(Received for publication, January 18, 1996; and in revised form, March 14, 1996)
From the
Basic fibroblast growth factor (bFGF), a potent mitogen for many cell types, is expressed by vascular smooth muscle cells and plays a prominent role in the proliferative response to vascular injury. Basic FGF has also been implicated as a survival factor for a variety of quiescent or terminally differentiated cells. Autocrine mechanisms could potentially mediate both proliferation and cell survival. To probe such autocrine pathways, endogenous bFGF production was inhibited in cultured rat vascular smooth muscle cells by the expression of antisense bFGF RNA. Inhibition of endogenous bFGF production induced apoptosis in these cells independent of proliferation, and apoptosis could be prevented by exogenous bFGF but not serum or epidermal growth factor. The induction of apoptosis was associated with an inappropriate entry into S phase. These data demonstrate that interruption of autocrine bFGF signaling results in apoptosis of vascular smooth muscle cells, and that the mechanism involves disruption of normal cell cycle regulation.
Basic fibroblast growth factor (bFGF) ()belongs to a
multigene family with important roles in embryonic mesodermal and
neuroectodermal development as well as angiogenesis and wound repair in
the mature organism(1, 2) . It is a potent mitogen for
many cell types, including vascular smooth muscle cells(3) .
Basic FGF is found in quiescent smooth muscle cells of the arterial
media(4) , and its release following vascular injury has been
implicated in driving the initial proliferative response of medial
smooth muscle cells, at least by a paracrine mechanism(5) . The
concomitant expression of both bFGF and cell surface FGF receptors by
smooth muscle cells suggests that autocrine FGF signaling has important
phenotypic consequences. Autocrine FGF signaling has been reported to
contribute to smooth muscle cell migration (6) and may also
participate in proliferation, as has been reported for other cell
types(7, 8, 9, 10) . Other
consequences of autocrine FGF signaling in vascular smooth muscle
cells, such as the promotion of cell survival, have not been explored.
A variety of cell types that are either terminally differentiated or normally remain in a quiescent state in the adult organism express FGF, but the autocrine functions of FGF in this context have not been elucidated. There is good experimental evidence that FGF can reside in the extracellular matrix around nondividing cells as a storage form, with its mitogenic activity available in the event of tissue damage and a requirement for repair or scar formation(11, 12, 13) . However, the widespread expression and intracellular location of FGF in nondividing cells suggests other functions, and has spurred studies of the ability of FGF to promote cell survival. The survival-promoting activity appears to be distinct from the mitogenic effects of FGF, as it has been documented in nonproliferating cells(14, 15) , including terminally differentiated cells of neuronal origin(16, 17) . How FGFs promote survival through an autocrine mechanism in this context has not been defined but may be through the prevention of apoptosis, or programmed cell death. That FGFs are autocrine survival factors has been suggested by studies in vitro(15) and in vivo(18) . The notion that exogenous FGFs promote survival by preventing apoptosis, at least through a paracrine mechanism, has been suggested by studies in PC12 cells (19) and endothelial cells(20, 21) . However, these studies do not distinguish between the survival function of FGFs and either their ability to modulate phenotype or stimulate mitogenesis.
These observations, coupled with the presence of bFGF in medial smooth muscle cells of the arterial media, prompted the hypothesis that autocrine bFGF supports smooth muscle cell proliferation, survival, or both. Vascular smooth muscle contributes to normal vessel function, the development of vascular diseases, and the response to vascular injury. There is a growing appreciation that smooth muscle cell apoptosis contributes to vascular development, physiologic adaptation, and vascular remodeling in response to the natural progression or invasive treatment of human vascular disease(22, 23, 24, 25) . The role of autocrine FGF signaling in smooth muscle cell proliferation or survival has not been clearly defined. In order to probe the autocrine function of bFGF in vascular smooth muscle cells, endogenous expression was inhibited using an antisense strategy, and the functional consequences were examined.
Recombinant adenovirus was identified by PCR amplification of
putative recombinant plaques using one vector-specific and one
insert-specific primer. The vector-specific primer
(5`-AGACATGATAAGATACAT-3`) corresponds to a region of the shuttle
vector upstream of the insert cloning site but contains no adenoviral
genomic sequence. The insert-specific primer (5`-GCTTCTTCCTGCGCATCC-3`)
corresponds to codons 37-42 of the 18-kDa rat bFGF coding
sequence. The recombinant virus was twice plaque-purified, and
expression of the antisense transcript was confirmed by reverse
transcription-PCR (RT-PCR). Total cellular RNA (1 µg) isolated from
uninfected smooth muscle cells or cells infected with either Ad.ASbFGF
or Ad5/RSV/GL2 was heat-denatured (65 °C for 5 min) and
reverse-transcribed using 0.5 pmol of a sequence-specific primer. This
primer contains 18 bases (5`-ACTTCGCTTCCCGCACTG-3`) of bFGF sequence
complementary to the antisense strand (corresponding to codons
7-12 of 18-kDa rat bFGF) at the 3` end, as well as a 30-base
random sequence at the 5` end (5`-CTTATACGGATATCCTGGCAATTCGGACTT-3`).
The 5` end random sequence tag permits subsequent PCR amplification
with a primer corresponding to this tag sequence only, thus rendering
the overall amplification both strand- and RNA-specific(30) .
The 5` primer used for the PCR amplification
(5`-GCACACACTCCCTTGATGGACAC-3`) corresponds to codons 71-78 of
18-kDa rat bFGF. As controls, each of the cellular RNAs was also
reverse-transcribed using random hexamer primers (1 pmol), and these
cDNA products were subsequently amplified using primers (5`-primer:
5`-CCTGAAGGGTGGTGCAAAAG-3`; 3`-primer: 5`-CCATCCACAGTCTTCTGAGTG-3`) for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Reverse transcription
was performed in a final reaction volume of 20 µl using 20 units of
avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim)
according to the supplier's recommendations, and controls
included ASbFGF and sense GAPDH RNA transcripts generated in vitro using cDNA templates and bacteriophage T7 RNA polymerase as well
as a no-template negative control. The ASbFGF in vitro transcript yields a 246-base pair RT-PCR amplification product
that is indistinguishable from that generated from the ASbFGF
recombinant adenoviral transcript; the GAPDH cDNA used to generate the in vitro transcript contains a 106-base deletion relative to
the native mRNA, which is flanked by the PCR primer sites and thus
generates a smaller RT-PCR amplification product (132 base pairs versus 238 base pairs, respectively). Ten percent of each cDNA
product was amplified using 1 pmol of each pair of relevant primers,
with one primer in each reaction 5`-end-labeled using T4 polynucleotide
kinase (New England Biolabs, Beverly, MA) and
[-
P]ATP (6000 Ci/mmol, Amersham Corp.). The
PCR was performed in a 20-µl volume using 1 unit/reaction of Taq polymerase (Perkin-Elmer) in an Ericomp thermal cycler
(San Diego, CA) for 25 cycles using 1-min steps. Denaturation was done
at 94 °C, annealing at 60 °C, and extension at 72 °C.
Products were separated on a 6% polyacrylamide, 7 M urea gel
and detected by autoradiography using a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA).
Figure 1:
Expression of an antisense bFGF RNA
inhibits endogenous bFGF expression. Rat aortic smooth muscle cells
were sham-infected or infected with either Ad.ASbFGF or Ad5/RSV/GL2
recombinant adenoviruses at a multiplicity of infection (MOI) of 1000.
RT-PCR analysis (A) was performed on total RNA isolated from
cells infected with either Ad.ASbFGF (lane 2, AS) or
Ad5/RSV/GL2 (lane 3, Luc) and from uninfected cells (lane 4, Un). Controls included a synthetic ASbFGF
transcript generated from a cDNA template in vitro (lane
5, +C) and no template (lane 1, -C). Each of the cellular RNAs was also analyzed for a control
mRNA transcript, GAPDH, using a synthetic GAPDH transcript containing a
short deletion (GAPDH) as a positive control. This
experiment was repeated at least five times using independent RNA
samples and alternative primer sets with similar results. Basic FGF
cell content was determined by Western (immunoblot) analysis (B). Both uninfected cells (lane 1) and cells
infected with Ad5/RSV/GL2 (lane 2) contain equivalent amounts
of all three isoforms of rat bFGF, whereas Ad.ASbFGF-infected cells (lane 3) contain much less immunoreactive bFGF. This analysis
was repeated three times with similar
results.
Figure 2: Antisense inhibition of endogenous bFGF synthesis provokes cell death in either high or low serum, which can be prevented by exogenous bFGF. Antisense inhibition of bFGF expression reduces the accumulation of cells cultured in 10% FBS (A). Cells were infected with Ad.CMVlacZ at an MOI = 1000 (open squares), or Ad.ASbFGF at an MOI = 50 (filled diamonds), 100 (filled triangles), or 1000 (filled circles) and maintained in 10% FBS. Cells were counted daily starting on the day after infection; individual data points in this and each of the experiments in this figure represent the mean ± S.D. of triplicate wells, and each experiment was repeated at least three times. For the bFGF rescue experiment (B), cells were infected with Ad.CMVlacZ or Ad.ASbFGF at an MOI = 100; bFGF was added daily starting 1 day following infection, and cell counts were monitored. Cells infected with Ad.CMVlacZ and cultured in the absence (open squares), or presence (open circles) of bFGF were compared with cells infected with Ad.ASbFGF in the absence (filled squares) or presence (filled circles) of bFGF. To distinguish between inhibition of proliferation and promotion of cell death, cells were cultured in 0.5% serum to maintain quiescence both prior to and following infection with recombinant adenoviruses (C). Cells were infected with either Ad5/RSV/GL2 at an MOI = 1000 (open squares) or with Ad.ASbFGF at an MOI = 100 (filled triangles) or 1000 (filled circles), and cell counts were monitored daily.
Figure 3: Antisense inhibition of bFGF expression induces apoptosis: morphologic and biochemical criteria. Cells infected at an MOI = 1000 with Ad.CMVlacZ (A and C) and Ad.ASbFGF (B and D) were photographed using either phase contrast or epifluorescence microscopy. Ad.CMVlacZ-infected cells showed normal cell morphology (A), whereas Ad.ASbFGF-infected cells displayed rounding and blebbing (B). Ad.CMVlacZ-infected cells (C) displayed normal nuclear morphology compared with condensation of nuclear chromatin and nuclear fragmentation in Ad.AsbFGF-infected cells (D). Cells stained using the TUNEL reaction (E-H) revealed positive staining in Ad.ASbFGF-infected cells (E). Negative controls included Ad.ASbFGF-infected cells incubated with reaction buffer lacking TdT (F), and both uninfected (G) and Ad5/RSV/GL2-infected cells (H) incubated with reaction buffer containing TdT.
Figure 4:
Antisense inhibition of bFGF expression
induces oligonucleosomal fragmentation. The low molecular weight DNA
fraction prepared from equal numbers of sham-infected and Ad5/RSV/GL2-
or Ad.ASbFGF-infected cells was displayed by agarose gel
electrophoresis. Lane 1, 123-base pair markers; lane
2, uninfected cells; lane 3, Ad5/RSV/GL2-infected cells; lane 4, Ad.ASbFGF-infected cells; lane 5, positive
control (v-myc-transformed rat embryo fibroblasts exposed to
10 Gy -radiation, total cellular DNA).
Apoptosis can be induced in some cell types by deprivation of specific growth factors(19, 20, 35, 36, 37) . Other cell types, including vascular smooth muscle cells(38) , survive well when cultured in serum-free medium but undergo apoptosis when forced into S phase in the absence of serum by constitutive expression of cell cycle transcription factors like c-myc(39, 40) or E2F(41, 42) . Apoptosis triggered by antisense inhibition of bFGF production might resemble a growth factor withdrawal model if it could be prevented by heterologous growth factor signaling pathways(43) . Alternatively, it might conform to the second type of apoptotic mechanism involving inappropriate S phase entry. To distinguish between these two possibilities, the ability of a different growth factor to prevent apoptosis following infection with Ad.ASbFGF was assessed, and the degree of S phase entry was estimated by flow cytometry, rates of DNA synthesis, and BrdU labeling of apoptotic nuclei in Ad.ASbFGF-infected cells.
Figure 5: Antisense inhibition of endogenous bFGF expression provokes inappropriate entry into S phase. Nuclei from SMCs infected with either Ad.ASbFGF (A), Ad5/RSV/GL2 (B), Ad.CMVlacZ (C), or uninfected cells (D) were prepared and analyzed for cell cycle distribution by flow cytometry as described in ``Experimental Procedures.'' Using curve-fitting software to estimate the cell cycle phase distributions, the estimated proportion of cells in S phase in each of the control samples (B, C, or D) ranged from 9-15%. In contrast, the estimated proportion of Ad.ASbFGF-infected cells in S phase was 53%.
Figure 6: Antisense inhibition of endogenous bFGF expression provokes inappropriate DNA synthesis in high or low serum. Rates of DNA synthesis were measured by thymidine incorporation in cells cultured in either 10% FBS (A) or 0.5% FBS (B). In panel A, cells were labeled for 18 h; in panel B, they were labeled for 12 h. The data, expressed as dpm/100 cells, represent the mean ± S.D. from triplicate wells normalized to cell number, determined by counting of companion wells.
Figure 7: Increased DNA synthesis is associated with apoptotic nuclei. Smooth muscle cells infected with either Ad.ASbFGF (A and C) or Ad5/RSV/GL2 (B and D) recombinant adenoviruses at an MOI of 1000 were pulse-labeled with BrdU for 2 h prior to fixation and staining with a fluorescent anti-BrdU antibody and counterstaining with propidium iodide. The fluorescein isothiocyanate-labeled anti-BrdU images (A and B) correspond to the same fields as the propidium iodide fluorescence images (C and D) and show that in addition to the greater proportion of labeled cells in the Ad.ASbFGF-infected sample, there is intense staining associated with nuclei displaying abnormal morphology, interpreted as indicating apoptosis.
This study demonstrates that inhibition of endogenous basic FGF synthesis using an antisense strategy triggers apoptosis in cultured vascular smooth muscle cells, supporting an essential survival function for autocrine FGF signaling in this cell type. Prior studies implicating fibroblast growth factors as survival factors in mesodermal and neuroectodermal cells (14, 16, 17, 19, 20, 31, 44) suggest that this survival function is related to prevention of apoptosis (19, 20, 44) but do not distinguish between mitogenic and survival-promoting activities.
Overexpression of FGF can lead to a transformed phenotype(45, 46, 47, 48, 49, 50) , supporting the idea that autocrine FGF signaling can drive proliferation, but interactions between FGFs and transmembrane FGF receptors may be required for FGF-stimulated mitogenesis(45, 46, 47, 48, 49) . On the other hand, selective overexpression of the high molecular weight isoforms of bFGF can either promote or inhibit proliferation without evidence of receptor interaction(50, 51) . None of these studies establish that autocrine FGF signaling is either necessary or sufficient for proliferation in cells capable of such autocrine loops. The mechanism of apoptosis in the present study appears to be independent of proliferation, as it occurs in cells cultured in either high (10%) or low (0.5%) serum. The ability of exogenous bFGF to prevent apoptosis may be related to activation of receptor-mediated signal transduction pathways or to internalization of ligand per se, although the inability of epidermal growth factor to block this mechanism of apoptosis suggests that receptor-mediated signaling may be less important. Thus the survival function of endogenous bFGF in smooth muscle cells appears to involve signal transduction pathways that may be distinct from those mediating mitogenesis. Experiments in transfected skeletal myoblasts (52) and fibroblasts (51) support important differences between FGF signaling pathways controlling mitogenesis and other cell functions.
The data presented establish that the pathway of apoptosis triggered by inhibition of autocrine FGF signaling involves an inappropriate entry into S phase. The possibility that DNA repair mechanisms contribute to the observed increase in total DNA synthesis has not been excluded, but the cell cycle phase analysis suggests that S phase entry is the predominant, if not exclusive, mechanism responsible. In this aspect it resembles other models of apoptosis that are due to disruption of normal cell cycle regulation. Unregulated expression of c-myc(39, 40) or E2F (41, 42) induces an inappropriate S phase entry and triggers apoptosis in the absence of serum. These pathways of apoptosis can be understood, at least in the case of E2F, by alteration in the regulatory balance between E2F and the retinoblastoma gene product, normally regulated by cyclins and cyclin-dependent kinase inhibitors. It is possible that expression of adenoviral genes in conjunction with the antisense bFGF transcript could be contributing to the observed phenotype. The adenoviral protein E1A interacts with cell cycle regulators such as the retinoblastoma protein and is a recognized inducer of apoptosis(22, 53) . Although the recombinant viruses used in this study are specifically E1-deleted, contamination with E1-expressing virus, although highly unlikely, has not been rigorously excluded. Although a direct relationship between FGF signaling and the activities of cell cycle regulatory proteins has not been demonstrated, the present study provides more direct evidence of the ability of autocrine FGF signaling to influence cell cycle regulation than has previously been reported.
Antisense inhibition of endogenous bFGF expression leads to an interruption of autocrine FGF signaling that appears to alter cell cycle regulation, resulting in apoptosis of vascular smooth muscle cells. These observations provide a mechanism that could explain the survival function of FGFs expressed in quiescent or terminally differentiated cells. The expression of FGFs is widely detectable in mesodermal and neuroectodermal lineages of adult organisms, and autocrine FGF signaling may well play a role in the cellular homeostasis and survival of these cell types. The present work provides insights into the autocrine functions of FGF and the signaling pathways whereby endogenous FGF prevents apoptosis are currently being explored.