P. Marlene Absher

Genealogies of clones of diploid fibroblasts: Cinemicrophotographic observations of cell division patterns in relation to population age☆

Direct assessment of cell division patterns of human diploid fibroblasts (WI-38) has been achieved using techniques of time-lapse cinemicrophotography. Pedigrees of progeny cells in clones from middle and late passage WI-38 cultures are presented. The passage 28 clone exhibited a division pattern that was highly synchronous through four generations and had an average interdivision time that increased linearly from the second through sixth generations. Division was essentially logarithmic through the fourth generation. The passage 53 clone was much more variable in interdivision time, less synchronous, and less motile than the passage 28 clone. While there is considerable overlap in the interdivision times of cells of the passage 28 and 53 clones, the late passage cells generally exhibit more variation in interdivision times. The average population doubling time was 16.8 h in thepassage 28 clone and 32.0 h in the passage 53 clone.

Clonal variation and aging of diploid fibroblasts: Cinematographic studies of cell pedigrees☆

Abstract Time-lapse cinematographic (TLC) analysis of clones of human diploid fibroblasts indicate heterogeneity in clonal division behaviour. Variations are noted in interdivision time, clone size and generations per clone. Correlation coefficients for interdivision times of sister pairs are high in young clones and generally low in aged clones. A consistent division pattern at all population doubling levels is one of low average interdivision time for early and late generations of a clone and high average interdivision time for the middle range of generations of a clone. The clonal division patterns observed experimentally have been duplicated in computer simulated pedigrees. The computer model is based on an oscillating system which allows for flux of regulator substances. The critical concentrations of regulator substances determine the clonal division pattern for a given progenitor cell.

Changes in Arterial Expression of Fibrinolytic System Proteins in Atherogenesis

Plasminogen activators (PAs) and their inhibitor, plasminogen activator inhibitor type-1 (PAI-1), have been implicated in modulation of luminal fibrinolysis and mural proteolysis contributing to atherogenesis. Expression of PAs/PAI-1 (normalized to extracted tissue protein) was delineated by assays of conditioned media and of extracts from walls of human arterial segments in culture. Arterial specimens (n = 39 from 26 subjects) were divided into four groups: normal (n = 14), fatty streak (n = 6), moderate atherosclerosis (mural thickening with < 70% lumen obstruction, n = 5), and severe atherosclerosis (mural thickening with > 70% lumen obstruction, n = 14). Paired samples from the same individual comprising a normal arterial segment and an atherosclerotic segment were evaluated also. A fourfold molar excess in PAI-1:t-PA was seen in conditioned media from samples with any evidence of atherosclerosis compared with normal specimens (normal 21 +/- 4, diseased 82 +/- 21, P < or = .05). Compared with normal pairs, the tissue content of PAI-1 (ng) was increased in fatty streak lesions (n = 3, normal 35 +/- 12, fatty streak 50 +/- 8, P < or = .05); stable to decreased in moderate atherosclerosis (n = 3, normal 34 +/- 3, moderate 22 +/- 7, P = .16); and increased in severe atherosclerosis (n = 6, normal 48 +/- 9, severe 85 +/- 19, P < or = .05). The tissue content of PAs (ng), though not increased in fatty streak lesions, was elevated in moderately and severely atherosclerotic segments (normal 0.7 +/- 0.2, moderate 1.6 +/- 0.1; normal 0.8 +/- 0.3, severe 2.1 +/- 0.3, P < or = .05 for each comparison). Atherogenesis is associated with decreased luminal fibrinolytic capacity that may exacerbate thrombosis. Decreased mural proteolysis in early atherogenesis may exacerbate matrix accumulation. Increased mural proteolysis later is associated with, and may potentiate, smooth muscle cell migration and proliferation.

Dependence of Augmentation of Arterial Endothelial Cell Expression of Plasminogen Activator Inhibitor Type 1 by Insulin on Soluble Factors Released From Vascular Smooth Muscle Cells

BACKGROUND Insulin-resistant states are characterized by accelerated atherosclerosis and are associated with increased plasma concentrations of insulin and plasminogen activator inhibitor type 1 (PAI-1). To determine whether arterial expression of PAI-1 in response to insulin contributes to the increased PAI-1 observed, human and porcine arteries in culture were exposed to insulin, and results were compared with responses of specific arterial cellular constituents maintained in culture and coculture. METHODS AND RESULTS Human and porcine arterial segments and cells obtained from arteries were maintained in culture. Insulin increased accumulation of PAI-1 in conditioned medium from arterial segments (ng PAI-1 [1 nmol/L insulin minus control]: human arteries 47+/-17, porcine arteries 3.1+/-1.2, P<.05 for each) and from endothelial cells (ECs) cocultured with smooth muscle cells (SMCs, ng PAI-1 [1 nmol/L insulin minus control]: human cells 43+/-8, porcine cells 0.5+/-0.1, P<.05 for each). Insulin had no effect on EC expression of PAI-1 when not cocultured with SMCs. Increased accumulation of PAI-1 was seen when ECs, in coculture chambers without SMCs, were cultured with medium previously conditioned by SMCs in the presence of insulin. The increased accumulation of PAI-1 in conditioned medium was secondary to both an increased transport of PAI-1 from the basal to the apical surface of ECs as well as an increased production of PAI-1 by ECs. CONCLUSIONS Insulin augments arterial expression of PAI-1 by stimulating release of a soluble factor(s) from SMCs. Accordingly, increased arterial elaboration of PAI-1 in response to insulin is likely to account, in part, for the elevated PAI-1 observed in the blood of subjects with insulin-resistant states.

Increased proliferation of explanted vascular smooth muscle cells: a marker presaging atherogenesis

The JCR:LA-cp homozygous cp/cp corpulent rat is genetically predisposed to develop atherosclerosis evident after 9 and 18 months of age in males and females and to manifest metabolic derangements resembling those seen in type II diabetes in humans (hyperinsulinemia, insulin resistance, hyperglycemia and hypertriglyceridemia). The present study was undertaken to determine whether vascular smooth muscle cells (SMCs) explanted from vessels destined to become atherosclerotic later in life exhibit intrinsic properties ex vivo that presage atherogenesis to provide a means for evaluating promptly intervention designed to modify it. SMCs were cultured from aortic explants of JCR:LA-cp corpulent (cp/cp) and lean control (+/+) rats of 4, 5, 6, and 9 months of age. Compared with SMCs from controls, SMCs from cp/cp rats exhibited increased proliferation, higher saturation density, increased augmentation of proliferation in response to selected mitogens and greater adherence to extracellular matrix proteins. The increased proliferative activity ex vivo anteceded by several months the development of atherosclerotic lesions in vivo. Thus, it is a promising marker in assessments of the efficacy of interventions designed to retard or prevent atherosclerosis.

The retardation of vasculopathy induced by attenuation of insulin resistance in the corpulent JCR:LA-cp rat is reflected by decreased vascular smooth muscle cell proliferation in vivo

Proliferation in vivo of vascular smooth muscle cells occurs early in the course of atherosclerosis. Cultured smooth muscle cells (SMCs) explanted from aortas of JCR:LA-cp corpulent rats known to exhibit metabolic derangements and insulin resistance typical of type II diabetes early in life and to develop atherosclerosis later in life exhibit increased proliferation compared with SMCs from lean, normal rats. Vascular smooth muscle proliferation in vitro was found to be positively and significantly correlated with plasma insulin levels in vivo. Proliferation of aortic SMCs from JCR:LA-cp cp/cp corpulent rats cultured in vitro exhibited increased proliferation in the presence of exogenous insulin. Exercise and diet, selected as interventions designed to ameliorate the insulin resistance and hyperinsulinemia in the JCR:LA-cp cp/cp rat, effectively lowered blood insulin levels and decreased subsequent proliferation in vitro of aortic SMCs explanted from these animals. The results indicate that assessment of proliferation of vascular smooth muscle cells ex vivo may provide insight into the presence and severity of atherogenicity in association with insulin resistance in diverse species under diverse circumstances. Accordingly, with appropriate controls, it may be possible to use SMC proliferation ex vivo as a marker of the extent to which an intervention such as administration of insulin sensitizers to experimental animals and human subjects results in a change in behavior of vessel wall elements potentially indicative of amelioration of atherogenicity and detectable as judged from reduced proliferative rates of the cells ex vivo when they have been harvested from vessels exposed to a milieu in which insulin resistance has been attenuated.

PRODUCTION OF INTERFERON FOLLOWING AN IMMUNE RESPONSE

The r e l a t i o n s h i p between i n t e r f e r o n product ion and immune phenomena -i n viva h a s been l i t t l e s tud ied . 1965 t h a t pas s ive admin i s t r a t ion of an t ibod ie s t o Newcastle d i s e a s e v i r u s (NDV) i n h i b i t e d t h e i n t e r f e r o n product ion of mice chal lenged with the same v i r u s . i n humans who had had a c t i v e measles i n f e c t i o n and who had subsequent ly been challenged wi th a t t enua ted measles v i r u s a s a means of s t i m u l a t i n g i n t e r f e r o n . Borecky , Lackovic, and Waschke3 found t h a t a n t i v i r a l a n t i bodies suppressed t h e a b l l i t y of NDV v i r h s t o cause i n t e r f e r o n appearance, b u t i n c o n t r a s t immunization aga ins t endotoxin enhanced t h e a b i l i t y of mice so immunized t o produce i n t e r f e r o n . Youngner and S t i n e b r i n g l r epor t ed i n

High glucose concentrations induce oxidative damage to mitochondrial DNA in explanted vascular smooth muscle cells

Oxidative stress is considered to be one of the mechanisms leading to atherosclerosis. It occurs in response to injury or to altered metabolic state. Alterations in cell growth (proliferation or apoptosis) can also contribute to the pathogenesis of atherosclerosis and is influenced by oxidative stress. Smooth muscle cells (SMC) from aortic explants of JCR:LA-cp homozygous cp/cp corpulent rats who are genetically predisposed to develop atherosclerosis exhibit increased SMC proliferation, which can be attenuated by exercise and food restriction. This study was conducted to characterize the effects fo oxidative stress and high glucose media on cell growth and its relationship to mitochondrial DNA integrity and gene expression in explanted aortic SMC from corpulent and lean JCR:LA-cp rats. The results show that SMC from the cp/cp rat appear to be resistant to oxidant-induced cell death and that they accumulate mitochondrial DNA mutations, probably as a result of a reduction in apoptosis. These data suggest that susceptibility to age- and glucose-related atherosclerosis may be related to alterations in redox signaling.

Time-Lapse Cinemicrophotographic Studies of Cell Division Patterns of Human Diploid Fibroblasts (WI-38) during their in Vitro Lifespan

Genealogies of human diploid embryonic lung fibroblasts, WI-38 were prepare from analysis of filmed sequences of clones at passages 20, 28 and 53. The results indicate heterogeneity in cell division patterns, interdivision time and migration activity. The relationship of the cell division patterns to age of culture is difficult to assess at this time because of the heterogeneity of the clones, however, the late passage culture appeared to be more variable in terms of sister-sister, and mother-daughter relationships. The passage 28 culture was representative of a highly proliferating clone, exhibiting short interdivision times and a synchronous division pattern. The passage 20 and 53 clones exhibited longer interdivision times and less synchronous division pattern than the passage 28 clone. A gradual lengthening of average interdivision time with successive generations has been observed in all genealogies regardless of passage level of the donor culture. A portion of daughter cells in the fifth and sixth generation exhibited lower interdivision time than the mother cell. The effects of nutrients, space, and mitotic inhibitors or stimulators on interdivision time of the cells within the clones is discussed.

Comparison of the use of isotopic proline vs leucine to measure protein synthesis in cultured fibroblasts

Compartmentation of the amino acid precursor pools for protein synthesis in cultured cells can substantially complicate measurements of synthesis rates. This is particularly true for nonessential amino acids such as proline, an amino acid often used in isotopic form to measure collagen synthesis. We have made a comparative study of this problem in cultured IMR-90 fibroblasts using isotopic proline and leucine to measure total protein and collagen synthesis. 3H-leucine in the extracellular (EC) medium equilibrates with tRNA-leucine at an EC concentration of 0.4 mM in both dividing and stationary cells. Thus, under these experimental conditions there is no complicating compartmentation of leucine for protein synthesis. Equilibration of EC and tRNA-bound 3H-proline, however, does not occur even when the EC concentration is in the mM range, based upon simultaneous measurements of synthesis rates using 3H-proline and 3H-leucine together. Furthermore, significant changes in EC proline concentration and specific activity occur over short time intervals (2 hr) if the initial EC proline concentration is below 0.2 mM. Thus, the use of isotopic proline to measure protein synthesis introduces substantial interpretive problems. Serum deprivation causes changes in both total collagen synthesis and the percent of protein synthesis devoted to collagen when measured with either 14C-leucine or 3H-proline. At the same time, isotopic proline remains the better choice for measuring percent collagen synthesis.