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Departments of Molecular and Cellular Physiology, and Endocrinology and Metabolism, University of Cincinnati, Cincinnati, Ohio 45267
Address all correspondence and requests for reprints to: James A. Fagin, M.D., Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, Vontz Center for Molecular Studies, 3125 Eden Avenue, Cincinnati, Ohio 45267. E-mail: faginja{at}uc.edu
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Abstract |
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-actin
promoter. Denuded aortas from SMP8-IGF-I mice generated increased force
in response to KCl or phenylephrine and had greater sensitivity to KCl
depolarization. This is not due to desensitization of a SM NO pathway,
as pretreatment with n-
-nitro-L-arginine affected both
wild-type and SMP8-IGF-I aortas to a similar degree. The increased
contractility ex vivo is not associated with changes in
heart rate or blood pressure. Total smooth muscle myosin heavy chain
(SMHC) messenger RNA (mRNA) was greater in SMP8-IGF-I aortas, with
preferential expression of SMHC-A. Reciprocal effects on contractility
and SMHC mRNA were observed in SMP8-IGFBP-4 animals, in which
IGF-binding protein-4 was overexpressed through the same promoter.
Also, SM -actin mRNA was increased in the aortas from SMP8-IGF-I
mice. In summary, chronic arterial overexpression of IGF-I is
associated with increased contractility. These effects differ from
those seen after acute exposure to the growth factor and may relate to
IGF-mediated changes in expression and relative isoform abundance of
critical contractile proteins. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is ample evidence that IGF-I stimulates smooth muscle cell (SMC) growth (1, 2) and inhibits apoptosis (3) in vitro. Until recently, information on the paracrine effects of IGF-I in SMC tissue beds in vivo was conjectural and based primarily on descriptions of the regulation of IGF-I gene expression in association with events that trigger smooth muscle (SM) hyperplasia or hypertrophy (4, 5, 6, 7, 8, 9, 10). Transgenic mice with targeted overexpression of IGF-I in SMC (SMP8-IGF-I) develop marked hyperplasia of the arterial media, bladder, and uterine SM layers, providing direct evidence that IGF-I is a paracrine growth factor for these cells in vivo (11).
The effects of IGF-I are modulated physiologically by a family of high
affinity IGFBPs, believed to modify the access and/or interactions of
IGFs with its receptors. Expression of IGFBPs is tissue specific
and developmentally regulated. IGFBPs are bound to the extracellular
matrix (i.e. IGFBP-5) (12, 13), to the cellular
membrane (i.e. IGFBP-3) (14), or, as in the
case of IGFBP-4, to an unknown site(s) in the extracellular milieu.
IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5 are, in turn, cleaved by
specific proteases that degrade the respective binding proteins to
fragments with low or absent affinity for the IGFs (reviewed in Ref.
15). The expression of the individual binding proteins is
subject to regulation, as is the activity of the IGFBP3, IGFBP-4, and
IGFBP-5 proteases. The major IGFBPs expressed in vascular tissues of
adult rodents are IGFBP-4 and IGFBP-5 (16, 17). IGFBP-4,
the most abundant member of this family expressed by rodent vascular
SMC, has been consistently found to antagonize IGF-I action in
vitro. Accordingly, we recently demonstrated that cell-specific
overexpression of IGFBP-4 in transgenic mice by means of a SM -actin
promoter (SMP8-IGFBP-4) results in SM hypoplasia (18).
IGF-I may also be involved in the regulation of vascular tone (19, 20, 21, 22, 23). Infusions of IGF-I decrease renal vascular resistance in rats and humans. Similarly, bolus injections of IGF-I decrease blood pressure in male Wistar rats (21) and increase forearm blood flow in humans (19). Like insulin, IGF-I regulates vascular tone at least in part by decreasing vascular reactivity to contractile agents. Preincubation of rat aortas with IGF-I decreases vascular contractile responses to both voltage-induced and receptor-mediated agents by more than 50% (21). This is attributed to both endothelium and SM generation of nitric oxide (No) (21, 24). These effects require gene expression, as preincubation of vessels with cycloheximide blocks the IGF-I-mediated increase in NO production. Indeed, IGF-I-induced NO production can also be blocked by pretreatment with the tyrosine kinase inhibitor genistein (25) and the phosphoinositide 3-kinase inhibitor wortmanin (26).
Thus, IGF-I appears to function as a vasodilator when it is infused in vivo or when arteries are bathed in this growth factor ex vivo. Here we report the effects of IGF-I on arterial contractility when it is chronically overexpressed within the vessel wall itself. This paradigm is relevant to a number of pathophysiological conditions in which arterial IGF-I levels are high: i.e. hypertension (27), aortic coarctation (28), and postangioplasty (29). As opposed to the acute effects of IGF-I on vascular reactivity, arteries from SMP8-IGF-I mice have greater contractile force and increased sensitivity to KCl depolarization. By contrast, arteries from SMP8-IGFBP-4 mice, in which the IGF-I antagonist IGFBP-4 is expressed in great abundance, have lower contractile responses. We demonstrate that sustained elevation of IGF-I within the vessel wall is associated with significant regulation of SMC differentiated properties, including a higher abundance and changes in the relative isoform distribution of critical myofibrillar proteins, which may at least in part account for the enhanced response to contractile stimuli.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-actin
promoter (SMP8) cloned upstream of either a rat IGF-I or IGFBP-4
complementary DNA (cDNA). Expression of the respective transgenes was
robust and was found exclusively in SM-rich tissues (artery, vein,
bladder, uterus, and myocyte layer of the gastrointestinal tract).
IGF-I messenger RNA (mRNA) levels in these tissues (i.e.
aorta and bladder) were equivalent or greater than endogenous IGF-I
mRNA levels in liver. IGF-I expression in SMP8-IGF-I mice remained
entirely paracrine, as there was no increase in plasma IGF-I levels.
Similarly, overexpression of IGFBP-4 was confined to SM in SMP8-IGFBP-4
mice and was not observed in plasma. SMP8-IGF-I mice had a striking
degree of SM cell hyperplasia, whereas animals with targeted
overexpression of IGFBP-4 had a reciprocal phenotype.
Vessel preparation
All animal studies were performed in accordance with and
after approval by the local insitutional animal care and use committee.
Ten-week-old SMP8-IGF-I, SMP8-IGFBP-4, or FVB/N (control) mice
(Taconic Farms, Inc., Germantown NY) were killed by
CO2 asphyxiation. Aortas were dissected and
prepared for analysis as previously described (30, 31).
Briefly, vessels were rinsed in cold bicarbonate-buffered physiological
saline solution (PSS), and loose fat and connective tissue were
removed. PSS contained 118 mmol/liter NaCl, 4.73 mmol/liter KCl, 1.2
mmol/liter MgSO4, 0.025 mmol/liter EDTA, 1.2
mmol/liter KH2PO4, 2.5
mmol/liter CaCl2, and 11 mmol/liter glucose and
was buffered with 25 mmol/liter
NaH2CO3; the pH, when
bubbled with 95%O2-5%
CO2, was 7.4 at 37 C. The endothelium was removed
by gently rubbing the ring between thumb and forefinger. The efficiency
of endothelium removal using this method was confirmed by demonstrating
the loss of an endothelium-dependent relaxation to acetylcholine.
Endothelium removal did not significantly affect the amount of force
generated in response to phenylephrine (PE) administration (data not
shown).
Aorta force measurements
Aortic rings were threaded with two triangular 100-µm
stainless steel wires; each completed mounting formed a double
triangle. The aorta and holder were then mounted on a hook that was
attached to a Harvard Apparatus Differential Capacitor Force
Transducer. Resting tension on each aorta was set at 30 mN, to
approximate an in vivo aortic pressure of approximately 100
mm Hg, and this passive tension was maintained throughout the
experiment. The effects of pharmacological agents were determined. Data
were obtained using MP100W hardware and were analyzed using
AcqKnowledge software (Biopac, Goleta, CA). Isometric force is
expressed in terms of the vessel wall cross-sectional area normalized
(perpendicular) to the direction of force generation. Our estimate of
this area (CSA) is based on vessel geometry and wet weight [CSA =
2 x wet weight/(circumference)]. As the widths of the aortic
rings were cut to the same size, differences in area are related to
those in vessel wall thickness. Our geometric estimate showed a 21%
greater thickness in the SMP8-IGF-I transgenic mouse aorta. This is
identical to the increase in medium thickness based on morphometry
reported earlier (11).
Bladder preparation and bladder force measurements
The urinary bladder was isolated and dissected under a
microscope at room temperature in PSS. The bladder was cleaned of
connective tissues and bisected along its medial axis. The mucosa was
removed by gentle rubbing, and loops of 7/0 nonabsorbable surgical
suture were tied at each end of the half-bladder to facilitate mounting
between two hooks of apparatus for isometric force measurement. The
tissue length was increased until a resting tension of 15 mN was
maintained. The bladder was challenged with 80 mM KCl for
two contraction/relaxation cycles or until reproducible forces were
maintained. KCl (10–80 mM) and carbachol
(10-9–10-5
M) concentration-response curves were obtained in a
cumulative fashion.
Noninvasive blood pressure measurements.
Noninvasive blood pressure measurements were determined as
described by Krege et al. (32). After a 5-day
training period, daily blood pressure measurements were performed in
conscious mice over a 5-day period using a computerized tail-cuff
system (Visitech Systems, Apex, NC). Animals were placed in a Lucite
restrainer with their tails protruding from a small opening in the
back. A balloon cuff was placed over the proximal portion of the tail,
and a more distal portion of the tail was draped over a photoelectric
sensor for detecting blood flow. For 10 consecutive cycles, the balloon
cuff was inflated by a small air pump until detectable blood flow in
the tail ceased; this pressure was taken as the end point. The cuff was
then immediately deflated, and the next cycle was started 10 sec later.
In each trial, the 10 measurement cycles were preceded by 10
preliminary cycles to acclimatize the mice to the apparatus.
RT-PCR cloning of the SM myosin heavy chain (SMHC) isoform cDNA
fragments
SMHCs, the motor proteins that power SM contraction, are
produced by alternative splicing from a single gene. A 39-nucleotide
insertion in the 3'-end results in SMHC-2, which differs from SMHC-1 in
the structure of the C-terminus. There is also a 21-nucleotide
insertion in the 5'-head region, giving rise to isoform B, which has 7
additional amino acids compared with isoform A. Both SMHC-1 and -2 have
A and B variants, thus resulting in the 4 isoforms. To distinguish SMHC
isoforms 1 from 2 or A from B, we used RT-PCR to amplify and clone
fragments of SMHC isoforms 2 and B from mouse stomach RNA using primers
complementary to published mouse myosin heavy chain gene sequences from
GenBank. PCR amplification was conducted on about 50 ng stomach cDNA
with 50 pmol of each primer, 0.2 mM of each deoxy-NTP, 1.5
mM MgCl2, and 5 U Taq DNA
polymerase. After an initial polymerase activation step (95 C for 10
min), samples were amplified through 35 cycles at 95 C for 1 min, 46 C
for 1 min, and 72 C for 1 min. Sequences of primers used were as
follows: 5'SMHC-2, ACCAAGTCGCTGAAGC; 3'SMHC-2, AATGAAGCCTCGTTTCC;
5'SMHC-B, AAGGGCAAGAAGAGG; and 3'SMHC-B, TGCCTGTAGAAGCTG. After PCR
amplification, the predicted 326-bp SMHC-2 cDNA and 258-bp SMHC-B cDNA
fragments were purified, ligated into a T-vector made from pBluescript
KS(-) by cutting the plasmid with EcoR V, and then tagging
a T residue with Taq DNA polymerase. The identity of the
cloned fragments was confirmed by sequencing.
Ribonuclease (RNase) protection assay (RPA)
Total RNA was extracted from adult SMP8-IGF-I or SMP8-IGFBP-4
and age/sex-matched wild-type mouse tissues using a single step acid
guanidinium thiocyanate-phenol-chloroform extraction method. RPA was
performed using the RPA II kit (Ambion, Inc.), following
the manufacturer’s instructions. Briefly, RNA (10 µg/sample) was
hybridized to [32P]complementary RNA (cRNA)
made by transcribing antisense RNA with T7 RNA polymerase for clone
SMHC-B or with T3 RNA polymerase for clone SMHC-2. An 80-base 18S cRNA
probe made from plasmid pT7 18S (Ambion, Inc.) was used as
a control. As 18S RNA abundance is in great excess compared with SMCH
mRNA, we generated 18S cRNA probe with much lower specific activity by
decreasing the [32P]UTP/cold UTP ratio in the
in vitro transcription reaction. In addition, we synthesized
unlabeled 18S cRNA, an aliquot of which was added to each sample.
Hybridization was performed at 42 C overnight. After RNase A/T1
digestion, the protected mRNAs were precipitated and analyzed by 5%
polyacrylamide/urea denaturing gel electrophoresis. A
-32P-labeled 123-bp DNA ladder was used for
size reference. The gel was then dried and autoradiographed. The
density of the protected bands and of 18S RNA in the gel was
quantitated by phosphorimaging.
Northern blot analysis
Northern blots were performed as previously described
(33). Briefly, 10 µg tissue total RNA were
gel-separated, transferred to a nylon membrane, and then hybridized
with [-32P]ATP end-labeled mouse SM
-actin oligonucleotide primers. For standardization, blots were
rehybridized with human 18S cDNA. Quantitation was performed by
phosphorimaging.
Statistics
Data from concentration-isometric force curves were compared
using two-way ANOVA. Student’s t test was used for isoforms
comparison. Significance was defined as P < 0.05 for
all tests.
Chemicals
Phenylephrine, potassium chloride, and
n--nitro-L-arginine (L-NNA) were obtained
from Sigma (St. Louis, MO).\.
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Results |
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, A and B). Additionally, IGF-I
overexpression was associated with an enhanced sensitivity of aortas to
KCl, whereas sensitivity to PE was unaffected (Fig. 1, C and D). As we
normalized force to area, and the number of cells per area was constant
(11), the increase in force observed in aorta from the
SMP8-IGF-I mice indicates that these changes took place at the cellular
level. SMP8-IGF-I bladders, on the other hand, generated approximately
the same amount of force as nontransgenic age- and sex-matched controls
(Fig. 2A). When the amount of force
generated by the bladders was normalized to cross-sectional area,
SMP8-IGF-I bladders generated significantly less force than their
nontransgenic counterparts (Fig. 2B).
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, A and
B). Sensitivity to contractile agents was unaffected by IGFBP-4
overexpression (Fig. 3, C and D).
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), L-NNA pretreatment
increased isometric force development in both WT and SMP8-IGF-I mice
aortas. For PE stimulation (Fig. 4B), the effects of L-NNA
pretreatment on force were smaller than that for KCl and were not
significant. Importantly, in both conditions L-NNA did not
reduce the difference in force between the WT and SMP8-IGF-I
mice.
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Effects of IGF-I overexpression on SM contractile proteins
IGFs have been shown to have effects on the differentiation
program of skeletal myocytes and also impact on differentiation of SMC
in vitro (34). A possible explanation for the
increased vascular contractility in SMP8-IGF-I mice is that chronic
paracrine expression of the growth factor may alter the relative
abundance of critical contractile proteins. As SMHC is the major
component of the contractile apparatus, and the abundance and type of
isoform have been shown to be important in determining shortening
velocity and Ca2+ sensitivity of the myofiber
(35, 36), the effect of IGF-I overexpression on SMHC
isoforms expression in SMC was determined by RPA. There are four
isoforms of SMHC that are produced by alternative splicing of a single
gene. A 39-nucleotide insertion in the 3'-end results in SM-2, which
differs from SM-1 in the structure of the C-terminus. The isoforms
generated from a 7-amino acid insertion in the head of the protein are
termed A and B. Both SM1 and 2 have A and B variants, thus resulting in
the four possible isoforms. The abundance of isoforms 1 and 2 were both
increased significantly in aorta of 10-week-old SMP8-IGF-I transgenic
mice, but there were no significant changes in bladder or uterus (Fig. 5A; data not shown). The ratio of SM1 to
SM2 mRNA in aorta was decreased significantly in SMP8-IGF-I mice (Fig. 5A). Consistent with this finding, the SM1/SM2 protein ratio was also
significantly decreased in aorta of transgenic mice, as determined by
silver staining of 5% SDS-PAGE of aortic tissue extracts (data not
shown). The abundance of SMHC mRNA isoforms A and B did not
significantly differ in aorta, bladder, or uterus; however, the ratio
of SM-A to SM-B was significantly higher in SMP8-IGF-I aorta (Fig. 5B).
As expected, overexpression of IGFBP-4 in SMC had reciprocal effects on
SMHC isoform abundance and ratios in aortas from SMP8-IGFBP4 mice (Fig. 6). The abundance of isoforms 1 and 2 was
decreased, and the ratio of SM-A to SM-B was significantly lower in
SMP8-IGFBP-4 aortas compared with WT controls. Consistent with the
evidence supporting a role for IGF-I in the regulation of contractile
protein gene expression, there was a significant increase in SM
-actin mRNA levels in aorta in transgenic mice compared with
nontransgenic controls (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGF-I exists as both a circulating hormone and an autocrine/paracrine factor, where it acts discretely to modulate local tissue responses. Strong support for the critical importance of the latter mode of action of IGF-I is provided by a study using the Cre/loxP recombination system to selectively delete the IGF-I gene from the liver, the major source of circulating or endocrine IGF-I (38). Deletion of the IGF-I gene in hepatocytes was associated with a profound decrease in the plasma IGF-I concentration without affecting IGF-I mRNA levels in peripheral tissues. These mice exhibited no obvious phenotypic abnormalities, suggesting that autocrine/paracrine IGF-I can support normal growth and development. The present study uses an in vivo SMP8-IGF-I overexpression model to determine the effects of manipulating IGF-I levels within the artery wall and explores the consequences of these prolonged elevations in IGF-I on SM contractility. Further rationale for the relevance of this model is provided by the fact that arterial IGF-I concentrations are subject to regulation in various physiological or pathological conditions: i.e. cyclic stretch (39), balloon angioplasty (4, 5, 29), and arterial hypertension (27).
The results clearly establish that sustained elevations of IGF-I levels in SM have profound tissue-specific effects on contractility and that these differ from acute effects of IGF-I in vivo and in vitro. In contrast to acute IGF-I exposure, our results demonstrate that prolonged elevation of IGF-I levels can result in greater force production in aorta. This increase was not due to IGF-I-mediated effects on aortic SMC mass, as differences remained after normalization to crosssectional area. Likewise, analysis of contractility in IGFBP-4-overexpressing mice revealed a reciprocal depression in force of contraction.
It is of interest to compare these chronic effects of IGF-I expression to the acute response to the growth factor. As reported by Sowers and colleagues (21), rat tail arteries pretreated with IGF-I either in vivo or ex vivo demonstrated decreased tension development in response to PE and KCl. We investigated the acute effects of IGF-I treatment by constructing cumulative concentration-force responses and repeating these measurements after preincubation with IGF-I (10-8–10-7 M) in aortas from SMP8-IGF-I and nontransgenic mice. In our studies, ex vivo IGF-I treatment (10-7 M) had no effect on KCl concentration-force relations for either endothelium-intact or denuded aortas (data not shown). This was also true for PE concentration-force relations (data not shown). This discrepancy in results may be attributable to species differences in the response of mouse vessels to IGF-I pretreatment or differences in the vessel examined (i.e. tail artery vs. aorta).
Numerous reports have implicated the NO pathway as an effector system modulated by IGF-I (21, 40). To test the hypothesis that this pathway is down-regulated in mice with chronically elevated levels of IGF-I, we eliminated the contribution of NO. Pretreatment with the NO synthase inhibitor L-NNA enhances the aortic contractility of wild-type and SMP8-IGF-I mice to a similar degree. We conclude that the enhanced contractility is not attributable to a reduction in NO production, although NO may contribute to enhanced sensitivity.
Although IGF-I has been demonstrated to play an important role in skeletal muscle differentiation (41), relatively little is known about how IGF-I may impact on the expression of SM-specific gene products. As opposed to striated myocytes, SMC are not terminally differentiated and retain the potential to replicate postnatally. Once placed in culture, vascular SMC irreversibly lose the contractile phenotype and are therefore of limited value to explore differentiated function. However, IGF-I has been demonstrated to maintain the differentiated phenotype of SMC cells in a gizzard primary culture system through the phosphoinositide 3-kinase pathway (34), supporting a role for IGF-I in SMC differentiation. The low body weight and the decreased survival rate in the IGF-I knockout mice make it difficult to specifically explore SMC function and properties in these animals (42). The SMP8-IGF-I transgenic mice are the first example of overexpression of a functional protein in SMC cells in vivo and therefore enable the examination of the putative effects of this growth factor on SMC cell differentiation in the intact animal. The fact that paracrine overexpression of IGF-I results in opposite effects on contractility from those seen after acute exposure may relate to the site of action of the growth factor. It is reasonable to propose that systemic administration of IGF-I is vasorelaxant through effects on endothelium-derived NO, whereas in the transgenic model IGF-I is acting directly on SMC by increasing myofibrillar protein gene expression, leading to a greater number of contractile units per cell.
To test the hypothesis that the increase in SMC contractility may be due to effects of IGF-I on contractile protein gene expression, we focused in particular on the relative abundance of the two C-terminal isoforms of SMHC, SM1 (204 kDa) and SM2 (200 kDa), that are generated by differential splicing of the same gene. There is an important precedent of hormonal regulation of these splice variants. The relative abundance of SM1 increases in uterine muscle of ovariectomized rats after estrogen administration, as do isometric force and maximum shortening velocity (43). Recent evidence indicates that the SM1 isoform may also differ from SM2 in its mechanical properties. Interestingly, IGF-I has been postulated to function as a mediator of estrogen action (estromedin) in the uterus (8, 9, 44), and it is tempting to consider that this may also apply to the regulation of myometrial contractility and perhaps of contractile function of other SMC cell tissue beds.
Modification of contractile protein gene expression in the vasculature of SMP8-IGF-I mice may explain in part their greater response to contractile stimuli. Putative paracrine effects of IGF-I on relative expression of SMHC isoforms have an added interest. SM1 is believed to be the fetal isoform, whereas SM2 appears in adult tissue. After vascular injury, SMC revert to expression of SM1 only, thus assuming the embryonal phenotype (45). Whether IGF-I may also elicit this phenotypic switch is unclear. Analysis of aortic SM1 and SM2 isoforms revealed that increased IGF-I levels causes a significant up-regulation of both SM1 and SM2 and an overall decrease in the SM1/SM2 ratio. Thus, although IGF-I levels increase in the vessel wall after arterial injury, it is unlikely that local expression of this growth factor is sufficient to account for the switch to the fetal SM myosin isoform. However, the presence of more total myosin may contribute to enhanced force development, and the decreased SM1/SM2 ratio may account for the sensitivity change. Bladder contractility was largely unaffected by IGF-I, and levels of SM1 and SM2 were also unchanged. Finally, local expression of IGF-I also appeared to alter the splicing efficiency, leading to generation of SM-A and SM-B in the aorta. These isoforms differ according to the presence or absence of a seven-amino acid insertion in the head of the protein. This has been postulated to have important functional consequences, as it alters Mg2+-adenosine triphosphatase activity and contractile velocity (36).
In summary, chronic overexpression of IGF-I within the vessel wall is associated with increased arterial contractility. These effects differ from those seen after acute exposure to the growth factor and may relate at least in part to IGF-mediated changes in expression and relative isoform abundance of critical contractile proteins. Thus, IGF-I may participate in the control of differentiated functions of SMC, as previously shown for other myocyte lineages.
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Footnotes |
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Received April 7, 2000.
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-actin-IGFBP-4 fusion gene induces smooth muscle hypoplasia.
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) aortas.
Denuded aortas from 10-week-old WT and SMP8-IGF-I mice were
isometrically mounted, and concentration-force curves were generated to
KCl (10–50 mM) and PE (1 nM to 10
µM). SMP8-IGF-I mice produced significantly more force in
response to both KCl (A) and PE (B). When force was expressed as a
percentage of the maximum force, an increased sensitivity to
KCl-mediated (C), but not PE-mediated (D), contractions was
observed. Data are the mean ± SEM (n = 5).
