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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 58:B1074-B1077 (2003)
© 2003 The Gerontological Society of America

Age-Dependent Decline of In Vitro Migration (Basal and Stimulated by IGF-1 or Insulin) of Human Vascular Smooth Muscle Cells

Antonio Ruiz-Torres1, Rosario Lozano1, José Melón2 and Raffaele Carraro1,3

1 University Research Institute of Ageing
2 Department of Cardiovascular Surgery
3 Department of Endocrinology, Hospital de la Princesa, Madrid, Spain.


   Abstract
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 Abstract
Methods
 Results
 Discussion
 References
 
Since biological aging causes a decrease in functions such as cell proliferation, we have studied the possible effect of age on the migration capacity of human vascular smooth muscle cells (SMCs). To this aim, the migration activity of cultured SMCs from arteries of male human donors ranging in age from 43–77 years was determined in a Boyden chamber, under basal conditions and after insulin-like growth factor-1 (IGF-1) or insulin stimulation. Migration activity decreased with donor age (r2 = 87%, 85%, and 78%, respectively). IGF-1 and insulin significantly reduced the age-dependent relationship observed in basal conditions, so that, comparing young with old, both IGF-1 and insulin stimulated SMC migration similarly, although the effect of age remained in absolute terms. In this article, we conclude that the age-dependent decline of migration activity—similar to what has already been shown for SMC proliferation—may be part of the biological ageing phenotype, which is not overcome by hormone stimulation.

DURING adulthood, vascular smooth muscle cells (SMCs) are in a postmitotic state in vivo. Therefore, in order to exhibit mitotic and migratory activities, these cells must change their "contractile" phenotype into a more undifferentiated "synthetic" one. This dedifferentiation process plays a basic role in atherogenesis, since SMC migration through the endothelium and eventually secretion of collagen are important contributions to atherom building (1). Independent of the cause that could activate the chain of events leading to atherom (2), it seems important to gather information on the role played by ageing itself to such phenotypic changes. Indeed, such a role could be a relevant one, keeping in mind that atherosclerosis could be considered as a normal manifestation of the ageing process, although such an assumption is taken from pathological studies, which suggest—but do not prove—a relationship between atherosclerosis and ageing. To date, no evidence exists that the process of atherom building is accelerated in advanced age. In fact, cell culture observations indicate that cell age and organism age are closely related (3). Therefore, in old age, senescent cells, which are not able to proliferate or migrate, should be more prevalent.

This article focuses on the issue of the relationship between atherosclerosis and biological ageing. To this aim, we have studied in vitro the migration capability of human vascular SMCs from donors of different ages, at basal conditions and after stimulation by insulin-like growth factor (IGF-1) or insulin. The former is a potent growth factor, but secretion drastically decreases with ageing (4). However, the opposite happens with insulin, whose mitogenic effect and atherogeneity are well known, which shows an inverse relationship with IGF-1 (5).


   METHODS
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 Abstract
 Methods
Results
 Discussion
 References
 
Chemical and Materials
Human recombinant insulin, IGF-1, and collagenase type II were purchased from Sigma Chemical Co. (St. Louis, MO), filters with an 8 µm pore size from Millipore (Marlborough, MA), fetal calf serum from Gibco (Gaithersburg, MD), and Dulbecco's modified Eagle medium (D-MEM) from Gibco Br. (Scotland). Boyden's chamber was provided by the technical department of the Autonoma University of Madrid.

Preparation and Culture of SMCs
Femoral and tibial artery fragments from the healthy proximal zone of amputated legs of 5 male individuals from 43 to 77 years of age, affected by foot gangrene and showing comparable metabolic states, were cut into small pieces for subsequent dispersion of SMCs with collagenase II (0.4%, 37°C, 120 minutes in 50 mmol/l HEPES buffer with 0.1% BSA). The dispersed cells were identified as SMCs by immunological detection of smooth muscle alpha-actin, according to Skalli and colleagues (6). After two washings and suspension of sediments in 5 ml of D-MEM with 10% FBS (fetal bovine serum) on a petri dish for 20 minutes, the cells were transferred with 15 ml of medium into 75 cm2 flasks where the culture was continued up to confluence. The first culture passage was started by seeding a new flask with half of the original amount of cells. In samples of SMC cultures from donors of different ages, beta-galactosidase staining at pH 6 (senescence associated) was performed to determine the proportion of senescent cells (7).

Cell mobility studies were performed with SMCs from the second to fifth culture passage. A final volume of 0.3 ml containing 70,000 cells in D-MEM was added into the upper compartment of a cell migration chamber as described by Boyden (8). PVPF filter with 8 µm pores separated the upper from the lower compartment, which contained PBS. Three hours later, the filter was fixed with 50% methanol for 5 minutes and with 75% ethanol for an additional 5 minutes. After washing with distilled water for 2 minutes, the cells that had migrated to the lower side of the filter were stained for 24 hours with hematoxylin and counted under a light microscope. The number of cells that migrated spontaneously was referred to as basal migration in contrast to stimulated migration obtained after addition of insulin or IGF-1 (1 nM each) into the lower chamber compartment. At least three migration experiments per donor for each design were performed. The effector concentration was the lowest most-effective concentration according to previous dose-dependent chemotaxis experiments using dispersed cells from human arteries (9). Considering the receptor number for IGF-1 and insulin found in these cells, the concentration used corresponds to that of a clear saturation for the IGF-1 receptor but not for the insulin receptor, as we have already published (10).

The statistical analysis of the results was performed using a simple linear regression model, taking the age of the donor as the independent variable and the number of migrating cells as the dependent one. The correlation coefficient (r), as well as the r2 and p levels of regression, were obtained from pooled results of all migration experiments performed for each donor. The statistical significance between two regression lines was calculated on the basis of the confidence intervals according to Armitage and Berry (11). Statistical analysis was performed by using the SPSS (version 10.0) computer program (SPSS, Inc., Chicago, IL).


   RESULTS
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 Abstract
 Methods
 Results
Discussion
 References
 
As Figure 1 shows, the basal migration activity of human SMCs decreases with increasing donor age according to a linear regression curve that crosses the x-axis at the age of 115 years, where, theoretically, no migration should be expected.



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  		Figure 1. Linear regression of migration activity of smooth muscle cells under basal conditions (without hormone stimulation) with donor age. The arrow shows the intercept with the x-axis. Each point is one migration experiment (bottom: n = total number per donor). Statistical analysis of the regression: r = 0.93, r2 = 87.3%, p <.001

 
When comparing the effect of insulin and IGF-1 on SMC migration activity in a younger (43-year-old) adult with that in a 77-year-old individual (Figure 2), a similar proportional increment above basal was seen at both ages. Nevertheless, there is an absolute reduction of the migratory capability both in basal conditions and after hormonal addition in SMCs from the older individual.



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  		Figure 2. Mean and standard deviation of migration activity of smooth muscle cells from a 43-year-old donor and a 77-year-old donor to show that, compared with the corresponding basal value, the stimulation by insulin or insulin-like growth factor-1 (IGF-1) is relatively similar independent of age, but absolute basal or stimulated amounts are clearly reduced in old age (p <.001). The induction in cells from both young and old donors is statistically significant (p <.05), but there are no differences between the effectors at each age. There is also a difference between the migration of cells from a young donor under basal conditions and that of insulin or IGF-1 induced in old donor cells (p <.05 and p <.006, respectively)

 
The effect of either insulin or IGF-1 addition to SMCs from donors of different ages results in similar relative increments, as shown in Figure 1, whereas the absolute value of migration decreases as donor age advances (Table 1). Comparing the mathematical functions, it may be seen that, when adding hormones, the slope becomes shallower than that under basal conditions, moving the x-axis crossing point into older ages. Moreover, the regression curves of insulin- or IGF-1-stimulated migration are statistically different from that of basal migration (p <.0001), but not different from each other.


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  		Table 1. Data From Regression Analysis of Relationship Between Vascular SMC Migration Rate Under Basal Conditions and Hormone Application and Age of Donor: Equations and Coefficients as Well as X-Axis Crossing Points of Extrapolated Curve.

 

   DISCUSSION
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 Abstract
 Methods
 Results
 Discussion
References
 
Although the results of the present study are based on a limited number of donors, three arguments support the reliability of our findings, besides methodological standardization. First, a satisfactory dispersion of age was present within a range of more than 30 years in adulthood. Second, health and metabolic status of the donors were comparable, including the local conditions of the artery from which the cells were taken (i.e., it was always the proximal healthy zone of the amputated leg that showed no wall alteration). Finally, as may be seen, the results showed little variance (both intra- and interexperimentally), which, together with clear significance in statistical analysis, were in logical agreement with the mentioned standardized conditions. All regression curves including the differences between slopes were statistically significant. A similar pattern was observed when comparing the differences between ages of the mean results obtained under basal or stimulated conditions.

The results presented here are in agreement with previous findings on proliferation activity (12). Nevertheless, contradictory results in SMCs from rat aorta have been reported when comparing young with old animals (13). Probably, species-specific differences and the use of only two different ages (6-month-old rats vs. 30-month-old rats) may explain the disagreement with our results. Furthermore, the agreement in our migration and published proliferation data is such that the linear regressions of these activities as a function of donor age show a parallel decay [slope (% of intercept): -0.84 vs. -0.80], resulting in very close x-axis crossing points at donor ages 117 and 115 years, respectively.

As the SMCs used were from a similar culture passage, the observed functional decline seems to be associated with in vivo ageing rather than to changes due to longer culture periods. This decline could be due to either an age-dependent reduction of metabolism or an increase in the number of senescent cells (14). Both processes would be consequences of in vivo cell ageing. Furthermore, it is known that senescence reduces the migration capacity of fibroblasts in vitro or in vivo (15,16), and their epidermal growth factor (EGF) responsiveness (17). In this regard, it seems of interest to note that, under comparable culture conditions [subconfluent, from similar (8th–9th) passage], the percentage of beta-galactosidase-related senescent SMCs in our samples increases with the age of the donor. The calculated slope indicates an increase of 1.7%–2.1% of positive cells per year of adult age. Nevertheless there is a great intraindividual variation (30% of mean value), so that comparing the results from donors with an age range difference of 12 years, there is no statistical significance [age 62 years, 33.3% (± 22.8) vs.74 years, 52% (± 3.3)]. This age difference is probably too slight, taking into account that the great variance is possibly due to the many factors in vitro that may influence beta-galactosidase activity (18). This screening test was later performed by us to support the above-mentioned hypothesis on the role played by senescent cells in the declining migration of SMCs. It is known that beta-galactosidase, at pH 6, is a marker of senescent cells (7), probably due to the increased lysosomal content from ageing (19), which becomes detectable in several cell types, especially in epithelial cells from different organs (20–23). Currently, there are few studies performed in SMCs from rabbits, but not from humans, where this staining method has been applied for in vitro evaluation (24). In summary, it seems that there is a relationship between beta-galactosidase-positive cells and donor age so that the donor age-dependent decrease of migration activity, as shown, may be due to the increasing numbers of senescent cells.

The donor age-dependent decline of migration capability involves both basal and insulin- or IGF-1-stimulated activity. The stimulation by either hormone is relatively similarly in SMCs from young and old donors, but the age-dependent decrease of the absolute value is not reversible. This could be related to the ageing process in vivo, whose irreversible characteristic is very well known. As mentioned above, the x-axis crossing point of basal condition experiments is very near to that calculated from our published proliferation studies, but both insulin and more so IGF-1, modify the migration decline, so that the intersection of the regression line with the x-axis, which points out where no migration should exist, occurs at later ages than under basal conditions. This shift could be interpreted as expression of an enhancement of the in vitro life potential of SMC functionality, which may be due to the fact that both insulin and IGF-1 are able to inhibit apoptosis (25).

Many questions remain unanswered. The simple extrapolation of our in vitro results to in vivo conditions such as atherogenesis may be misleading. Nevertheless, if any parallelism should exist, it could mean that, contrary to what is expected, SMC migratory activity in the artery wall is reduced in advanced age. In any case, many factors participating in the development of atheromatosis, such as those following vascular injury, interleukin-3 (26), chemoattractant protein-1 (27), or receptor-tyrosine kinase (28), interfering with migration, might minimize the hypothetic "protective" effect of ageing, turning old cells less sensitive to specific inhibitors from a contractile to a synthetic phenotype (29). Another issue that should be considered with caution is the meaning of the similarity of the insulin effect with that of IGF-1 on in vitro migration in relation to in vivo processes such as forming the neointima and upregulating tropoelastin synthesis through the functional cooperation of synthetic SMCs as has been described for IGFs (30). The last unanswered question is whether the in vitro effects of insulin and IGF-1 on migration and probably also on proliferation may also modulate cell life potential.

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   Acknowledgments
 
This research has been supported by a grant from the Fondo de Investigaciones Sanitarias (98/0177), Spanish Ministry of Health.

Address correspondence to Prof. Dr. Antonio Ruiz-Torres, Instituto Universitario de Investigación Gerontológica y Metabólica, Diego de León 62, 28006 Madrid, Spain. E-mail: aruto{at}arrakis.es

Received December 9, 2002

Accepted August 15, 2003


   References
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 Methods
 Results
 Discussion
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