|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
HEMATOPOIESIS
From the Departments of Pathology and Medicine,
University of Washington School of Medicine; the Division of Clinical
Research, Fred Hutchinson Cancer Research Center, Seattle, WA; the
Department of Medical Biochemistry, University of Göteborg; and
the Department of Clinical Chemistry, Sahlgrenska Hospital,
Göteborg, Sweden.
Platelet-derived growth factor (PDGF)-B and PDGF Platelet-derived growth factor (PDGF) constitutes a
family of mitogens, primarily for mesenchymal cells, that includes at least 4 dimeric forms: PDGF-AA, -AB, -BB, and -CC, products of 3 distinct but highly related genes, respectively encoding the A-, B-,
and C-chains.1-4 PDGF dimers activate 2 specific
receptors,4-6 the PDGF Targeted disruption of PDGF-B or PDGFR The hematopoietic defects may reflect a primary abnormality intrinsic
to the hematopoietic system, or it may be secondary to cardiovascular
or placental dysfunction. In principle, the hematopoietic defect in
PDGF-B and PDGFR Circulating cells, especially monocytes and platelets, are a major
source of PDGF after activation or injury.2 Cultured monocytes also express PDGFR In this paper, we address the nature and cause of the hematopoietic
abnormalities in PDGF-B and PDGFR Mice
Isolation of fetal liver cells and determination of PDGF-B and
PDGFR DNA was prepared from the tail of each embryo with a QIAamp tissue kit (Qiagen, Chatsworth, CA). The PDGF-B genotype was determined by polymerase chain reaction (PCR) using primers specific for the neomycin phosphotransferase cassette (5'-TGTTCTCCTCTTCCTCATCTCC-3', 5'-ATTGTCTGTTGTGCCCAGTC-3') and the wild-type exon 4-specific sequence (5'-AGCAGAGCCTGCTGTAATCGCCGAG-3', 5'-TTGCACATTGCGGTTATTGCAGCAG-3'). Separate amplifications with each primer pair were performed for 25 to 30 cycles (96°C, 30 seconds; 61°C, 30 seconds; 72°C, 30 seconds), respectively, yielding 140-bp (neo) and 157-bp (exon 4) diagnostic amplification products. In the most recent transplantation experiments, genotyping of PDGF-B was performed using a polymerase chain reaction (PCR) mix containing one forward primer (5'-GGGTGGGACTTTGGTGTAGAGAGG-3') and 2 reverse primers (5'-TTGAAGCGTGCAGAATGCC-3', 5'-GGAACGGATTTTGGAGGTAGTGTC-3'), respectively, yielding 265-bp (wild-type-specific) and 624-bp (neo-specific) products (40 cycles: 96°C, 30 seconds; 59.5°C, 30 seconds; 64°C, 120 seconds). The PDGFR Phenotypic appearance and genotyping results from each embryo were compared with specific attention to the presence of focal renal hemorrhage and the reduced liver size characteristic of null mutants. Only wild-type and null mutant fetal livers that showed concordant results were used for transplantation or stem cell assay. Concordance was high, and only the absence of an interpretable PCR result was grounds for excluding an embryo with a mutant phenotypic appearance. In the initial series of transplantation experiments, male and female fetal liver cells were transplanted into recipients of the same sex. In some later experiments, pools of male and female fetal liver cells were prepared. In vitro colony-forming assays and erythropoietin enzyme-linked immunosorbent assay Colony assays were performed as previously described for burst-forming unit-erythroid (BFU-E) and colony-forming units containing erythroid cells (CFU-E), granulocytes and macrophages (CFU-GM),24 and megakaryocytes (CFU-Meg).25 Lysates of embryonic day (E)14.5 and E16.5 embryos were prepared and evaluated in a sensitive enzyme-linked immunosorbent assay for mouse erythropoietin26 using recombinant mouse erythropoietin as a standard.Transmission electron microscopic analysis of fetal liver megakaryocytes Half the E16.5 fetal livers were fixed overnight at 4°C with Karnovsky fixative, osmicated (2% OsO4 in veronal-acetate buffer, pH 7.4), stained in block with uranyl acetate, dehydrated in a graded series of ethanol, infiltrated with propylene oxide, and embedded in Epon. Thin sections were cut, stained with uranyl acetate and lead citrate, and examined with a JEOL transmission electron microscope (JEOL, Peabody, MA). At least 25 megakaryocytes were examined per fetal liver; in total, 6 fetal livers were examined (2 PDGFR  /, 1 PDGF-B/, and 3 wild-type
littermate controls).
Total body irradiation and fetal liver cell transplantation Female and male Ly-5a recipients 8 to 12 weeks of age were prepared by total body irradiation in a single 14-Gy fraction from dual-opposed cobalt Co60 sources at an exposure rate of 20 cGy/min on the day before transplantation. Fetal cells were stained with propidium iodide (Sigma, St Louis, MO), and cell viability was analyzed by flow cytometry using a FACScan (Becton Dickinson, San Jose, CA). Aliquots containing 5 × 106 to 10 × 106 viable fetal liver cells were injected into each recipient through the lateral tail vein.Bone marrow transplantation Bone marrow was flushed from the femurs23 of wild-type and PDGF-B/ chimeras 20 weeks after fetal
liver transplantation. Complete replacement of host with donor
hematologic cells was confirmed in each donor before sacrifice. Marrow
cells were washed in phosphate-buffered saline and injected into
lethally irradiated (14 Gy) B6.Ly-5a recipients
(10 × 106 viable cells/recipient).
Assessment of chimerism The degree of chimerism after fetal liver cell or bone marrow transplantation was assessed at 28 days and at time points up to 107 days. Heparinized blood was obtained from the orbital venous plexus, and red blood cells were lysed in NH4Cl buffer. Leukocytes were stained with biotinylated Ly-5.1- and Ly-5.2-specific antibodies, as described previously.23 The Ly-5.1 antibody stained leukocytes derived from Ly-5a recipients, whereas the Ly-5.2 antibody stained leukocytes derived from Ly-5b donors. In some experiments, 2-color analysis was performed with fluorescein isothiocyanate -conjugated antibodies against CD3+ cells (hamster IgG mAb 145-2C11), B lymphocytes (rat B220, clone RA3-6B2; Pharmingen, San Diego, CA), CD4 (rat L3T4, clone H129.19; Pharmingen), and CD8 (rat Ly2, clone 53-6.7; Pharmingen). Stained cells were fixed in 1% paraformaldehyde and analyzed by flow cytometry. Scatter plots for monocytes, granulocytes, and lymphoid cells were determined by forward and side scatter characteristics, and the percentage of donor cells within each window was enumerated. Results were rounded to the nearest integer and were not corrected for background staining. For each experiment, thresholds for delineating positive and negative cells were determined by staining samples from appropriate positive and negative controls.23Blood analyses Venous blood (400 µL) from chimeras was obtained from the orbital venous plexus, and peripheral blood parameters were determined by using a Celldyn 3500 instrument (Abbott Diagnostics, Abbott Park, IL). Blood from decapitated E17.5 embryos was collected into microtiter EDTA tubes (Becton Dickinson, Bedford, MA) containing phosphate-buffered saline. Differential counting of nucleated cells was performed on May-Grünwald-stained blood films.In situ hybridization We applied a nonradioactive protocol for in situ hybridization using digoxigenin-labeled RNA probes (Boehringer Mannheim [now Roche Molecular Biochemicals], Indianapolis, IN) and for their detection on sections using alkaline phosphatase-conjugated antibodies.27 PDGF-B and PDGFR sense and antisense
probes were generated as described previously.10 The
results shown were obtained using 14-µm thick sections and
interference contrast microscopy. As negative controls, the
corresponding sense probes were used.
Anemia, erythroblastosis, thrombocytopenia, and hepatic
hypocellularity in PDGF-B was
embryonic lethal, and null embryos displayed anemia and
thrombocytopenia when evaluated at E18.7,8 To assess more
completely the effect of full or partial disruption of the PDGF-B gene
on the hematologic profile in embryos, we evaluated peripheral blood
from decapitated E17.5 PDGF-B null mutant, heterozygous, and wild-type
embryos (Figure 1).
PDGF-B/ mutant embryos showed a 50% reduction in the
mean hemoglobin concentration (52 ± 11 g/L, n = 8,
P < .0001) compared with that of wild-type controls
(101 ± 7 g/L, n = 14), whereas heterozygous mutant embryos
appeared mildly anemic (92 ± 11 g/L, n = 20,
P = .0104 versus wild-type). Erythroblast counts
( × 109/L) were increased 15-fold in
PDGF-B/ mutants (21.8 ± 10.6, n = 7) compared with
wild-type embryos (1.47 ± 1.54, n = 13, P < .0001)
and 10-fold over heterozygous embryos (2.19 ± 1.62, n = 13,
P < .0001). PDGF-B/ mutants had low
platelet counts (178.5 ± 69 × 109/L) compared with
wild-type controls (345 ± 71 × 109/L,
P < .0001). Heterozygous embryos showed a nonsignificant
reduction in circulating platelets (331 ± 46 × 109/L,
P = .587). The hematologic data in E17.5
PDGF-B/ mutants are consistent with anemia,
erythroblastosis, and thrombocytopenia.7,8
The fetal liver is the major site of hematopoiesis during midgestation
in mice.28,29 We analyzed total cellularity and cell
viability of mutant and wild-type livers after limited storage ex vivo
(Table 1). Storage of individual fetal
liver suspensions from E16.5 PDGF-B or PDGFR
Comparable hematopoietic progenitor colony formation with
fetal liver cells from PDGF-B+/+ and
PDGF-B /
and PDGFR/ embryos had been properly populated by
hematopoietic progenitor cells, fetal liver cells were used for
hematopoietic progenitor cell assays. As shown in Table
2, we observed comparable hematopoietic progenitor colony formation for CFU-GM and CFU-Meg when equivalent numbers of fetal liver cells were assayed. A modest, but statistically significant (P = 0.04), reduction in BFU-E colony
formation was seen with fetal liver cells from
PDGFR/ embryos (Table 2). No differences were
observed in CFU-E in fetal liver cells from PDGF-B null (n = 3) and
PDGFR null (n = 2) chimeras and wild-type (data not shown)
littermate controls (n = 6). These results suggested that the
expression of PDGF-B or PDGFR was not required for the development
of these lineages.
Because megakaryocytes are a major source of PDGF and platelets have
been shown to express PDGF Generation of hematopoietic chimeras by transplantation of
PDGF-B , we constructed chimeras in which either PDGF-B or
PDGFR was selectively disrupted in the hematopoietic compartment.
The chimeras were prepared by transferring fetal liver cells into
irradiated, H-2-compatible, wild-type recipients. In pilot
dose-response experiments, the correlation between the administered
irradiation exposure and the percentage donor cell reconstitution after
28 days was established for B6.Ly-5a recipients
transplanted with wild-type B6.Ly-5b fetal liver cells.
Complete (more than 98%) repopulation by donor granulocytes was
achieved at an irradiation dose of 13 Gy or greater (data not shown).
Therefore, we routinely administered a dose of 14 Gy in all
transplantation experiments.
In 6 independent experiments, 5 × 106 to
10 × 106 viable fetal liver cells (E16.5,
Ly-5b) from wild-type and PDGF-B Kinetics of peripheral leukocyte reconstitution after
transplantation of wild-type and PDGF-B / (Figure 3A)
or wild-type and PDGFR/ (Figure 3B) donors.
Engraftment of donor-derived cells remained stable throughout
observation periods up to 107 days, indicating that long-term
reconstitution occurred. Results with wild-type and null mutant donors
were indistinguishable.
Throughout the first 100 days after transplantation, the
proportion of donor-derived T cells in the blood was lower than the proportion of donor-derived B cells and myeloid cells, but the proportion did not depend on donor genotype (Figure 3). The proportion of donor-derived T cells increased from 42.5% ± 6.9%
(PDGF-B+/+ donors) and 39.7% ± 8.8%
(PDGF-B Normal peripheral blood parameters in PDGF-B / and PDGFR/
chimeras were healthy after they recovered from acute irradiation
sickness, approximately 14 days after transplantation. Recipients
transplanted from PDGF-B/ and PDGFR/
donors and their respective wild-type controls had identical appearances, weights, and behaviors at all time points after fetal liver transplantation. Bone marrow cross-sections from the chimeric mice 14-18 weeks after transplantation showed no overt phenotypic differences between the different chimeric combinations, which suggested that the hematopoietic activity in the bone marrow of null
mutants was normal. No significant differences in red cell counts,
platelet counts, or differential counts of granulocytes, lymphocytes,
or monocytes were found between PDGF-B/ and
PDGF-B+/+ chimeras (Table 3)
or PDGFR/ and PDGFR+/+ chimeras
(Table 4). These results indicated that
normal hematopoietic functions of all major lineages were maintained in
the absence of PDGF-B or PDGFR expression in hematopoietic
stem cells.
Identical reconstitution kinetics of wild-type and
PDGF-B / hematopoietic stem cells. The second generation
chimeras appeared healthy, and at 28 days after transplantation, the
proportions of donor-derived B- and T-lymphoid and myeloid cells were
comparable to those found after fetal liver transplantation (Figure
4; compare with Figure 3A). No
significant differences were observed in the reconstitution of
wild-type and PDGF-B/ second-generation chimeras
(Figure 4).
Expression of PDGF-B and PDGFR / and PDGFR/ fetal livers
reflected a direct role of PDGF in the liver, we determined the
expression patterns of PDGF-B and PDGFR in the fetal mouse liver by
nonradioactive in situ hybridization. PDGF-B mRNA was strongly
expressed by a subpopulation of cells in the developing liver
(Figure 5A). These cells had the
morphologic hallmarks of megakaryocytes large and characterized by
multilobulated nuclei (Figure 5B). In
hematoxylin-eosin-stained sections, fetal livers
contained comparable proportions of megakaryocytes in E16.5 to E18.5
PDGF-B+/+ and PDGF-B/ livers (data not
shown). We concluded that the only cell type with detectable PDGF-B
mRNA expression in the fetal mouse liver was the megakaryocyte
and that PDGF-B deficiency did not block megakaryocyte
development in PDGF-B/ embryos.
PDGFR We concluded that PDGF-B and PDGFR
What role does PDGF play in hematopoiesis? Previous in vitro and in vivo studies have suggested a role for PDGF in erythropoiesis17,18,20,21 and in stimulation of pluripotent stem cells.19,21,32-34 PDGF promotes in vitro proliferation of erythropoietic progenitors,20 but stimulation requires the presence of adherent stromal cells,21 presumably because of PDGF induction of colony-stimulating factor (CSF) release from the stromal cells. Mouse splenic erythroid cells express PDGF in vivo after stimulation with erythropoietin,17,18 demonstrating that precursors of the erythroid lineages are a source of PDGF. In mixed marrow cultures, PDGF stimulates the growth of primitive hematopoietic progenitor cells, such as colony-forming units containing granulocytes, erythroid cells, macrophages, megakaryocytes (CFU-mix, CFU-GEMM),19,32,33 and lineage-restricted hematopoietic progenitors.34 Adherent accessory cells are also required for the stimulation of primitive and lineage-restricted hematopoietic progenitors by PDGF, and growth enhancement appears to occur as an indirect effect through the stimulation of interleukin-1 (IL-1) production by PDGFR-positive stromal cells, including macrophages.19 All the above in vitro studies were performed with PDGF that contained PDGF-AA, -AB, and -BB. However, no specific effects of PDGF-AA or PDGF-CC have been reported. Overexpression of PDGF-B in murine hematopoietic cells induces a lethal myeloproliferative syndrome in vivo.35 Taken together, in vitro and in vivo studies suggest that PDGF enhances hematopoiesis by stimulating stromal cells to produce a variety of factors that act directly on hematopoietic progenitors.Bone marrow stromal cells play a crucial role in sustaining
proliferation, differentiation, and self-renewal of hematopoietic stem
cells.36,37 It has also been shown that hematopoietic stem
cells, not committed progenitor cells, mediate early hematopoietic reconstitution.38 Within the marrow, the principal
sources of PDGF are megakaryocytes, platelets, erythroid precursors,
and activated monocytes-macrophages.3 Populations known
to express PDGF receptors are predominantly stromal cells,
including fibroblasts, smooth muscle cells, osteoblasts, early
macrophage precursors, and macrophages. Our finding that PDGF-B
null mutant chimeras have a normal hematologic phenotype demonstrates
that neither the release of PDGF from the hematopoietic cells nor the
response of PDGFR Our results with PDGF null hematopoietic chimeras contrast with the
results of studies of mice with targeted deletion of growth factors
known to stimulate hematopoiesis directly, such as
erythropoietin,30,40 M-CSF,41 and
thrombopoietin,42,43 which are required for the normal
development of specific hematologic lineages. Some hematopoietic growth
factors appear to serve redundant functions in hematopoiesis, as
indicated by the observation that GM-CSF null mutant mice have normal
hematopoietic parameters44,45 in spite of more subtle
functional defects.46 Our results suggest that PDGF may
also have a redundant function in hematopoiesis. Stromal production of
hematopoietic cytokines can be stimulated not only by PDGF but also by
other growth factors and cytokines, such as fibroblast growth factor,
heparin-binding epidermal growth factor, epidermal growth factor, IL-1,
and tumor necrosis factor- Pathogenesis of disorders in PDGF-B- and
PDGFR / embryos has
demonstrated a severe pericyte deficiency in capillaries at many sites
and smooth muscle hypoplasia in many larger vessels.10,11
It is likely that the pericyte loss leads to the microaneurysms, tissue
edema, and petechial hemorrhage characteristic of these mice. It is
possible that the bleeding may contribute to the anemia seen in the
mutants, but 2 observations suggest other principal causes of the
anemia. First, anemia is present at E17.5, at which time most mutants have not yet developed significant hemorrhaging.7 Second,
the anemia correlates with a consistent, approximately 50%, reduction in the size of the liver, the major hematopoietic organ at mid to late
gestation. The size reduction is balanced (ie, normal ratios between
the different cell types are preserved) and is apparent by E14.5, far
earlier than the development of microaneurysms, tissue edema, and
petechial hemorrhage. Vascular leakage leading to reduced fetal liver
cellularity has also been observed in mice with null mutations in
Tie1, a receptor tyrosine kinase essential for vascular cell
integrity.55
The reduced liver size may be sufficient to explain the anemia and
thrombocytopenia, but what is causing the reduction in liver size? Our
in situ hybridization analysis demonstrates PDGF-B expression in
megakaryocytes in E14.5 fetal livers, but PDGFR Two defects might be relevant to the reduction in liver size. First,
PDGF-B and PDGFR In summary, the hematopoietic chimera analysis, the normal embryonic
PDGF-B and PDGFR PDGF-B
gene deletions that are otherwise lethal during gestation. After lethal
total body irradiation, recipient granulocytes, monocytes, and B cells
are replaced rapidly by donor-derived populations, whereas recipient T
cells are replaced more slowly. We surmise that some T cells are
resistant to irradiation-induced interphase death.59
However, as judged by the absence of major histocompatibility complex-incompatible marrow graft rejection, it appears that most recipient T cells remaining after 14-Gy total body irradiation are
functionally incompetent, presumably because they die whenever they are
stimulated to replicate. Therefore, their persistence after
transplantation probably reflects the long life span characteristic of
certain T-cell populations.60 The slower repopulation of T
cells in this model will have to be considered in experiments to test
immune and inflammatory responses.
With the exception of the platelet, whose
We thank Kelli McIntyre, Karen Engel, Roderick Browne, and
Li-Chuan Huang for expert technical assistance, Philippe Soriano (Fred
Hutchinson Cancer Research Center, Seattle, WA) for provision of
PDGFR
Submitted April 19, 2000; accepted November 21, 2000.
Supported by National Institutes of Health grants HL18645 (R.R., E.W.R.), HL55257 (P.J.M.), HL03174 (D.F.B.-P.), CA31615 (V.C.B.); Merck (R.R.); Swedish Medical Research Council, Cancer Foundation, Inga-Britt and Arne Lundberg Foundation, Novo Nordisk Foundation (C.B.); and Deutsche Forschungsgemeinschaft Ka 1078/1 (W.E.K.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Elaine W. Raines, Dept of Pathology, University of Washington, HMC, 325 9th, Box 359675, Seattle, WA 98104-2499; e-mail: ewraines{at}u.washington.edu.
1. Heldin C-H, Westermark B. Platelet-derived growth factor: three isoforms and two receptor types. Trends Genet. 1989;5:108-111[CrossRef][Medline] [Order article via Infotrieve]. 2. Raines EW, Bowen-Pope DF, Ross R. Platelet-derived growth factor. In: Sporn MB,Roberts AB, eds. Handbook of Experimental Pharmacology: Peptide Growth Factors and Their Receptors I. New York, NY: Springer-Verlag; 1990:173-262. 3. Raines EW, Ross R. Platelet-derived growth factor in vivo. In: Westermark B,Sorg C, eds. Biology of Platelet-derived Growth Factor 5. Basel, Switzerland: Karger; 1993:74-114. 4. Li X, Ponten A, Aase K, et al. PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor. Nat Cell Biol. 2000;2:302-309[CrossRef][Medline] [Order article via Infotrieve]. 5. Heldin C-H, Bäckström G, Östman A, et al. Binding of different dimeric forms of PDGF to human fibroblasts: evidence for two separate receptor types. EMBO J. 1988;7:1387-1393[Medline] [Order article via Infotrieve].
6.
Seifert RA, Hart CE, Phillips PE, et al.
Two different subunits associate to create isoform-specific PDGF receptors.
J Biol Chem.
1989;264:8771-8778
7.
Levéen P, Pekny M, Gebre-Medhin S, Swolin B, Larsson E, Betsholtz C.
Mice deficient in PDGF B show renal, cardiovascular, and hematological abnormalities.
Genes Dev.
1994;8:1875-1887
8.
Soriano P.
Abnormal kidney development and hematological disorders in PDGF
9.
Lindahl P, Hellström M, Kalén M, et al.
Paracrine PDGF-B/PDGF-R
10.
Lindahl P, Johansson BR, Levéen P, Betsholtz C.
Pericyte loss and microaneurysm formation in PDGF-B-deficient mice.
Science.
1997;277:242-245
11.
Hellström M, Kalén M, Lindahl P, Abramsson A, Betsholtz C.
Role of PDGF-B and PDGFR- 12. Ohlsson R, Falck P, Hellström M, et al. PDGF-B regulates the development of the labyrinthine layer of the mouse fetal placenta. Dev Biol. 1999;212:124-136[CrossRef][Medline] [Order article via Infotrieve].
13.
Inaba T, Shimano H, Gotoda T, et al.
Expression of platelet-derived growth factor 14. Gersuk GM, Westermark B, Mohabeer AJ, Challita PM, Pattamakom S, Pattengale PK. Inhibition of human natural killer cell activity by platelet-derived growth factor (PDGF), III: membrane binding studies and differential biological effects of recombinant PDGF isoforms. Scand J Immunol. 1991;33:521-532[CrossRef][Medline] [Order article via Infotrieve].
15.
Daynes RA, Dowell T, Araneo BA.
Platelet-derived growth factor is a potent biologic response modifier of T cells.
J Exp Med.
1991;174:1323-1333
16.
Wickenhauser C, Hillienhof A, Jungheim K, et al.
Detection and quantification of transforming growth factor beta (TGF- 17. Sytkowski AJ, O'Hara C, Vanasse G, Armstrong MJ, Kreczko S, Dainiak N. Characterization of biologically active, platelet-derived growth factor-like molecules produced by murine erythroid cells in vitro and in vivo. J Clin Invest. 1990;85:40-46.
18.
Keutzer JC, Sytkowski AJ.
Regulated production of a pleiotropic cytokine 19. Yan X-Q, Brady G, Iscove NN. Platelet-derived growth factor (PDGF) activates primitive hematopoietic precursors (pre-CFCmulti) by up-regulating IL-1 in PDGF receptor-expressing macrophages. J Immunol. 1993;150:2440-2448[Abstract]. 20. Dainiak N, Davies G, Kalmanti M, Lawler J, Kulkarni V. Platelet-derived growth factor promotes proliferation of erythropoietic progenitor cells in vitro. J Clin Invest. 1983;71:1206-1214. 21. Delwiche F, Raines E, Powell J, Ross R, Adamson J. Platelet-derived growth factor enhances in vitro erythropoiesis via stimulation of mesenchymal cells. J Clin Invest. 1985;76:137-142. 22. Yang M, Chesterman CN, Chong BH. Recombinant PDGF enhances megakaryocytopoiesis in vitro. Br J Haematol. 1995;91:285-289[Medline] [Order article via Infotrieve].
23.
Martin PJ.
Donor CD8 cells prevent allogeneic marrow graft rejection in mice: potential implications for marrow transplantation in humans.
J Exp Med.
1993;178:703-712
24.
Broudy VC, Lin NL, Priestley GV, Nocka K, Wolf NS.
Interaction of stem cell factor and its receptor c-kit mediates lodgment and acute expansion of hematopoietic cells in the murine spleen.
Blood.
1996;88:75-81
25.
Broudy VC, Lin NL, Kaushansky K.
Thrombopoietin (c-mpl ligand) acts synergistically with erythropoietin, stem cell factor, and interleukin-11 to enhance murine megakaryocyte colony growth and increases megakaryocyte ploidy in vitro.
Blood.
1995;85:1719-1726 26. Rinaudo D, Toniatti C. Sensitive ELISA for mouse erythropoietin. Biotechniques. 2000;29:218-220[Medline] [Order article via Infotrieve]. 27. Boström H, Willetts K, Pekny M, et al. PDGF-A signaling is a critical event in lung alveolar myofibroblast development and alveogenesis. Cell. 1996;85:863-873[CrossRef][Medline] [Order article via Infotrieve]. 28. Borghese E. The present state of research on WW mice. Acta Anat. 1959;36:185-220. 29. Orkin SH. Hematopoiesis: how does it happen? Curr Opin Cell Biol. 1995;7:870-877[CrossRef][Medline] [Order article via Infotrieve].
30.
Lin C-S, Lim S-K, D'Agati V, Costantini F.
Differential effects of an erythropoietin receptor gene disruption on primitive and definitive erythropoiesis.
Genes Dev.
1996;10:154-164
31.
Vassbotn FS, Havnen OK, Heldin C-H, Holmsen H.
Negative feedback regulation of human platelets via autocrine activation of the platelet-derived growth factor alpha-receptor.
J Biol Chem.
1994;269:13874-13879 32. Michalevicz R, Francis GE, Price GM, Hoffbrand AV. The role of platelet-derived growth factor on human pluripotent progenitor (CFU-GEMM) growth in vitro. Leuk Res. 1985;9:399-405[CrossRef][Medline] [Order article via Infotrieve]. 33. Michalevicz R, Katz F, Stroobant P, Janossy G, Tindle RW, Hoffbrand AV. Platelet-derived growth factor stimulates growth of highly enriched multipotent haematopoietic progenitors. Br J Haematol. 1986;63:591-598[Medline] [Order article via Infotrieve].
34.
Cashman JD, Eaves AC, Raines EW, Ross R, Eaves CJ.
Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures, I: stimulatory role of a variety of mesenchymal cell activators and inhibitory role of TGF- 35. Yan XQ, Brady G, Iscove NN. Overexpression of PDGF-B in murine hematopoietic cells induces a lethal myeloproliferative syndrome in vivo. Oncogene. 1994;9:163-173[Medline] [Order article via Infotrieve]. 36. Whetton AD, Dexter TM. Influence of growth factors and substrates on differentiation of haemopoietic stem cells. Curr Opin Cell Biol. 1993;5:1044-1049[CrossRef][Medline] [Order article via Infotrieve]. 37. Clark BR, Keating A. Biology of bone marrow stroma. Ann NY Acad Sci. 1995;V770:70-78[Medline] [Order article via Infotrieve].
38.
Zijlmans JM, Visser JW, Laterveer L, et al.
The early phase of engraftment after murine blood cell transplantation is mediated by hematopoietic stem cells.
Proc Natl Acad Sci U S A.
1998;95:725-729 39. Simmons PJ, Przepiorka D, Thomas ED, Torok-Storb B. Host origin of marrow stromal cells following allogeneic bone marrow transplantation. Nature. 1987;328:429-432[CrossRef][Medline] [Order article via Infotrieve]. 40. Wu H, Liu X, Jaenisch R, Lodish HF. Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor. Cell. 1995;83:59-67[CrossRef][Medline] [Order article via Infotrieve].
41.
Wiktor-Jedrzejczak W, Bartocci A, Ferrante AW Jr, et al.
Total absence of colony-stimulating factor 1 in the macrophage deficient osteopetrotic (op/op) mouse.
Proc Natl Acad Sci U S A.
1990;87:4828-4832
42.
Gurney AL, Carver-Moore K, de Sauvage FJ, Moore MW.
Thrombocytopenia in c-mpl-deficient mice.
Science.
1994;265:1445-1447
43.
Kimura S, Roberts AW, Metcalf D, Alexander WS.
Hematopoietic stem cell deficiencies in mice lacking c-Mpl, the receptor for thrombopoietin.
Proc Natl Acad Sci U S A.
1998;95:1195-1200
44.
Dranoff G, Crawford AD, Sadelain M, et al.
Involvement of granulocyte-macrophage colony-stimulating factor in pulmonary homeostasis.
Science.
1994;264:713-716
45.
Stanley E, Lieschke GJ, Grail D.
Granulocyte/macrophage colony-stimulating factor-deficient mice show no major perturbation of hematopoiesis but develop a characteristic pulmonary pathology.
Proc Natl Acad Sci U S A.
1994;91:5592-5596
46.
Scott CL, Hughes DA, Cary D, Nicola NA, Begley CG, Robb L.
Functional analysis of mature hematopoietic cells from mice lacking the beta chain of the granulocyte-macrophage colony-stimulating factor receptor.
Blood.
1998;92:4119-4127
47.
Broudy VC, Kaushansky K, Segal GM, Harlan JM, Adamson JW.
Tumor necrosis factor type alpha stimulates human endothelial cells to produce granulocyte/macrophage colony-stimulating factor.
Proc Natl Acad Sci U S A.
1986;83:7467-7471 48. Falkenburg JH, Harrington MA, Walsh WK, Daub R, Broxmeyer HE. Gene expression and release of macrophage-colony-stimulating factor in quiescent and proliferating fibroblasts: effects of serum, fibroblast growth-promoting factors, and interleukin-1. J Immunol. 1990;144:4657-4662[Abstract].
49.
Leizer T, Cebon J, Layton JE, Hamilton JA.
Cytokine regulation of colony-stimulating factor production in cultured human synovial fibroblasts, I: induction of GM-CSF and G-CSF production by interleukin-1 and tumor necrosis factor.
Blood.
1990;76:1989-1996
50.
Inaba T, Ishibashi S, Harada K, et al.
Induction of macrophage colony-stimulating factor receptor (c-fms) expression in vascular medial smooth muscle cells treated with heparin-binding epidermal growth factor-like growth factor.
J Biol Chem.
1996;271:24413-24417 51. Rothe J, Lesslauer W, Lötscher H, et al. Mice lacking the tumour necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature. 1993;364:798-802[CrossRef][Medline] [Order article via Infotrieve]. 52. Erickson SL, de Sauvage FJ, Kikly K, et al. Decreased sensitivity to tumour-necrosis factor but normal T-cell development in TNF receptor-2-deficient mice. Nature. 1994;372:560-563[CrossRef][Medline] [Order article via Infotrieve].
53.
Fantuzzi G, Zheng H, Faggioni R, et al.
Effect of endotoxin in IL-1
54.
Metcalf D.
Hematopoietic regulators: redundancy or subtlety?
Blood.
1993;82:3515-3523 55. Rodewald HR, Sato TN. Tie1, a receptor tyrosine kinase essential for vascular endothelial cell integrity, is not critical for the development of hematopoietic cells. Oncogene. 1996;12:397-404[Medline] [Order article via Infotrieve]. 56. Crosby JR, Seifert RA, Soriano P, Bowen-Pope DF. Chimaeric analysis reveals role of Pdgf receptors in all muscle lineages. Nat Genet. 1998;18:385-388[CrossRef][Medline] [Order article via Infotrieve].
57.
Tamura K, Sudo T, Senftleben U, Dadak AM, Johnson R, Karin M.
Requirement for p38
58.
Barak Y, Nelson MC, Ong ES, et al.
PPAR 59. Anderson RE, Warner NL. Ionizing radiation and the immune response. Adv Immunol. 1976;24:215-335[Medline] [Order article via Infotrieve].
60.
Tough DF, Sprent J.
Turnover of naive- and memory-phenotype T cells.
J Exp Med.
1994;179:1127-1135
© 2001 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  J. Andrae, R. Gallini, and C. Betsholtz Role of platelet-derived growth factors in physiology and medicine Genes & Dev., May 15, 2008; 22(10): 1276 - 1312. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. David, N. C. P. Cross, S. Burgstaller, A. Chase, C. Curtis, R. Dang, M. Gardembas, J. M. Goldman, F. Grand, G. Hughes, et al. Durable responses to imatinib in patients with PDGFRB fusion gene-positive and BCR-ABL-negative chronic myeloproliferative disorders Blood, January 1, 2007; 109(1): 61 - 64. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Armulik, A. Abramsson, and C. Betsholtz Endothelial/Pericyte Interactions Circ. Res., September 16, 2005; 97(6): 512 - 523. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Tang, K. Kozaki, A. G. Farr, P. J. Martin, P. Lindahl, C. Betsholtz, and E. W. Raines The Absence of Platelet-Derived Growth Factor-B in Circulating Cells Promotes Immune and Inflammatory Responses in Atherosclerosis-Prone ApoE-/- Mice Am. J. Pathol., September 1, 2005; 167(3): 901 - 912. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Bjarnegard, M. Enge, J. Norlin, S. Gustafsdottir, S. Fredriksson, A. Abramsson, M. Takemoto, E. Gustafsson, R. Fassler, and C. Betsholtz Endothelium-specific ablation of PDGFB leads to pericyte loss and glomerular, cardiac and placental abnormalities Development, April 15, 2004; 131(8): 1847 - 1857. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. V. Hoch and P. Soriano Roles of PDGF in animal development Development, October 15, 2003; 130(20): 4769 - 4784. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Bondjers, M. Kalen, M. Hellstrom, S. J. Scheidl, A. Abramsson, O. Renner, P. Lindahl, H. Cho, J. Kehrl, and C. Betsholtz Transcription Profiling of Platelet-Derived Growth Factor-B-Deficient Mouse Embryos Identifies RGS5 as a Novel Marker for Pericytes and Vascular Smooth Muscle Cells Am. J. Pathol., March 1, 2003; 162(3): 721 - 729. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kozaki, W. E. Kaminski, J. Tang, S. Hollenbach, P. Lindahl, C. Sullivan, J.-C. Yu, K. Abe, P. J. Martin, R. Ross, et al. Blockade of Platelet-Derived Growth Factor or Its Receptors Transiently Delays but Does Not Prevent Fibrous Cap Formation in ApoE Null Mice Am. J. Pathol., October 1, 2002; 161(4): 1395 - 1407. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. S. Buetow, J. R. Crosby, W. E. Kaminski, R. K. Ramachandran, P. Lindahl, P. Martin, C. Betsholtz, R. A. Seifert, E. W. Raines, and D. F. Bowen-Pope Platelet-Derived Growth Factor B-Chain of Hematopoietic Origin Is Not Necessary for Granulation Tissue Formation and Its Absence Enhances Vascularization Am. J. Pathol., November 1, 2001; 159(5): 1869 - 1876. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
-receptor null mice
Top
Abstract
Introduction
-receptor (PDGFR
P = .01 for comparison between PDGF-B+/+
and PDGF-B+/
80°C until analysis after
lysis and homogenization in 150 mM NaCl, 66 mM EDTA, 10 mM Tris HCl (pH
7.4), 0.4% Na deoxycholate, 1% NP-40, and protease inhibitor cocktail
(P-2714; Sigma). Erythropoietin activity was quantified using a
combination of 2 commercially available erythropoietin enzyme-linked
immunosorbent assay kits.
-deficient
mice,