Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2897-2903

This Article
Right arrow Abstract Freely available
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 PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Krettek, A.
Right arrow Articles by Lustig, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Krettek, A.
Right arrow Articles by Lustig, F.
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2897-2903.)
© 1997 American Heart Association, Inc.

Articles

Effect of Phenotype on the Transcription of the Genes for Platelet-Derived Growth Factor (PDGF) Isoforms in Human Smooth Muscle Cells, Monocyte-Derived Macrophages, and Endothelial Cells In Vitro

Alexandra Krettek; Gunnar Fager; Helena Lindmark; Carolina Simonson; ; Florentyna Lustig

From the Wallenberg Laboratory for Cardiovascular Research, Göteborg University, Sahlgrenska University Hospital, Göteborg, Sweden.

Correspondence to Alexandra Krettek, Wallenberg Laboratory for Cardiovascular Research, Göteborg University, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden. E-mail alexandra.krettek{at}wlab.wall.gu.se


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract Proliferation of arterial smooth muscle cells (ASMCs) contributes considerably to enlargement of the arterial wall during atherosclerosis. The platelet-derived growth factor (PDGF) is a well-known mitogen and chemoattractant for ASMCs. Quantitative reverse transcription–polymerase chain reaction showed that cells appearing in atherosclerotic lesions, such as ASMCs, endothelial cells, and monocytes/macrophages, expressed mRNAs for both PDGF A and B chains in vitro, with the highest expression in endothelial cells. On proliferation, ASMCs and endothelial cells upregulated PDGF A mRNA. Differentiation of macrophages increased the amount of both mRNAs. Thus, the regulation of PDGF A- and B-chain expression depends on cell types and phenotypic states of the cells, which have also been found in vivo in human atherosclerotic lesions. PDGF A can be produced as short and long isoforms. The latter binds with high affinity to glycosaminoglycans. Irrespective of phenotype, only the minor part of total PDGF A mRNA consisted of the long variant in ASMCs, while endothelial cells produced 40% of total PDGF A as the long form. The differentiation of macrophages increased the production of the long PDGF A mRNA from 10% to 40%. Thus, increasing numbers of stimulated cells in the atherosclerotic lesion may increase the transcription of PDGF isoforms, and particularly of the long PDGF A isoform. Together with increasing amounts of ASMC-derived proteoglycans in developing lesions, this may contribute to accumulation of PDGF in the arterial wall matrix, resulting in prolonged stimulation of ASMCs.


Key Words: smooth muscle cells • macrophages • endothelial cells • differentiation • platelet-derived growth factor


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Several processes contribute to the formation of atherosclerotic lesions: accumulation of lipid, intimal proliferation of ASMCs, and formation of new connective tissue matrix by these cells. Disorders of lipid metabolism accelerate the formation of lesions, and interventions against high cholesterol levels have successfully decreased the incidence of atherosclerotic coronary complications by about 30%.1 2 3 On the other hand, up to 50% of the volume of the atherosclerotic wall enlargement consists of ASMCs and ASMC-derived intercellular tissue. Presently, there is no effective therapy to control the latter phenomenon.

In the lesions of experimentally induced atherosclerosis, ASMCs appear simultaneously with or after the appearance of monocyte-derived macrophages.4 5 6 7 ASMCs, endothelial cells, and macrophages are three cell types that localize within the atherosclerotic lesion and may influence each other by the growth factors/cytokines they secrete. Thus, the release of biologically active mitogens by these cells in the arterial intima is likely to play a role on target cells expressing cognite receptors. One of these mediators is PDGF, which, together with the fibroblast growth factors, is a major mitogen for mesenchymal cells, including SMCs from all species. PDGF is regarded as an incomplete mitogen (competence factor), which induces proliferation only in the simultaneous presence of cofactors (commitment factors) such as the insulin-like growth factors.8 Further, the effects of stimulatory cytokines are balanced by inhibitory cytokines such as transforming growth factors9 and by binding to GAGs.10 11 Consequently, the regulation of ASMC proliferation is a complex network of interactions that has been only partially penetrated.

The mature and active forms of PDGF occur as homodimers or heterodimers of two disulfide-linked polypeptide chains, A and B. These chains have a high amino acid homology of approximately 50%. All PDGF chimeras (AA, AB, and BB) have been isolated from several sources, including platelets, vascular SMCs, endothelial cells, and macrophages.12 13 14 15 16 The gene for the human PDGF A chain has been mapped to chromosome 7,17 while the gene for the PDGF B chain was mapped to chromosome 22.18 19 The PDGF A- and B-chain genes have similar organization and consist of seven analogous exons spaced by differently sized introns. The PDGF A chain occurs as two isoforms due to alternative mRNA splicing of exon 6.17 The longer PDGF A form contains a highly basic amino acid C-terminal extension corresponding to exon 6. This sequence has cellular retention properties,20 presumably due to high-affinity binding to heparin-like GAGs.10 21 22 We have shown previously that hASMCs are stimulated by PDGF BB and long and short PDGF AA isoforms in vitro.23 One can assume that the shorter form of the A-chain lacking the basic domain may diffuse away from its site of synthesis. However, the long form of PDGF A may accumulate on GAGs and affect the cells in the arterial wall long after its synthesis by release from GAGs through enzymatic cleavage24 or displacement by other GAG-binding proteins.11 Proliferation of cells may subsequently be induced through paracrine or autocrine mechanisms long after the initial PDGF secretion.

The accumulation of cells in the intima after arterial injury is accompanied by a change in cell phenotype. In the present study we wanted to investigate the effect of phenotypic state on the transcription of the genes for PDGF A and PDGF B isoforms in hASMCs, hAVECs, and hM{Phi} in vitro. We also examined the effect of cell phenotype on the transcription of long and short PDGF A isoforms. Due to the lack of antibodies, there is presently no method available for the estimation of the expression of long and short PDGF A peptides. We thus decided to evaluate the expression of all PDGF isoforms on the mRNA level to obtain a complete picture. Here, applying an RT-PCR method, we quantified the expression of different PDGF isoforms in hASMCs, monocytes/hM{Phi}, and hAVECs.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Cell-Culture Conditions and RNA Preparations
Human arterial SMCs. Human arterial smooth muscle cells from three donors were isolated from the inner media of human uterine arteries as described previously25 and studied in passage 5-8. The establishment of hASMCs of differentiated (quiescent) and dedifferentiated (proliferating) cells in sparse cultures and differentiated confluent phenotypes in vitro has been described and characterized elsewhere.23 25 26 As demonstrated before, positive immunofluorescent staining for smooth muscle {alpha}-actin confirmed the identity of the cells (not shown).

Mononuclear cells. Mononuclear cells were isolated from buffy coats from three blood donors by a Ficoll-Hypaque discontinuous gradient.27 Buffy coats were obtained from the blood bank at Sahlgrenska University Hospital/Sahlgrenska (Göteborg, Sweden). Monocytes at day 0 were obtained without plating the cell suspension after gradient centrifugation. These cells were isolated either with Dynabeads Pan-T/CD2 (one donor), which removes CD2-positive T cells from the cell suspension, or with Dynabeads M-450/CD14 (two donors), which specifically isolates monocytes/macrophages. Beads were used according to the manufacturer's instructions (Dynal AS). The purity of the mononuclear cell isolates was warranted by the specificity of the techniques. Isolated monocytes were cultured for 1, 3, 5, and 7 days in culture conditions as described.28 Viability of mononuclear cells was between 90% to 95% as seen with trypan blue staining. Morphological characterization of mature hM{Phi} was made after hematoxylin staining (not shown).

Human adult vein ECs. hAVECs were a generous gift from Professor Bo Risberg at the Department of Surgery, Sahlgrenska University Hospital/Östra (Göteborg, Sweden). Cells were isolated from stripped varicose veins and cultured in M199 medium (BioWhittaker) containing 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 20% (vol/vol) fetal bovine serum, 90 µg/mL heparin, and 20 µg/mL endothelial cell growth factor (Sigma). Proliferating and confluent cells (passage 4) from one donor were obtained by growing sparse cultures for 1 and 2 weeks, respectively. The dedifferentiated and differentiated phenotypes have been characterized previously.29 Cells showed positive fluorescence when stained for von Willebrand Factor (not shown).

Total RNA. Total RNA was isolated according to Chirgwin et al.30

Quantitative RT-PCR
PDGF A- and B-chain mRNA expressions were studied with quantitative RT-PCR31 and a Gene Amp RNA PCR kit (Perkin-Elmer Cetus). The PCR Kit contains the internal standard pAW109RNA, which is transcribed from plasmid pAW109. This standard contains a linear array of synthetic 5' primers followed by the complementary sequences of the 3' primers of 12 different target genes. Among these are primer sequences corresponding to mRNA for PDGF A and B chains.

For amplification of PDGF isoforms, two pairs of oligonucleotide primers (TableDown) were synthesized on a DNA synthesizer (Applied Biosystems Inc). To prevent amplification of genomic DNA,31 at least one primer in each primer pair spanned the junction between two exons (TableDown). The primer pair used for amplification of PDGF A allowed us to quantitate the total amount of PDGF A mRNA without consideration of splicing phenomena.13


View this table:
[in this window]
[in a new window]
  		Table 1. Sequences of Oligonucleotide Primers Used in RT-PCR of PDGF Isoforms

The cell-derived RNA (100 ng) was reversely transcribed into cDNA, together with the pAW109RNA standard (defined number of molecules) and random hexamer primer (final concentration, 2.5 µmol/L) and 2.5 U/µL SuperScript reverse transcriptase (Life Technologies). The RT step was incubated and processed for amplification as described previously.32 The cDNA mixture obtained from the RT step was further amplified by PCR using specific primer pairs for either PDGF A or PDGF B cDNAs. The 5' primer was labeled by T4 polynucleotide kinase (Boehringer Mannheim) with [{gamma}-32P]ATP (specific activity 5000 Ci/mmol; Amersham Sweden AB). PCR amplifications, recovery of amplification products, and determination of RNA copy numbers were done as described for PDGF receptors.23 The results from the different donors were averaged for each cell type and culture condition.

RT-PCR of PDGF A-Chain Isoforms
Splicing of exon 6 in the PDGF A-chain mRNA was studied with primers according to Matoskova et al33 corresponding to sequences in exons 4 and 7 of the human PDGF A-chain gene (TableUp). The rates of amplification were exponential at 35 cycles and approached a plateau at 45 cycles.

Statistical Methods
Means, variances, and 95% CI were calculated using standard procedures. Two-way ANOVA was used to test differences between donors and culture conditions. This test also accounts for methodological and other variabilities in its residual error against which any additional variation provided by donor and/or culture condition is tested. The methodological variability (coefficient of variation) was 15%, 23%, and 21% for the determinations of PDGF A mRNA, PDGF B mRNA, and the ratio short PDGF A/long PDGF A, respectively. Paired data were tested using Students' paired t test. A value of P<.05 (two-sided tests) was regarded as statistically significant.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Expression of PDGF mRNAs in hASMCs
Quantitative Evaluation of Total PDGF A and PDGF B mRNA Expression
Human ASMCs from all donors (n=3) expressed both PDGF A and PDGF B mRNAs (Fig 1Down). Across all donors and all culture conditions, there was consistently much more PDGF A than -B transcripts (P=.006). The average copy number per nanogram RNA for the three culture conditions was about 1900 for PDGF A and 11 for PDGF B, suggesting at least 100 times higher expression of PDGF A compared with PDGF B mRNA. Confluent cultures showed approximately 50% lower copy numbers of both PDGF A (P=.116) and -B (P=.130) mRNAs than sparse cultures. Neither these differences nor the differences between donors (P>.05) were statistically significant.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 1. Summary of PDGF A- and B-chain mRNA expression in hASMCs. Results are given as the mean of three donors, each experiment run in duplicate. Means for mRNA values (copies per nanogram RNA) ±SEM are shown for PDGF A chain (open bars) and PDGF B chain (shaded bars). Copy numbers for each PDGF chain are given within the corresponding bars.

Comparisons based on results of PCR amplifications of different target genes needs precaution. Priming efficiency varies with primers, sequence, and size of the amplified fragments. Such variations make it difficult to compare the expression of different genes. For this reason, the priming efficiency of our A- and B-chain primers was compared with that of the internal standard pAW109, in which both primer pairs amplify almost the same DNA sequence. The B-chain primers were about six times more efficient than the A-chain primers. Thus, priming efficiency cannot explain the higher A- than B-chain mRNA concentrations seen in hASMCs with quantitative RT-PCR. Furthermore, the amplified fragments from cell-derived RNA were almost of the same size (224 versus 227 bp) and had a 62% sequence homology. Also, both amplified sequences for PDGF A and B chains contained almost equal amounts of GC nucleotides (63% and 65%, respectively), which contributes to a similar amplification efficiency of these two sequences. Consequently, the larger abundance of A- than B-chain transcripts in hASMCs may seem reliable even if not completely precise.

Expression of Long and Short PDGF A mRNA Isoforms
Since these experiments were based on semiquantitative RT-PCR analyses only, the concentration of long and short PDGF A chains could not be expressed as copies per nanogram RNA. On the other hand, both isoforms were determined simultaneously on transcripts from the same gene using the same primer pairs and differed only in length of the amplified fragments. This procedure enabled us to estimate the relative abundance of PDGF A isoforms. Due to the low expression of the long PDGF A mRNA, PCR amplifications were run for 45 cycles. This high cycle number enabled visualization of both long and short A-chain variants after gel electrophoresis.

The results showed two amplification products of expected sizes; one of 180 bp for the shorter message of the PDGF A chain and one of 250 bp for the longer one (data not shown). The main amplification product in all hASMC phenotypes was the short isoform lacking exon 6. The short A-chain transcript was 8 (CI; 6-10) times more abundant than that of the long form. It was almost the same irrespective of phenotype (P=.599) (Fig 2Down). The ratio differed between donors (P=.010), and the possibility of a true interindividual variability cannot be excluded from our results.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 2. Semiquantitation of PDGF A isoform mRNAs in hASMCs. Diagram of both long (L) and short (S) PDGF A isoforms' expression representing the mean of three donors. Results are given as cpm per PCR product. SEM values are included in each bar. The main amplification product in hASMCs from all donors was the short isoform lacking the exon 6–coded sequence. PCR amplifications were run for 45 cycles. The exact ratio of short/long (S/L) is shown as a table incorporated in the figure.

In proliferating and confluent hASMCs, the ratio between the short and long PDGF A mRNAs was further investigated by PCR amplifications run for 35 cycles. The obtained radiolabeled products were mixed with unlabeled products resulting from a PCR run for 45 cycles before electrophoretic separation of the products. In these experiments, the short/long mRNA ratio was approximately the same as in experiments run for 45 cycles.

Considering the higher priming efficiency of the PDGF B primers and that the long PDGF A isoform mRNA comprised only the minor part of the total A-chain mRNAs, one can conclude that the long A-chain transcripts were somewhat more abundant than the B-chain mRNA (Figs 1Up and 2Up).

Expression of PDGF mRNAs in hM{Phi}
Quantitative Evaluation of Total PDGF A and PDGF B mRNA Expression
Monocytes from all donors (n=3) expressed both PDGF A and PDGF B mRNAs. The amount of PDGF A mRNA was higher than that of PDGF B mRNA across donors and days in culture (P=.014). However, in unplated monocytes (day 0), the copy numbers of PDGF A (mean 246, CI 38-454 copies per nanogram RNA) and B (mean 77, CI 52-103 copies per nanogram RNA) did not differ significantly (P=.40). On differentiation in culture, marked increases in copy numbers of both PDGF mRNAs were seen already after 1 day. Maximal stimulation was obtained after 3 days, with about 1200 copies of PDGF A mRNA and about 2600 copies of PDGF B mRNA per nanogram RNA. Clearly, these numbers were outside the CI of day 0 (see above) and statistically significant. Thus, during the first 3 days of monocyte differentiation, the amount of PDGF A mRNA increased 5-fold and that of PDGF B mRNA approximately 30-fold compared with monocytes at day 0 (Fig 3Down). After day 3, the expression of both mRNAs tended to decrease. Nevertheless, after 7 days in culture, the fully differentiated hM{Phi} still expressed higher amounts of both PDGF mRNAs compared with monocytes, ie, twice as much PDGF A mRNA and 20 times more PDGF B mRNA than in monocytes. The error bars in Fig 3Down reflect mainly differences between donors (P=.014 for PDGF A and P=.054 for PDGF B).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 3. PDGF mRNA expression during monocyte differentiation to macrophages. Fold increase from day 0 for PDGF A-chain ({bullet}), B-chain ({blacksquare}), and long PDGF A-chain ({circ}) mRNAs. Fold increase is given as a mean of three donors, each donor run in triplicate. T bars represent SEM.

Expression of Long and Short PDGF A mRNA Isoforms
Since the long and short PDGF A mRNAs were expressed at similar high levels in macrophages in contrast to hASMCs, 35 cycles were used for PCR amplification. However, monocytes at day 0 expressed 9% (CI; 4-14) of PDGF A mRNA as the long form. Clearly, this amount differed very significantly from the short form and from differentiated hM{Phi} and corresponded to a short/long ratio of about 8. Already, after 1 day in culture, the expression of long A-chain mRNA was upregulated to approximately 40% (S/L ratio=1.6) and remained at a similar level throughout the differentiation (day 7) (Fig 3Up). In conclusion, the overall expression pattern in differentiating monocytes shifted to a fourfold increase in the proportion of long A-chain mRNA after 7 days in culture. Given the simultaneous increase in total A-chain mRNA, the absolute increase in long A-chain mRNA was considerable.

Expression of PDGF mRNAs in hAVECs
Quantitative Evaluation of Total PDGF A and PDGF B mRNA Expression
hAVECs expressed both PDGF A and PDGF B mRNAs in high amounts compared with other cells (Figs 4Down and 5Down). This expression was dependent on cell phenotype. On proliferation, hAVECs increased the expression of PDGF A mRNA by a factor of three and decreased the expression of PDGF B mRNA by a similar factor (Fig 4Down).



View larger version (38K):
[in this window]
[in a new window]
  		Figure 4. Summary of PDGF mRNA expression in hAVECs. Bars represent the mRNA expression of PDGF A (open bars) and PDGF B chains (shaded bars). Mean±SEM for each PDGF isoform are included in the corresponding bar. PCR amplifications were run in triplicate.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 5. Comparative summary of PDGF mRNA expression in three cell types. Diagram showing the expression of PDGF A- and B-chain mRNAs in hASMCs, monocytes/hM{Phi}, and hAVECs in vitro. Values represent the mean of proliferating and confluent cultures (hASMCs and hAVECs) or of day 0 and day 3 (monocytes and hM{Phi}).

Expression of Long and Short PDGF A mRNA Isoforms
The PCR amplifications (n=9) were run for 35 cycles, and the relative abundance of the short and long PDGF A mRNA isoforms in proliferating and confluent hAVECs was determined. The ratio of short/long seemed to be stable and grossly independent of cell phenotype, with a mean value of 1.5 (proliferating cells, 1.6±0.23; confluent cells, 1.4±0.18). The results showed a constant and comparatively high production of the long PDGF A mRNA, representing approximately 44% of the total PDGF A mRNA expression.

A summary of average PDGF A- and B-chain expression in hASMCs, monocytes/hM{Phi}, and hAVEC is given in Fig 5Up.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Human ASMCs, blood-derived monocytes/hM{Phi}, and hAVECs express mRNAs for both PDGF A and PDGF B chains with the highest expression of both isoforms in hAVECs. Our results further suggest at least 100 times higher expression of PDGF A than PDGF B mRNA in hASMCs, while both isoforms were expressed at similar levels in hAVECs and hM{Phi}.

Furthermore, we have shown that cell phenotype affects the total mRNA expression of PDGF A and B. The confluent phenotype tended to downregulate transcripts for PDGF A in hAVECs and for both PDGF isoforms in hASMCs. Since we have shown previously that also the ß-subunit of the PDGF receptor is downregulated in confluent hASMCs,23 these observations taken together may be of potential importance. Consequently, it cannot be excluded that growth arrest during confluence is due at least partly to a reduction of endogenous production of both PDGF isoforms and at least one of their receptors. On proliferation, hAVECs upregulated the expression of PDGF A and downregulated that of PDGF B mRNA. In concordance with these results, it has been shown previously that in vitro proliferation and differentiation of human endothelial cells regulate the levels of the sis (PDGF B) mRNA.34 Compared with confluent cells, proliferating hASMCs showed a tendency toward increase in the production of PDGF A but not of PDGF B mRNA. Previous studies with less sensitive Northern blot analyses on adult rat ASMCs were only able to demonstrate expression of transcripts for the PDGF A chain.35 Furthermore, the expression of PDGF A- and B-chain genes seems to be age related since aortic SMCs from adult rats expressed mainly PDGF A-chain mRNA, while newborn rat SMCs also expressed PDGF B-chain transcripts.36 PDGF A-chain expression is also increased in human uterine muscle during the physiological hypertrophy of pregnancy.37 Studies with in situ hybridization on human atherosclerotic plaques have shown the presence of PDGF A- but not B-chain transcripts in mesenchyme-like cells, which could not be definitely classified as ASMCs. Thus, our results are fully compatible with these observations. They demonstrate more clearly that ASMCs from humans express PDGF A-chain mRNA, but also that they express a low abundance of B-chain mRNA that is likely not detected by less sensitive techniques.

On differentiation of hM{Phi}, both PDGF A and B mRNAs were upregulated. The activating signal in our case seems to be the adherence to the culture dish. Our general findings concerning macrophages are consistent with previously published results by Nagaoka et al.38 According to their experiments after adherence of monocytes to plastic dishes (corresponding to day 1 in Fig 3Up), the expression of PDGF B but not of PDGF A increased both on the mRNA and the protein level during further differentiation.

Thus, we can conclude that both hM{Phi} differentiation and proliferation of hAVECs and hASMCs grossly increase the total production of PDGF mRNAs.

PDGF A and B chains may be transcriptionally regulated through 5'-untranslated GC-rich sequences39 or posttranscriptionally regulated through mRNA stability in addition to splicing of primary transcripts.40 Our results suggest an independent regulation of both PDGF genes. Independently regulated expression of PDGF A and B transcripts in other cell types has been reported previously. Thus, while many tumor cell lines express either one or both of the isoform mRNAs,41 42 the PDGF A mRNA seems to be predominantly expressed, for example, in skeletal myoblasts and fibroblasts,35 43 and PDGF B mRNA in placental cytotrophoblasts.44 Several different cytokines have been shown to regulate the expression of PDGF genes. The cytokine effect seems to be dependent on cell type. It has been reported that transforming growth factor-ß increases PDGF A mRNA levels in human fibroblasts45 and vascular SMCs,46 while it stimulates PDGF B mRNA expression47 or the expression of both isoforms48 in other cells. Furthermore, it has been shown that transforming growth factor-ß can either inhibit or promote the proliferation of rat aortic SMCs depending on their phenotype.46 It has also been shown that mechanical injury, shear stress, and oxidative products from lipoprotein oxidation within plaques may influence PDGF expression.49 50 51

The PCR amplifications showed that the short/long A-chain mRNA ratio varied between the different cell types. According to our results in hASMCs, only the minor part of the total PDGF A mRNA consisted of long PDGF A mRNA. Similar conclusions have been reported for human skin fibroblasts, human umbilical vein endothelial cells, and several transformed cell lines.33 Human AVECs produced 40% of the total PDGF A mRNA as the long isoform. Furthermore, the distribution of long and short PDGF A mRNA isoforms was stable irrespective of the phenotypic state of hAVECs. In contrast to these results, the 7-day differentiation of hM{Phi} drastically increased the amount of the long PDGF A mRNA. These results are consistent with a previous observation demonstrating that resting monocytes express only short PDGF A mRNA species, while in vitro maturated hM{Phi} expressed both short and long mRNA species.38

The long PDGF A chain may exhibit a higher mitogenic activity,52 probably due to increased efficiency of assembly and secretion.53 Primarily the long A-chain is upregulated during active wound healing in response to PDGF BB treatment.54 The long PDGF A isoform, which is not expressed in normal hearts, is present in cardiac allografts.55 In contrast to our results on hASMCs and hAVECs, in which phenotypic change did not affect the expression of the long PDGF A, the experiments with human monocyte-derived macrophages in vitro suggested a regulation of this PDGF A isoform during differentiation.

The functional significance of the long PDGF A chain is not clear. Our observations could possibly explain an accumulation of the long PDGF A isoform in the arterial wall after release from adhering platelets, local macrophages, and endothelial cells at the site of a growing atherosclerotic lesion. Indeed, others have shown that the long but not the short A-chain isoform accumulated outside cells associated with cell surface proteoglycans and extracellular matrix.20 We have shown previously that the long PDGF A isoform binds with high affinity to GAGs22 and that proliferating SMCs increase twice their production of GAGs compared with SMCs of differentiated phenotype.11 Particularly highly sulfated GAGs bind the long PDGF A isoform with high affinity.22 Although hASMCs express low concentrations of long A chain, their total contribution to the long A-chain pool may be substantial due to the large number of these cells in the arterial wall. The comparatively high expression of the long PDGF A mRNA in endothelial cells and its upregulation during monocyte differentiation further contributes to this storage.

Assuming no further translational control, all these phenomena would presumably promote the accumulation of PDGF, and particularly of PDGF isoforms binding to matrix components, in the arterial wall at the site of a growing atherosclerotic lesion. Release of PDGF from this pool would explain a prolonged stimulation of ASMC proliferation. However, this possibility needs further confirmation at the protein level in experimental systems as well as in vivo.


*    Selected Abbreviations and Acronyms
 
ASMC = arterial SMC
GAG = glycosaminoglycan
hASMC = human ASMC
hAVEC = human adult vein endothelial cell
hM{Phi} = human monocyte-derived macrophage
PDGF = platelet-derived growth factor
RT-PCR = reverse transcription–polymerase chain reaction
SMC = smooth muscle cell


*    Acknowledgments
 
This study was supported by grants from the Swedish Medical Research Council (project No. 19X-04531) and the Swedish Heart Lung Foundation (project No. 61551). Alexandra Krettek is the recipient of a PhD grant from the Medical Faculty at Göteborg University. We thank Bo Risberg and Peter Falk at the Fibrinolys Laboratory, Sahlgrenska University Hospital/Östra, Östra Hospital (Göteborg, Sweden), for kindly providing the hAVECs used in this study.

Received March 14, 1997; accepted July 1, 1997.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Sacks FM, Pfeffer MA, Moye LA, Rouleau JL, Rutherford JD, Cole TG, Brown L, Warnica JW, Arnold JMO, Wun C-C, Davis BR, Braunwald E. The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. N Engl J Med. 1996;335:1001-1009.[Abstract/Free Full Text]
  2. Scandinavian Simvastatin Survival Study Group. Randomized trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet. 1994;344:1383-1389.[Medline] [Order article via Infotrieve]
  3. Shepherd J, Cobbe SM, Ford I, Isles CG, Lorimer AR, MacFarlane PW, McKillop JH, Packard CJ. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. N Engl J Med. 1995;333:1301-1307.[Abstract/Free Full Text]
  4. Masuda J, Ross R. Atherogenesis during low-[level hypercholesterolemia in the nonhuman primate, I: fatty streak formation. Arteriosclerosis. 1990;10:164-177.[Abstract/Free Full Text]
  5. Masuda J, Ross R. Atherogenesis during low-level hypercholesterolemia in the honhuman primate, II: fatty streak conversion to fibrous plaque. Arteriosclerosis. 1990;10:178-187.[Abstract/Free Full Text]
  6. Rosenfeld ME, Tsukada T, Gown AM, Ross R. Fatty streak initiation in Watanabe heritable hyperlipemic and comparably hypercholesterolemic fat-fed rabbits. Arteriosclerosis. 1987;7:9-23.[Abstract]
  7. Rosenfeld ME, Tsukada T, Chait A, Bierman EL, Gown AM, Ross R. Fatty streak expansion and maturation in Watanabe heritable hyperlipemic and comparably hypercholesterolemic fat-fed rabbits. Arteriosclerosis. 1987;7:24-34.[Abstract]
  8. Clemmons DR. Exposure to PDGF modulates the porcine aortic smooth muscle response to somatomedin. Endocrinology. 1985;117:77-83.[Abstract]
  9. Bobik A, Campbell JH. Vascular derived growth factors: cell biology, pathophysiology, and pharmacology. Pharmacol Rev. 1993;45:1-42.[Medline] [Order article via Infotrieve]
  10. Fager G, Camejo G, Bondjers G. Heparin-like glycosaminoglycans influence growth and phenotype of human arterial smooth muscle cells in vitro, I: evidence for reversible binding and inactivation of the platelet-derived growth factor by heparin. In Vitro Cell Dev Biol. 1992;28A:168-175.
  11. Fager G, Camejo G, Olsson U, Östergren-Lundén G, Lustig F, Bondjers G. Binding of platelet-derived growth factor and low density lipoproteins to glycosaminoglycan species produced by human arterial smooth muscle cells. J Cell Physiol. 1995;163:380-392.[Medline] [Order article via Infotrieve]
  12. Stroobant P, Waterfield MD. Purification and properties of porcine platelet-derived growth factor. EMBO J. 1984;3:2963-2967.[Medline] [Order article via Infotrieve]
  13. Collins T, Ginsburg D, Boss JM, Orkin SH, Pober JS. Cultured human endothelial cells express platelet-derived growth factor B chain: cDNA cloning and structural analysis. Nature. 1985;316:748-750.[Medline] [Order article via Infotrieve]
  14. Heldin C-H, Johnsson A, Wennergren S, Wernstedt C, Betsholtz C, Westermark B. A human osteosarcoma cell line secretes a growth factor structurally related to a homodimer of PDGF A-chains. Nature. 1986;319:511-514.[Medline] [Order article via Infotrieve]
  15. Barrett TB, Benditt EP. Platelet-derived growth factor gene expression in human atherosclerotic plaques and normal artery wall. Proc Natl Acad Sci U S A. 1988;85:2810-2814.[Abstract/Free Full Text]
  16. Hammacher A, Hellman U, Johnsson A, Östman A, Gunnarsson K, Westermark B, Wasteson Å, Heldin C-H. A major part of platelet-derived growth factor purified from human platelets is a heterodimer of one A and one B chain. J Biol Chem. 1988;263:16493-16498.[Abstract/Free Full Text]
  17. Betsholtz C, Johnsson A, Heldin C-H, Westermark B, Lind P, Urdea MS, Eddy R, Shows TB, Philpott K, Mellor AL, Knott TJ, Scott J. cDNA sequence and chromosomal localization of human platelet-derived growth factor A-chain and its expression in tumour cell lines. Nature. 1986;320:695-699.[Medline] [Order article via Infotrieve]
  18. Swan DC, McBridge OW, Robbins KC, Keithley DA, Reddy EP, Aaronson SA. Chromosomal mapping of the simian sarcoma virus onc gene analogue in human cells. Proc Natl Acad Sci U S A. 1982;79:4691-4695.[Abstract/Free Full Text]
  19. DallaFavera R, Gallo RC, Giallongo A, Croce CM. Chromosomal localization of the human homologue (c-sis) of the simian sarcoma virus onc gene. Science. 1982;218:686-688.[Abstract/Free Full Text]
  20. Andersson M, Östman A, Westermark B, Heldin C-H. Characterization of the retention motif in the C-terminal part of the long splice form of the platelet-derived growth factor A-chain. J Biol Chem. 1994;269:926-930.[Abstract/Free Full Text]
  21. Khachigian LM, Chesterman CN. Synthetic peptides representing the alternatively spliced exon of the platelet-derived growth factor A-chain modulate mitogenesis stimulated by normal human serum and several growth factors. J Biol Chem. 1992;267:7478-7482.[Abstract/Free Full Text]
  22. Lustig F, Hoebeke J, Östergren-Lundén G, Velge-Roussel F, Bondjers G, Olsson U, Rüetschi U, Fager G. Alternative splicing determines the binding of platelet-derived growth factor (PDGF AA) to glycosaminoglycans. Biochemistry. 1996;35:12077-12085.[Medline] [Order article via Infotrieve]
  23. Krettek A, Fager G, Jernberg P, Östergren-Lundén G, Lustig F. Quantitation of platelet-derived growth factor (PDGF) receptors in human arterial smooth muscle cells in vitro. Arterioscler Thromb Vasc Biol. 1997;17:2395-2404.[Abstract/Free Full Text]
  24. Ko FN, Yang YC, Huang SC, Ou JT. Coagulation factor Xa stimulates platelet-derived growth factor release and mitogenesis in cultured vascular smooth muscle cells of rat. J Clin Invest. 1996;98:1493-1501.[Medline] [Order article via Infotrieve]
  25. Fager G, Hansson GK, Ottosson P, Dahllöf B, Bondjers G. Human arterial smooth muscle cells in culture: effects of platelet-derived growth factor and heparin on growth in vitro. Exp Cell Res. 1988;176:319-335.[Medline] [Order article via Infotrieve]
  26. Fager G, Hansson GK, Gown AM, Larson DM, Skalli O, Bondjers G. Human arterial smooth muscle cells in culture: inverse relationship between proliferation and expression of contractile proteins. In Vitro Cell Dev Biol. 1989;25:511-520.[Medline] [Order article via Infotrieve]
  27. Böyum A. Isolation of lymphocytes, granulocytes and macrophages. Scand J Immunol. 1976;5(suppl 5):9-15.
  28. Hurt E, Bondjers G, Camejo G. Interaction of LDL with human arterial proteoglycans stimulates its uptake by human monocyte-derived macrophages. J Lipid Res. 1990;31:443-454.[Abstract]
  29. Van Hinsbergh VWM. Propagation and morphologic phenotypes of human umbilical cord artery endothelial cells. Eur J Cell Biol. 1986;42:101-110.[Medline] [Order article via Infotrieve]
  30. Chirgwin JM, Przybyla AM, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979;18:5294-5298.[Medline] [Order article via Infotrieve]
  31. Wang AM, Doyle MV, Mark DF. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci U S A. 1989;86:9717-9721.[Abstract/Free Full Text]
  32. Mattsson L, Johansson H, Ottosson M, Bondjers G, Wiklund O. Expression of lipoprotein lipase mRNA and secretion in macrophages isolated from human atherosclerotic aorta. J Clin Invest. 1993;92:1759-1765.
  33. Matoskova B, Rorsman F, Svensson V, Betsholtz C. Alternative splicing of the platelet-derived growth factor A-chain transcript occurs in normal as well as tumour cells and is conserved among mammalian species. Mol Cell Biol. 1989;9:3148-3150.[Abstract/Free Full Text]
  34. Jaye M, McConathy E, Drohan W, Tong B, Deuel T, Maciag T. Modulation of the sis gene transcript during endothelial cell differentiation in vitro. Science. 1985;228:882-885.[Abstract/Free Full Text]
  35. Sejersen T, Betsholtz C, Sjölund M, Heldin C-H, Westermark B, Thyberg J. Rat skeletal myoblasts and arterial smooth muscle cells express the gene for the A chain but not for the B chain (c-sis) of platelet derived growth factor (PDGF) and produce a PDGF like protein. Proc Natl Acad Sci U S A. 1986;83:6844-6848.[Abstract/Free Full Text]
  36. Majesky MW, Benditt EP, Schwartz SM. Expression and developmental control of platelet-derived growth factor A-chain and B-chain/sis genes in rat aortic smooth muscle cells. Proc Natl Acad Sci U S A. 1988;85:1524-1528.[Abstract/Free Full Text]
  37. Mendoza AE, Young R, Orkin SH, Collins T. Increased platelet-derived growth factor A-chain expression in human uterine smooth muscle cells during the physiologic hypertrophy of pregnancy. Proc Natl Acad Sci U S A. 1990;87:2177-2181.[Abstract/Free Full Text]
  38. Nagaoka I, Honma S, Someya A, Iwabuchi K, Yamashita T. Differential expression of the platelet-derived growth factor-A and -B genes during maturation of monocytes to macrophages. Comp Biochem Physiol. 1992;103B:349-356.
  39. Rao CD, Pech M, Robbins KC, Aaronson SA. The 5'-untranslated sequence of the c-sis/platelet-derived growth factor 2 transcript is a potent translator inhibitor. Mol Cell Biol. 1988;8:284-292.[Abstract/Free Full Text]
  40. Betsholtz C. The PDGF genes and their regulation. In: Westermark B, Sorg C, eds. Biology of Platelet-Derived Growth Factor: Cytokines. Basel, Switzerland: Karger; 1993: chap 5.
  41. Heldin C-H, Betsholtz C, Cleasson-Welsh L, Westermark B. Subversion of growth regulatory pathways in malignant transformation. Biochim Biophys Acta. 1987;907:219-244.[Medline] [Order article via Infotrieve]
  42. Westermark B, Heldin C-H. Platelet-derived growth factor in autocrine transformation. Cancer Res. 1991;51:5087-5092.[Free Full Text]
  43. Paulsson Y, Hammacher A, Heldin C-H, Westermark B. Possible positive autocrine feedback in the prereplicative phase of human fibroblasts. Nature. 1987;328:715-717.[Medline] [Order article via Infotrieve]
  44. Goustin AS, Betsholtz C, Pfeifer-Ohlsson S, Persson H, Rydnert J, Bywater M, Holmgren G, Heldin C-H, Westermark B, Ohlsson R. Co-expression of the sis and myc proto-oncogenes in developing human placenta suggests autocrine control of trophoblast growth. Cell. 1985;41:301-312.[Medline] [Order article via Infotrieve]
  45. Paulsson Y, Beckmann MP, Westermark B, Heldin C-H. Density-dependent inhibition of cell growth by transforming growth factor-ß1 in normal human fibroblasts. Growth Factors. 1988;1:19-27.[Medline] [Order article via Infotrieve]
  46. Majack RA, Majesky MW, Goodman LV. Role of PDGF A expression in the control of vascular smooth muscle cell growth by transforming growth factor-ß. J Cell Biol. 1990;111:239-247.[Abstract/Free Full Text]
  47. Leof EB, Proper JA, Goustin AS, Shipley GD, Dicorleto PE, Moses HL. Induction of c-sis mRNA and activity similar to platelet-derived growth factor by transforming growth factor ß: a proposed model for indirect mitogenesis involving autocrine activity. Proc Natl Acad Sci U S A. 1986;83:2453-2457.[Abstract/Free Full Text]
  48. Kavanaugh WM, Harsh GRI, Starksen NF, Rocco CM, Williams LT. Transcriptional regulation of the A and B chain genes of platelet-derived growth factor in microvascular endothelial cells. J Biol Chem. 1988;263:8470-8472.[Abstract/Free Full Text]
  49. Khachigian LM, Resnick N, Gimbrone MA Jr, Collins T. Nuclear factor-{kappa}B interacts functionally with the platelet-derived growth factor B-chain shear-stress response element in vascular endothelial cells exposed to fluid shear stress. J Clin Invest. 1995;96:1169-1175.
  50. Khachigian LM, Lindner V, Williams AJ, Collins T. Egr-1-induced endothelial gene expression: a common theme in vascular injury. Science. 1996;271:1427-1431.[Abstract]
  51. Kume N, Gimbrone MA Jr. Lysophosphatidylcholine transcriptionally induces growth factor gene expression in cultured human endothelial cells. J Clin Invest. 1994;93:907-911.
  52. Collins T, Bonthron DT, Orkin SH. Alternative RNA splicing affects function of encoded platelet-derived growth factor A-chain. Nature. 1987;328:621-624.[Medline] [Order article via Infotrieve]
  53. Bonthron DT, Morton CC, Orkin SH, Collins T. Platelet-derived growth factor A chain: gene structure, chromosomal location and basis for alternative mRNA splicing. Proc Natl Acad Sci U S A. 1988;85:1492-1496.[Abstract/Free Full Text]
  54. Pierce GF, Tarpley JE, Tseng J, Bready J, Chang D, Kenney WC, Rudolph R, Robson MC, Vande Berg J, Reid P, Kaufman S, Farrell CL. Detection of platelet-derived growth factor (PDGF)-AA in actively healing human wounds treated with recombinant PDGF BB and absence of PDGF in chronic nonhealing wounds. J Clin Invest. 1995;96:1336-1350.
  55. Zhao X-M, Frist WH, Yeoh T-K, Miller GG. Confirmation of alternatively spliced platelet-derived growth factor-A chain and correlation with expression of PDGF receptor-{alpha} in human cardiac allografts. Transplantation. 1995;59:605-611.[Medline] [Order article via Infotrieve]



This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
S. Ling, G. Deng, H. E. Ives, K. Chatterjee, G. M. Rubanyi, P. A. Komesaroff, and K. Sudhir
Estrogen inhibits mechanical strain-induced mitogenesis in human vascular smooth muscle cells via down-regulation of Sp-1
Cardiovasc Res, April 1, 2001; 50(1): 108 - 114.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
A. Dobrian, S. S. Wade, and R. L. Prewitt
PDGF-A expression correlates with blood pressure and remodeling in 1K1C hypertensive rat arteries
Am J Physiol Heart Circ Physiol, June 1, 1999; 276(6): H2159 - H2167.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
E. Feyzi, T. Saldeen, E. Larsson, U. Lindahl, and M. Salmivirta
Age-dependent Modulation of Heparan Sulfate Structure and Function
J. Biol. Chem., May 29, 1998; 273(22): 13395 - 13398.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
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 PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Krettek, A.
Right arrow Articles by Lustig, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Krettek, A.
Right arrow Articles by Lustig, F.

ATVB Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 1997 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.