and proliferation or hypertrophy of renal cells, making the peptide a possible common denominator for the multiple pathophysiological alterations recognized in chronic renal disease.22 23 24 We designed the present studies to test the hypothesis that Ang II has potential growth-stimulatory effects on primary cultures of rat GECs. Our results demonstrate that Ang II induces MAP kinase 2 and the immediate early gene Egr-1 in rat GECs and is a mitogen for these cells.
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Methods |
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Isolation and Characterization of Rat GECs
Rat GECs were isolated from Sprague-Dawley rats. Kidneys from two adult female rats (100 to 120 g body weight) were pooled and glomeruli isolated by differential sieving as previously described.25 The resulting preparation contained greater than 95% glomeruli as judged by light microscopy. Glomeruli were treated with 0.1% collagenase for 20 minutes (type IV collagenase, Sigma Chemical Co) and washed twice in DMEM (with 450 mg/dL glucose; GIBCO-BRL) with 10% fetal calf serum (GIBCO), 2 mmol/L supplemental glutamine, 5 µg/mL insulin, 100 U/mL penicillin, and 100 µg/mL streptomycin. Fifty microliters of the cell suspension was then plated into each well of a 96-well cell culture plate (Nunc) previously covered with fibronectin. The microtiter plates were screened by phase-contrast microscopy for the presence of cells with a cobblestone appearance, which is one characteristic feature of cultured endothelial cells.2 9 20 Cells fulfilling this criterion were subsequently cloned twice by limiting dilution, and a homogeneous cell line was obtained. For all experiments, cells between passages 10 and 25 were used.
GECs were directly photographed in cell culture flasks by phase
contrast. Cells were further characterized by
immunofluorescence. For these studies, cells were
grown in glass slide chambers until they were subconfluent. The cells
were fixed at -20°C in acetone for 10 minutes before staining.
Cells were stained with the following antibodies with the use of
indirect immunofluorescence: monoclonal anti-Thy
1.1 (Pharmingen), polyclonal anti–CD 3126 (Pharmingen),
anti–human factor VIII (Miles), and anti–ELAM-127
(Immunotech). For expression of ELAM-1, GECs were also pretreated for
24 hours with 10 ng/mL recombinant murine tumor necrosis factor-
(Genzyme) before staining with the anti–ELAM-1 antibody. Moreover,
GECs were stained with fluoresceinated BSI lectin (Sigma),
a previously described marker of a rat GEC line.20
ACE activity was measured in cell lysates with a commercial assay system (Sigma). This assay is a spectrophotometric method utilizing the hydrolysis of the synthetic peptide FAPGG by ACE. Human serum of known ACE activity served as a standard. Total protein content was determined by a modification of the Lowry method, and ACE activity was expressed per milligram total protein. For these experiments, primary cultures of mesangial cells (passages 10 to 12) isolated from Sprague-Dawley rats were used as an additional control.
Ang II Receptor Binding Studies
Binding studies were performed on whole cells grown to
subconfluence in 24-well culture plates in assay buffer (150 mmol/L
NaCl, 50 mmol/L Tris-HCl [pH 7.1], 5 mmol/L EDTA, 5 mmol/L
MgCl2, 1 mmol/L phenylmethylsulfonyl
fluoride, 0.7% bovine serum albumin, 0.5% aprotinin)
on a shaking platform at 22°C for 2 hours. For binding curves, the
amount of specific binding (total nonspecific) was determined with 0.5
pmol/L
125I–[Sar1,Ile8]Ang II
(2000 Ci/mmol, Amersham) in the presence of
10-12 to
10-7 mol/L unlabeled
[Sar1,Ile8]Ang II (Sigma). Nonspecific
binding was determined in the presence of
10-5 mol/L
[Sar1,Ile8]Ang II and was less than
15% of total binding. For characterization of receptor subtypes, cells
were preincubated for 30 minutes with either
10-7 mol/L of the AT1
receptor blocker losartan (gift of Merck, Sharp & Dohme) or
10-7 mol/L of the AT2
receptor blocker PD 123177 (gift of Parke-Davis). Subsequently, 0.5
pmol/L
125I–[Sar1,Ile8]Ang II
was added, and specific binding in the absence of Ang II receptor
antagonists was considered 100%. After incubation, plates
were washed three times with ice-cold PBS for removal of unbound
[Sar1,Ile8]Ang II. The cells were
dissolved in 1N NaOH, and the amount of radioactivity was counted in a
gamma scintillation counter.
ET-1 ELISA
For measurement of ET-1 in culture supernatants, 105
GECs were plated into each well of a 24-well plate, made quiescent for
24 hours in serum-free DMEM, and subsequently stimulated with a
single dose of 10-10 to
10-5 mol/L Ang II acetate salt (Sigma)
redissolved in medium. After 24 hours, the supernatant was collected
and cells were trypsinized, scraped off the plate, and counted in a
Neubauer chamber. ET-1 was measured in 100 µL of cell culture
supernatant with a commercial two-site sandwich ELISA for ET-1
(Amersham) according to the manufacturer's specifications. ET-1 was
expressed as femtomoles per 104 cells. This experiment was
independently repeated six times, with duplicates for each assay.
Cell Proliferation Assays
A total of 104 GECs were transferred to each well of
a 96-well plate and were made quiescent in DMEM without serum. Cells
were then incubated for 24 hours with a single dose of
10-10 to
10-6 mol/L Ang II. Additional cells were
treated with Ang II in the presence of
10-6 mol/L losartan or
10-6 mol/L of the AT2
antagonist CGP 42112A (Neosystems). Cells were pulsed with
1 µCi per well [3H]thymidine (5 Ci/mmol, Amersham)
during the last 6 hours of culture. At the end of the incubation
period, GECs were washed twice in PBS, trypsinized for 10 minutes at
37°C, and finally collected on glass-fiber paper with an
automatic cell harvester.25 Radioactivity of dry filters
was determined by liquid scintillation spectroscopy in the presence of
5 mL per vial Rotiszint (Roth) scintillation cocktail.
[3H]Thymidine incorporation experiments were
independently repeated 10 times in duplicate. For direct cell counts,
105 GECs were plated in each well of a 24-well plate,
rested for 24 hours, and stimulated for another 24 hours with
10-10 to
10-6 Ang II. At the end of the incubation
period, cells were washed in PBS, scraped off the plate, and counted in
a Neubauer chamber. Direct cell count experiments were repeated
independently six times in duplicate.
Northern Hybridizations
Quiescent GECs were stimulated for 5 to 60 minutes with a single
dose of 10-7 mol/L Ang II in the presence
or absence of 10-6 mol/L
losartan. After being washed in RNAse-free PBS, cells were
directly lysed with acid guanidinium thiocyanate, and total RNA was
isolated by repeated phenol-chloroform extractions.28
Equal amounts of total RNA (20 µg per lane) were denatured in
formamide-formaldehyde at 65°C and electrophoresed through a
1.2% agarose gel containing 2.2 mol/L formaldehyde. The RNA was then
transferred to nylon membranes (Zetabind, Cuno) by vacuum blotting, and
filters were UV cross-linked with 120 000 µJ/cm2
with Stratalinker (Stratagene). Prehybridizations were performed for 2
hours at 70°C in a rotating drum with the Rapid-hyb buffer system
(Amersham) according to the manufacturer's recommendations. The
Egr-1 probe was a 300-bp MsP-1 fragment of the mouse
Egr-1 cDNA.29 For control hybridizations, a
2.0-kb cDNA insert of the plasmid pMCI encoding the murine ribosomal
18S band was used. Probes were labeled with 5 µCi
[32P]dATP (3000 Ci/mmol, Amersham) using a nick
translation kit (Amersham). The labeled fragments were separated from
unincorporated nucleotides by Sephadex G-50
chromatography, and membranes were hybridized with
106 cpm probe per milliliter hybridization buffer
(Rapid-hyb system from Amersham) for 24 hours at 70°C. After
hybridization, membranes were washed in 2x SSC (20x SSC: 3 mol/L
sodium chloride, 0.3 mol/L sodium citrate) and 0.5% SDS at room
temperature and subsequently twice for 30 minutes in 0.4x SSC and
0.5% SDS at 65°C. Autoradiography was performed
with intensifying screens at -70°C for 24 hours
(Egr-1) or 1 hour (18S). Blots were stripped for 60 minutes
in 5 mmol/L Tris-HCl (pH 8.0), 0.5% sodium pyrophosphate, 5x
Denhardt's (100x Denhardt's: 2% Ficoll 400, 2%
polyvinylpyrrolidone, 2% bovine serum albumin), and 0.2 mmol/L
EDTA (pH 8.0) at 65°C and rehybridized with the cDNA probe for the
ribosomal 18S band. Exposed films were scanned with a laser
densitometer (GS 300, Hoefer Scientific Instruments) connected to a
computer system. The areas under the curves were calculated by gaussian
integration with the program GS 365 W (Hoefer). Relative changes in RNA
were calculated after hybridization in control lanes was assigned an
arbitrary value of 1.28 Samples were normalized for the
signal intensity of the 18S hybridization. Northern blots were repeated
three times with qualitatively similar results.
Western Blotting and Immunoprecipitations
A total of 106 quiescent GECs were treated
for 5 to 60 minutes with 10-7 mol/L Ang
II in the presence or absence of 10-6
mol/L losartan. After being washed in PBS, cells were directly
lysed in 150 µL of electrophoresis buffer (125 mmol/L Tris-HCl [pH
6.8], 2% SDS, 5% glycerol, 100 mmol/L dithiothreitol), and the
protein content was measured by a modification of the Lowry method that
is insensitive to the concentrations of SDS and dithiothreitol
used.30 After addition of dye (0.003% bromophenol blue),
samples were boiled and centrifuged, and equal amounts of
protein (50 µg per lane) were loaded onto a denaturing 8%
SDS-polyacrylamide gel. Molecular weight markers (Rainbow
markers, Amersham) that comprise 14 300 to 200 000 D served as
molecular weight standards. After completion of electrophoresis,
proteins were electroblotted onto a nitrocellulose membrane
(Highland-N, Americium) in transfer buffer (50 mmol/L Tris-HCl [pH
7.0], 380 mmol/L glycine, 0.1% SDS, 20% methanol). Membranes were
blocked for 1 hour at room temperature with 5% nonfat dry milk
redissolved in PBS with 0.1% Tween 20. For the detection of MAP kinase
2, an affinity-purified polyclonal rabbit antibody (Transduction
Laboratories) was used in a 1:1000 dilution. A horseradish
peroxidase–conjugated polyclonal anti-rabbit antibody was used
as a secondary antibody. The ECL reagent (Amersham) was applied as a
detection system. Western blots were repeated four times with
qualitatively similar results.
Since activation of MAP kinase 2 involves autophosphorylation as well as phosphorylation by MAP kinase kinases,31 immunoprecipitation studies were performed. Cells were stimulated as described above and lysed at the end of the incubation period in 500 µL cold immunoprecipitation buffer (1% Triton X-100, 150 mmol/L NaCl, 10 mmol/L Tris-HCl [pH 7.4], 1 mmol/L EDTA, 1 mmol/L EGTA, 0.2 mmol/L sodium vanadate, 0.2 mmol/L phenylmethylsulfonyl fluoride, and 0.5% NP-40). Cells were scraped from the culture flask and passed twice through a 26-gauge needle to disperse any large aggregates. After centrifugation for 15 minutes at 4°C, 100 µL of supernatant was transferred to new tubes, and 5 µg polyclonal anti–MAP kinase 2 antibody, 400 µL distilled water, and 500 µL immunoprecipitation buffer were added. After vortexing and incubation for 1 hour at 4°C, 50 µL S. aureus Cowan strain (Calbiochem) was added, and tubes were incubated with agitation for another 30 minutes at 4°C. Precipitates were then washed three times with immunoprecipitation buffer, and recovered pellets were suspended in 30 µL electrophoresis buffer, boiled for 5 minutes, and centrifuged, and supernatants were finally loaded onto an 8% SDS-polyacrylamide gel. After Western blotting as described above, phosphorylated tyrosine residues were detected with a monoclonal anti-phosphotyrosine antibody (PY20, Transduction Laboratories). For these experiments, a horseradish peroxidase–conjugated anti-mouse antibody was used as a secondary antibody. Immunoprecipitation experiments with subsequent Western blotting were performed three times.
Statistical Analysis
Results are expressed as mean±SE. Statistical significance
between multiple groups was first tested with the
nonparametric Kruskal-Wallis test for multiple comparisons.
Individual groups were then tested with the
Wilcoxon-Mann-Whitney test. A value of P<.05 was
considered significant.
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Isolation and Characterization of Rat GECs
One goal of the present study was to isolate a rat GEC line. Although methods for the isolation and growth of bovine and rat endothelial cells requiring specific growth conditions have been published,9 18 19 32 33 we selected a somewhat different approach. Collagenase-treated glomeruli were plated in two 96-well plates and screened for the growth of cells with a cobblestone appearance. Cobblestone-like outgrowth was directly observed from the middle of the glomeruli remnants, making contamination with endothelial cells of nonglomerular origin very unlikely. At least three different clones were isolated and cloned to homogeneity by limiting dilution, and one clone was propagated for further studies.
Phase-contrast microscopy of vital cells in a culture flask
demonstrates the typical cobblestone appearance of GECs being grouped
in growth islands (Fig 1A
). Fig 1B shows
immunofluorescence expression of factor VIII, a
typical feature of endothelial cells.1 3
Moreover, our GECs expressed CD 31 (also called PECAM-1), a marker of
endothelial cells and platelets (Fig 1C).23 Treatment of GECs with 10 ng/mL recombinant tumor
necrosis factor- induced strong expression of ELAM-1 (Fig 1E),
whereas expression of this endothelial-specific
adhesion molecule was weak in unstimulated cells (Fig 1D). Fig 1F
reveals positive staining with the lectin BSI, which has been
previously described as a marker of rat GECs.20 However,
as previously described,20 BSI also reacts with
endothelial cells from other vascular beds, such as rat
endothelial cells derived from aorta (data not shown).
In addition, GECs bound fluorescent acetylated
low-density lipoprotein but failed to stain with an anti-Thy 1.1
antibody, a usual marker of rat mesangial cells (data not
shown).
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We next determined the presence of ACE, a typical endothelial marker, in cellular lysates of unstimulated GECs.5 8 9 In contrast to syngeneic mesangial cells, GECs expressed a considerable amount of active ACE (GECs, 150±7.8 mU/mg protein; mesangial cells, 12±1.3; n=8, P<.01).
To further characterize possible Ang II receptor subtypes, we performed some limited receptor binding studies. Competition studies with unlabeled [Sar1,Ile8]Ang II revealed a Ki value of 5±0.8x10-10 mol/L (n=4, data not shown). Preincubation of GECs with 10-7 mol/L losartan replaced 75±2.4% of 125I–[Sar1,Ile8]Ang II (n=4). In contrast, 10-7 mol/L PD 123177 replaced only 20±1.8% of 125I–[Sar1,Ile8]Ang II (n=4).
Since it has been previously described that endothelial
cells may synthesize endothelins after Ang II treatment,34
we measured ET-1 in culture supernatant of quiescent GECs treated for
24 hours with different doses of Ang II. As shown in Fig 2, Ang II dose-dependently stimulated the synthesis and
release of ET-1 in cell culture supernatants, with a maximal response
at 10-9 to
10-8 mol/L Ang II.
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Effect of Ang II on Proliferation
A single dose of 10-9 to
10-7 mol/L Ang II for 24 hours
significantly stimulated incorporation of [3H]thymidine
into quiescent GECs (Fig 3). Maximal stimulation was
observed with 10-8 mol/L Ang II, which
was as high as the proliferation induced by 10% fetal calf serum (Fig 3). This Ang II–stimulated incorporation of
[3H]thymidine was attenuated by the AT1
receptor blocker losartan but not by an AT2
receptor blocker (Table 1). In addition, treatment with
the Ang II receptor blocker did not significantly influence baseline
proliferation (Table 1). The Ang II–induced proliferation was also
reflected in a significant increase in total cell counts (Table 2).
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Induction of Egr-1 mRNA
Transcripts for the immediate early gene Egr-1 were
induced after 30 minutes of treatment with
10-7 mol/L Ang II (control, 1.00 relative
changes in mRNA level; 5 minutes, 0.98; 10 minutes, 1.51; 30 minutes,
15.21; 60 minutes, 10.30; Fig 4, left). This increase at
30 minutes was partly abolished by coincubation with
10-6 mol/L losartan (control,
1.00 relative changes in mRNA level; Ang II, 8.43; Ang II plus
losartan, 5.03; Fig 4, right).
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Phosphorylation of MAP Kinase 2
Since its has been described in other cells that Ang II–mediated
proliferation is associated with phosphorylation and
activation of MAP kinases,35 36 37 38 we immunoprecipitated MAP
kinase 2 from total cell lysates and probed the precipitates after
Western blotting with a specific anti-tyrosine antibody. As shown
in Fig 5, 10-7 mol/L Ang
II stimulated after 10 minutes the phosphorylation of
MAP kinase 2. This effect was maximal after 30 minutes of treatment.
Coincubation with 10-6 mol/L
losartan abolished this Ang II–induced
phosphorylation (data not shown). Ang II–induced
phosphorylation of MAP kinase 2 was also demonstrated
by a shift in the electrophoretic mobility when total cell lysates were
directly subjected to Western blot analysis with the anti–MAP
kinase 2 antibody (Fig 6).
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Discussion |
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GECs are at the interface between blood and adjacent cell populations such as mesangial cells.5 They probably play an important role in the interaction between circulating cytokines and the proliferation of intrinsic glomerular cells. For study of the effects of single cytokines and growth factors on GECs, a cell culture approach is mandatory, and one major goal of the present study was to establish and characterize a rat GEC line. However, the culture of microvascular endothelium-like GECs still remains difficult. Although the landmark studies by Ballermann9 and Striker and Striker18 provided detailed instructions on how to cultivate bovine and human GECs, these methods are rather complex and may not be applicable to rats with the same efficiency. Therefore, we selected a simple approach to isolate GECs. Collagenase-treated glomeruli were plated in 96-well plates, and wells were microscopically screened for the growth of cells with a cobblestone appearance. After identification and subcloning, homogeneous cell populations were obtained. Positive staining for factor VIII, CD 31, ELAM-1, and the lectin BSI as well as the presence of ACE clearly identified these cells as endothelial cells.1 20 26 27 These markers were still expressed in later passages (tested at passage 30) of this primary culture. We have also generated SV 40 transformed cell lines from the primary culture, and these transformed cells express the same markers as the parental cells although they exhibit a different, more fibroblast-like growth pattern (G.W. and F.N.Z., unpublished observations, 1995). Thus, we believe that our simplified method may generally facilitate the isolation and characterization of GECs.
We have a long-term interest in possible growth-stimulatory effects of Ang II on renal cells. There is considerable recent interest in the role of Ang II in the progression of chronic renal disease, and the growth-stimulatory properties of this vasoactive peptide play a pivotal role in Ang II–mediated renal injury.21 22 23 24 25 We and others have previously shown that Ang II stimulates hypertrophy of cultured proximal tubular cells, whereas it has a mitogenic action on more distal tubular cells.28 38 39 Our present study reveals that Ang II has a reproducible mitogenic effect on quiescent GECs. This Ang II–induced proliferation was comparable to that caused by 10% fetal calf serum. Under the growth conditions used, GECs exhibit only a weak basal proliferation compatible with their nontransformed origin. The Ang II–mediated proliferation in GECs is transduced by AT1 receptors because losartan but not the AT2 blocker CGP 42112A attenuated the effects of Ang II. A dose of 10-8 to 10-7 mol/L Ang II exhibited the maximal proliferative effect. Higher doses of Ang II may downregulate receptor expression, as described in other cell systems,40 and therefore may partly abolish the mitogenesis with higher concentrations of Ang II. Moreover, an optimal growth-stimulatory effect of 10-8 to 10-7 mol/L Ang II has been demonstrated on several other cell lines.28 38 41
We have performed some limited receptor binding studies on GECs. Competition studies revealed the presence of high-affinity Ang II receptors on the surface of whole GECs. Although we have not performed dose-dependent competition studies with the respective AT1 and AT2 antagonists, the observation that losartan replaced 75% and PD 123177 only 20% of 125I–[Sar1,Ile8]Ang II binding strongly suggests the presence of AT1 and AT2 receptors on GECs. Further studies are currently in progress to analyze the presence of transcripts for AT1 and AT2 receptors in the cells.
Although growth-promoting actions of Ang II on vascular smooth muscle cells have been described in great detail,41 42 43 44 only a few studies have addressed the potential effects of this octapeptide on endothelial growth.44 45 More than a decade ago, Fernandez et al45 provided evidence that Ang II stimulated neovascularization in rabbit corneas. This effect was most likely due to Ang II–mediated proliferation and differentiation of endothelial cells.45 More recent evidence suggests that this angiogenesis may be due to Ang II–stimulated expression of vascular endothelial growth factor.46 Is has been reported in preliminary form that Ang II is a potent mitogen for bovine GECs, with a maximal effect at 10-7 mol/L.44 This proliferation was mediated through the AT1 receptor and was associated with tyrosine phosphorylation of cellular proteins.44 Evidence exists from cells such as vascular smooth muscle and mesangial cells that the Ang II–mediated mitogenic effects may be due to the synthesis and autocrine action of ET-1.34 41 47 Although we have not investigated this possible autocrine mechanism in the present study, the fast induction of MAP kinase 2 and Egr-1 as well as the proliferation observed within 24 hours argue for a direct mitogenic effect of Ang II on rat GECs.
In a recent article, Stoll et al48 reported that in cultured rat coronary endothelial cells, Ang II only stimulated proliferation after pretreatment with an AT2 receptor antagonist. These interesting observations suggest that antimitogenic effects of Ang II may be mediated through AT2 receptors, a finding of potential therapeutic interest.48 However, although our limited receptor binding studies clearly suggest the presence of AT1 and AT2 receptors in GECs, we did not observe an increase in Ang II–mediated proliferation after blocking of AT2 receptors. Differences in the expression of Ang II receptor subtypes and/or coupling of the AT2 receptor to different signal transduction pathways in coronary endothelial cells and our GECs may account for this difference. More studies are certainly necessary to address this fascinating issue of potential antimitogenic effects of Ang II under specific conditions.
Over the past years, a group of MAP kinases have been identified which are important mediators of signal transduction from cell surface receptors to the nucleus.31 49 MAP kinases are activated by dual phosphorylation on threonine and tyrosine in response to a wide array of extracellular mitogens and physical stimuli.49 Ang II–stimulated activation and phosphorylation of MAP kinases has been described in mesangial cells, vascular smooth muscle cells, and neonatal cardiac fibroblasts.35 36 37 47 In vascular smooth muscle cells, an Ang II–induced signal transduction pathway has been defined: After binding of Ang II to AT1 receptors and coupling with G protein–mediated signal transduction, phospholipase C is activated, resulting in formation of diacylglycerol and inositol trisphosphate.36 41 Subsequent signal transduction steps involve the activation of protein kinase C, tyrosine kinases, MAP kinase kinase, and MAP kinase. MAP kinase itself may phosphorylate nuclear transcription factors, which in turn bind to the promoters of immediate early genes, whose gene products are important in the initiation of the G0-G1 transition of the cell cycle.36 41 Two main isoforms of MAP kinases have been described, p44 (Erk1, MAP kinase 1) and p42 (Erk2, MAP kinase 2).31 49 We have studied the phosphorylation and expression of MAP kinase 2 in GECs after a single dose of 10-7 mol/L Ang II. In agreement with observations obtained from other cell systems,35 36 47 50 Ang II rapidly stimulates phosphorylation of MAP kinase 2 as measured by specific immunoprecipitation with an anti–MAP kinase 2 antibody and probing with an anti-phosphotyrosine antibody as well as by electrophoretic shifts in direct Western blotting analysis. Similar to our observations, Yamada et al50 have reported in abstract form that Ang II activates MAP kinase in renal opossum kidney (OK) cells, a renal tubular cell line. This phosphorylation of MAP kinase was blocked in the presence of losartan, suggesting signal transduction through the AT1 receptor.50 A recent study has addressed the effects of Ang II on MAP kinases in a rat mesangial cell line in which the peptide induced cellular hypertrophy rather than proliferation.51 Although Ang II as well as the mitogen platelet-derived growth factor stimulated both MAP kinase isoforms with comparable kinetics and potencies, Ang II caused, in contrast to platelet-derived growth factor, only a transient phosphorylation of the upstream activator MAP kinase kinase.51 These studies suggest that upstream activators of MAP kinases may be differentially regulated by various growth factors.
Induction of the immediate early gene Egr-1 occurs after hyperplastic and hypertrophic stimuli in different cells.29 However, expression of Egr-1 transcripts in rat mesangial cells, in contrast to fetal human or transformed murine mesangial cells,21 25 in which Ang II does not induce proliferation but does stimulate cellular hypertrophy51 has been linked to mitogenicity of putative growth factors.52 53 Thus, these observations are in agreement with Ang II–mediated proliferation and Egr-1 expression in our GECs. Whether Egr-1 transcription is activated through the stimulated MAP kinase 2 signaling cascade remains to be tested.
The renin-angiotensin system contributes substantially to the development of glomerulosclerosis after reduction of renal mass.21 54 55 In a recent study, Lee and associates,54 using in situ reverse transcription, found an early increase of angiotensinogen and transforming growth factor-ß expression in GECs after renal ablation. These observations may suggest that GECs produce their own Ang II. Moreover, since endothelial cells including GECs express functional ACE, it is possible that Ang I is locally converted to Ang II.56 Thus, in addition to systemic concentrations of Ang II in the blood, GECs may also be influenced by the local generation of Ang II. Indeed, micropuncture studies revealed that Ang II concentrations in the glomerular ultrafiltrate were approximately 100 times higher than systemic levels, indicating glomerular generation of Ang II.57
Although frank proliferation of endothelial cells is not an obvious feature in most forms of glomerulonephritis, mild mitogenesis of these cells is observed in many types of human glomerulonephritis, in models of proliferative glomerulonephritis like the Habu snake venom–induced glomerular injury, and during transplant rejection.11 12 58 59 Evidence also exists that proliferation of GECs occurs during malignant hypertension and partly contributes to structural glomerular damage seen in these situations.16 17 In addition, Ang II infusion into rats for 14 days causes endothelial proliferation of the larger vessels and glomeruli.60 However, it remains to be determined whether this proliferation is due to Ang II–mediated hypertension or to the growth-stimulatory effects of the peptide per se.
We believe that the Ang II–induced proliferation of GECs is part of a general activation of these metabolically active cells.1 2 3 For example, we have obtained preliminary evidence that Ang II stimulates the synthesis and release of chemoattractive factors for macrophages/monocytes (unpublished observations, 1995). Similar observations have previously been made with Ang II–treated bovine aortic and human umbilical vein endothelial cell cultures.61 In addition, it is likely that Ang II stimulates the production of many other cytokines and growth factors that in turn may influence the proliferation and function of adjacent mesangial cells. In light of the many diverse mechanisms of how Ang II contributes to the progression of chronic renal injury,21 the induced proliferation of GECs may be only one of several deleterious effects of the octapeptide on glomerular function.
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Acknowledgments |
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Part of these studies was supported by the Deutsche Forschungsgemeinschaft (Wo 460/2-1, 2-2). We thank Regine Schroeder for technical help.
Received September 7, 1995; first decision October 23, 1995; accepted January 9, 1996.
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1. Pober JS, Cotran RS. Cytokines and endothelial cell biology. Physiol Rev. 1990;70:427-451.
2. Cotran RS. New roles for the endothelium in inflammation and immunity. Am J Pathol. 1987;129:407-413. [Medline] [Order article via Infotrieve]
3. Mantovani A, Bussolino F, Dejana E. Cytokine regulation of endothelial cell function. FASEB J. 1992;6:2591-2599. [Abstract]
4. Gerritsen ME, Bloor CM. Endothelial cell gene expression in response to injury. FASEB J. 1993;7:523-532. [Abstract]
5. Savage CO. The biology of the glomerulus: endothelial cells. Kidney Int. 1994;45:314-319. [Medline] [Order article via Infotrieve]
6. Nitta K, Simonson MS, Dunn MJ. The regulation and role of prostaglandin biosynthesis in cultured bovine glomerular endothelial cells. J Am Soc Nephrol. 1991;2:156-163. [Abstract]
7.
Canfield AE, Wren FE, Schor SL, Grant ME, Schor
AM. Aortic endothelial cell
heterogeneity in vitro. J Cell
Sci. 1992;102:807-814.
8. Marsden PA, Goligorsky MS, Brenner BM. Endothelial cell biology in relation to current concepts of vessel wall structure and function. J Am Soc Nephrol. 1991;1:931-948. [Abstract]
9.
Ballermann BJ. Regulation of bovine
glomerular endothelial cell growth in
vitro. Am J Physiol. 1989;256:C182-C189.
10. Page C, Rose M, Yacoub M, Pigott R. Antigenic heterogeneity of vascular endothelium. Am J Pathol. 1992;141:673-683. [Abstract]
11. Cotran RS, Pober JS. Effects of cytokines on vascular endothelium: their role in vascular and immune injury. Kidney Int. 1989;35:969-975. [Medline] [Order article via Infotrieve]
12. Hostetter TH. Chronic transplant rejection. Kidney Int. 1994;46:266-279. [Medline] [Order article via Infotrieve]
13. Matsuo S, Fukatsu A, Taub ML, Caldwell PR, Brentjens JR, Andres G. Glomerulonephritis induced in the rabbit by antiendothelial antibodies. J Clin Invest. 1987;79:1798-1811.
14. Schwartz SM, Majesky MW, Dilley RJ. Vascular remodeling in hypertension and atherosclerosis. In: Laragh JH, Brenner BM, eds. Hypertension: Pathophysiology, Diagnosis and Management. New York, NY: Raven Press Publishers; 1990:521-539.
15. Schwartz SM. Biology of the neointima. Exp Nephrol. 1994;2:63-77. [Medline] [Order article via Infotrieve]
16. Crane WAJ, Dutta LP. The utilisation of tritiated thymidine for deoxyribonucleic acid synthesis by the lesions of experimental hypertension in rats. J Pathol Bacteriol. 1963;86:83-97. [Medline] [Order article via Infotrieve]
17. Jones DB. Arterial and glomerular lesions associated with severe hypertension. Lab Invest. 1974;31:303-313. [Medline] [Order article via Infotrieve]
18. Striker GE, Striker LJ. Glomerular cell culture. Lab Invest. 1985;53:122-131. [Medline] [Order article via Infotrieve]
19. Green DF, Hwang KH, Ryan UN, Bourgoigne JJ. Culture of endothelial cells from baboon and human glomeruli. Kidney Int. 1992;41:1506-1516. [Medline] [Order article via Infotrieve]
20. Laulajainen T, Julkunen I, Haltia A, Knuutila S, Miettinen A, Holthöffer H. Establishment and characterization of a rat glomerular endothelial cell line. Lab Invest. 1993;69:183-192. [Medline] [Order article via Infotrieve]
21. Wolf G, Neilson EG. Angiotensin II as a renal growth factor. J Am Soc Nephrol. 1993;3:1531-1540. [Abstract]
22. Ichikawa I, Harris RC. Angiotensin actions in the kidney: renewed insight into the old hormone. Kidney Int. 1991;40:583-596. [Medline] [Order article via Infotrieve]
23. Wolf G. Vasoactive substances as regulators of renal growth. Exp Nephrol. 1993;1:141-151. [Medline] [Order article via Infotrieve]
24. Harris RC, Martinez-Maldonado M. Angiotensin II-mediated renal injury. Miner Electrolyte Metab. 1995;21:328-335. [Medline] [Order article via Infotrieve]
25. Wolf G, Haberstroh U, Neilson EG. Angiotensin II stimulates the proliferation and biosynthesis of type I collagen in cultured murine mesangial cells. Am J Pathol. 1992;140:95-107. [Abstract]
26.
Newman PJ, Berndt MC, Gorski J, White GC II, Lyman S,
Paddock C, Muller WA. PECAM-1 (CD 31) cloning and relation to
adhesion molecules of the immunoglobulin gene superfamily.
Science. 1990;247:1219-1222.
27.
Bevilacqua MP, Stengalin S, Gimbrone MA, Seed B.
Endothelial leukocyte adhesion molecule 1: an inducible
receptor for neutrophils related to complement regulatory proteins and
lectins. Science. 1989;243:1160-1165.
28.
Wolf G, Neilson EG. Angiotensin II
induces cellular hypertrophy in cultured murine proximal
tubular cells. Am J Physiol. 1990;259:F768-F777.
29. Sukhatme VP, Cao X, Chang LC, Tsai-Morris CH, Stamenkovich D, Ferreira PC, Cohen DR, Edwards SA, Shows TB, Curran T, Le Beau MM, Adamson ED. A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization. Cell. 1988;53:37-43. [Medline] [Order article via Infotrieve]
30. Stoscheck CM. Quantitation of protein. Methods Enzymol. 1990;182:50-68. [Medline] [Order article via Infotrieve]
31.
Pelech SL, Sanghera JS. MAP kinases: charting
the regulatory pathways. Science. 1992;257:1355-1356.
32.
Marsden PA, Dorfman DM, Collins T, Brenner BM, Orkin
SH, Ballermann BJ. Regulated expression of endothelin 1 in
glomerular capillary endothelial
cells. Am J Physiol. 1991;261:F117-F125.
33. Troyer DA, Kreisberg JI. Isolation and study of glomerular cells. Methods Enzymol. 1990;191:141-152. [Medline] [Order article via Infotrieve]
34.
Imai T, Hirata Y, Emori T, Yanagisawa M, Masaki T,
Marumo F. Induction of endothelin-1 gene by
angiotensin and vasopressin in endothelial
cells. Hypertension. 1992;19:753-757.
35.
Schorb W, Peeler TC, Madigan NN, Conrad KM, Baker
KM. Angiotensin II-induced protein tyrosine
phosphorylation in neonatal rat cardiac
fibroblasts. J Biol Chem. 1994;269:19626-19632.
36.
Molloy CJ, Taylor DS, Weber H.
Angiotensin II stimulation of rapid protein tyrosine
phosphorylation and protein kinase activation in rat
aortic smooth muscle cells. J Biol Chem. 1993;268:7338-7345.
37. Schorb W, Conrad KM, Singer HA, Dostal DE, Baker KM. Angiotensin II is a potent stimulator of MAP-kinase activity in neonatal rat cardiac fibroblasts. J Mol Cardiol. 1995;27:1151-1160. [Medline] [Order article via Infotrieve]
38.
Harris RC. Regulation of S6 kinase activity in
renal proximal tubule. Am J Physiol. 1992;263:F127-F134.
39.
Wolf G, Ziyadeh FN, Helchen U, Zahner G, Schroeder R,
Stahl RAK. Ang II is a mitogen for a murine cell line isolated
from medullary thick ascending limb of Henle's loop. Am
J Physiol. 1995;268:F940-F947.
40. Ullian ME, Linas SL. Role of receptor cycling in the regulation of angiotensin II surface receptor number and angiotensin II uptake in rat vascular smooth muscle cells. J Clin Invest. 1989;84:840-846.
41.
Weber H, Webb ML, Serafino R, Taylor DS, Moreland S,
Norman J, Molloy CJ. Endothelin-1 and angiotensin-II
stimulate delayed mitogenesis in cultured rat aortic smooth muscle
cells: evidence for common signaling mechanisms. Mol
Endocrinol. 1994;8:148-158.
42.
Daemen MJ, Lombardi DM, Bosman FT, Schwartz SM.
Angiotensin II induces smooth muscle cell proliferation in
the normal and injured arterial wall.
Circ Res. 1991;68:450-456.
43.
Stouffer GA, Owens GK. Angiotensin
II-induced mitogenesis of spontaneously hypertensive rat-derived
cultured SMC is dependent on autocrine production of
TGF-ß. Circ Res. 1992;70:820-828.
44. Riesco A, Gomez J, Millas I, Casado S, Lopez Farre A. Angiotensin II and endothelin 1 induced mitogenic response on endothelial cells: differences in signal-transduction mechanisms. Kidney Int. 1995;47:687. Abstract.
45. Fernandez LA, Twickler J, Mead A. Neovascularization produced by angiotensin II. J Lab Clin Med. 1985;105:141-145. [Medline] [Order article via Infotrieve]
46.
Gröne HJ. Angiogenesis and vascular
endothelial growth factor (VEGF): is it relevant in
renal patients? Nephrol Dial Transplant. 1995;10:761-763.
47.
Force T, Kyriakis JM, Avruch J, Bonventre JV.
Endothelin, vasopressin, and angiotensin II enhance
tyrosine phosphorylation by protein kinase C-dependent
and -independent pathways in glomerular
mesangial cells. J Biol Chem. 1991;266:6650-6656.
48. Stoll M, Steckelings UM, Paul M, Bottari SP, Metzger R, Unger T. The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells. J Clin Invest. 1995;95:651-657.
49. Davis RJ. MAPKs: new JNK expands the group. Trends Biochem Sci. 1994;19:470-473. [Medline] [Order article via Infotrieve]
50. Yamada T, Terada Y, Tomita K, Homma M, Nonoguchi H, Yang T, Krebs E, Yuasa Y, Marumo F. Angiotensin II (AT II) stimulation of c-raf-1 kinase, MAP kinase kinase, MAP kinase, and S6 kinase activities in OK (opossum kidney) cells. J Am Soc Nephrol. 1993;4:505. Abstract.
51. Huwiler A, Stabel S, Fabbro D, Pfeilschifter J. Platelet-derived growth factor and angiotensin II stimulate the mitogen-activated protein kinase cascade in renal mesangial cells: comparison of hypertrophic and hyperplastic agonists. Biochem J. 1995;305:777-784.
52. Schulze-Lohoff E, Köhler M, Fees H, Reindl N, Sterzel RB. Divergent effects of arginine vasopressin and angiotensin II on proliferation and expression of the immediate early genes c-fos, c-jun and Egr-1 in cultured rat glomerular mesangial cells. J Hypertens. 1993;11:127-134. [Medline] [Order article via Infotrieve]
53.
Rupprecht HD, Dann P, Sukhatme VP, Sterzel RB, Coleman
DL. Effect of vasoactive agents on induction of
Egr-1 in rat mesangial cells:
correlation with mitogenicity. Am J Physiol. 1992;263:F623-F636.
54. Lee LK, Meyer TW, Pollock AS, Lovett DH. Endothelial cell injury initiates glomerular sclerosis in the rat remnant kidney. J Clin Invest. 1995;96:953-964.
55. Jardine AG. Angiotensin II and glomerulonephritis. J Hypertens. 1995;13:487-493. [Medline] [Order article via Infotrieve]
56.
Gohlke P, Bünning P, Unger T. Distribution
and metabolism of angiotensin I and II in the
blood vessel wall. Hypertension. 1992;20:151-157.
57. Seikaly MG, Arant BS, Seney FD. Endogenous angiotensin concentrations in specific intrarenal fluid compartments of the rat. J Clin Invest. 1990;86:1352-1357.
58. Lorenzi M, Cagliero E. Pathobiology of endothelial and other vascular cells in diabetes mellitus: call for data. Diabetes. 1991;40:653-659. [Abstract]
59. Barnes JL, Hevey KA, Hastings RR, Bocanegra RA. Mesangial cell migration precedes proliferation in Habu snake venom-induced glomerular injury. Lab Invest. 1994;70:460-467. [Medline] [Order article via Infotrieve]
60.
Johnson RJ, Alpers CE, Yoshimura A, Lombardi D, Pritzl
P, Floege J, Schwartz SM. Renal injury from
angiotensin II–mediated hypertension.
Hypertension. 1992;19:464-474.
61. Farber HW, Center DM, Rounds S, Danilov SM. Components of the angiotensin system cause release of a neutrophil chemoattractant from cultured bovine and human endothelial cells. Eur Heart J. 1990;11(suppl B):100-107.
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