Changes in biological characteristics during the cellular aging of ligament fibroblasts derived from patients with prolapsus uteri

Prolapsus uteri in pelvic support disorders are common in elderly women. The etiology is unclear and more likely to be multifactorial. We examined changes in biological characteristics and responsiveness to growth factors during the in vitro cellular aging of cardinal ligamental fibroblasts derived from patients with prolapsus uteri (HPLiF), and compared them with those of cells from age-matched control subjects (HCLiF). HPLiF and HCLiF had almost the same in vitro life span and the age-related patterns of biological parameters were essentially the same. However, the saturation density was significantly higher in HPLiF than in HCLiF. Furthermore, the high proliferative activity of HPLiF to serum mitogens, especially to platelet-derived growth factor, was retained throughout the in vitro life span. p53 protein levels in HPLiF increased at late passages, but were significantly less than in aged HCLiF. These results indicate that the higher proliferative activity in prolapsus fibroblasts may result from the decreased expression of p53 protein and may lead to a decrease in the synthesis and deposition of extracellular matrix components. These results support the hypothesis that functional alterations in ligament fibroblasts are involved in the mechanism of the development of prolapsus uteri.     

Tumor necrosis factor alpha and interleukin 1 alpha enhance lipopolysaccharide-mediated bovine endothelial cell injury.

Alveolar macrophages (AMs) are important in the host response to aerogenous pulmonary bacterial infections, such as Pasteurella haemolytica-induced pneumonia in cattle. Previous work has shown that AMs enhance P. haemolytica-mediated pulmonary endothelial cell (EC) damage in vitro. The purpose of this study was to determine the mechanism of AM-enhanced EC damage using an in vitro AM-EC coculture system consisting of AMs cultured on culture plate insert membranes and ECs in the underlying chamber. The addition of lipopolysaccharide (LPS) to the culture plate insert chamber resulted in EC damage indicated by 51Cr release, which was enhanced in the presence of AMs. To determine the role of AM-secreted cytokines, recombinant human interleukin 1 alpha (IL-1) or tumor necrosis factor alpha (TNF) was added to ECs simultaneously with varying concentrations of LPS. Although TNF and IL-1 alone had only marginal toxic effects on ECs, the simultaneous treatment of TNF or IL-1 with LPS greatly increased the LPS cytotoxic effect on ECs. In addition, IL-1 receptor antagonist eliminated the IL-1 enhancement of LPS-mediated EC toxicity. These results suggest that macrophage-secreted cytokines synergistically enhance LPS-mediated pulmonary EC damage.     

Cell-surface changes accompanying aging in human diploid fibroblasts: Effects of tissue,donor age and genotype

The generality of age-related changes in concanavalin A (Con A)-mediated red blood cell (RBC) adsorption to human diploid fibroblasts was investigated on fibroblast-like cells from fetal lung, heart, liver, skin and muscle tissues. All the cells examined showed the continuous increase from early passages in RBC adsorption with the RBC coating method (in which Con A-coated RBCs are adsorbed to fibroblasts) and the increase only at phase III with the fibroblast coating method (in which RBCs are adsorbed to Con A-coated fibroblasts). All of the four strains of lung fibroblasts gave nearly the same extent of the age-related change in RBC adsorption, when expressed as a function of percentage life span consumed, indicating that the change in RBC adsorption is independent of genetic heterogeneity and the conditions of primary culture. Liver and heart fibroblasts also gave results similar to those of lung fibroblasts. However, skin and muscle fibroblasts were lower in their RBC adsorption capacity throughout the life span. The continuous age-related increase in RBC adsorption to these cells could be sensitized by using glutaraldehyde-prefixed RBCs, trypsinized RBCs or phytohemagglutinin P in place of Con A. The relevance of the phenotype of in vitro aging revealed by RBC adsorption to in vivo aging was also demonstrated on skin fibroblasts from different ages of donors using glutaraldehyde-prefixed RBCs. In addition, fibroblasts from patients with Werner's syndrome, an hereditary disease manifested by early and widespread degenerative changes, showed senescent phenotype in RBC adsorption even at early passages.     

Neurotropin increases in vitro life span of human fibroblasts

Fibroblasts isolated from human fetal tissues (skin, lung and heart) exhibited the following population doubling levels (PDL): about 40 PDL for skin; 60 PDL for lung; 10 PDL for heart. Neurotropin extract from vaccinia-virus infected skin tissues of rabbits increases the growth rate of the fibroblast when added to the old lung cell culture (PDL 40, 70% of the maximum life span) but not when added to the young lung (PDL 5, 8% of the maximum life span). Neurotropin (40 or 80 micrograms/ml) increased the replicative life span of the three lines of fibroblasts (skin, lung and heart) when added to the young cell cultures. The increase of the maximum PDL of skin, lung and heart fibroblasts with the Neurotropin treatment was by 19%, 5% and 17%, respectively. When Neurotropin was added to the old lung cell culture (PDL 44, 73% of the maximum life span), the maximum PDL was increased by 9%. Since the existence of hydrocortisone, known to extend the in vitro life span, was negligible in the fluorimetric test of the medium containing Neurotropin, this agent may belong to the class other than hydrocortisone, for increasing in vitro life span.     

Tumor-secreted vascular permeability factor increases cytosolic Ca2+ and von Willebrand factor release in human endothelial cells.

Vascular permeability factor (VPF), a tumor-secreted heparin-binding protein (Mr approximately 38,000), is responsible for increased vessel permeability and fluid accumulation associated with tumor growth. Vascular permeability factor also promotes the growth of human umbilical vein endothelial cells (EC) and bovine pulmonary ECs in vitro. It is shown for the first time that guinea pig VPF (half-maximal and maximal dose approximately 0.4 and 22 pmol/l (picomolar), respectively), as well as human VPF, are potent stimuli for human ECs resulting in [Ca2+]i increases (maximal three- to fourfold) and inositol triphosphate (IP3) formation. Unlike the maximal responses to thrombin and histamine, the [Ca2+]i response to a maximal VPF dose was preceded by a characteristic 10- to 15-second delay. Guinea pig VPF also selectively increased [Ca2+]i in cultured aortic and pulmonary artery ECs, but not aortic smooth muscle cells, human fibroblasts, or neutrophils. Affinity-purified rabbit antibody (raised to a synthetic peptide representing VPF N-terminal amino acids 1 to 24) adsorbed all vessel permeability-increasing activity, EC growth-promoting activity, and specifically all activity responsible for increasing EC [Ca2+]i. Similar to other mediators that increase [Ca2+]i in cultured ECs, VPF also induced a 200% increase in von Willebrand factor release. Together these data indicate that VPF acts directly on ECs and that rapid cellular events in its in vivo/in vitro actions are likely to involve phospholipase C activation, [Ca2+]i increase, and von Willebrand factor release.     

Senescent fibroblasts resist apoptosis by downregulating caspase-3

In replicative senescence, cells undergo permanent exit from cell cycle traverse; this is traditionally thought to occur at the end of a culture's in vitro life span, after serial passaging. In general, the checkpoint for replicative senescence is found at the G(1)/S border, controlled by the modulation of a battery of proteins, typified by gaining inhibitors of cell cycle traverse, such as cyclin-dependent kinases or RB hyperphosphorylation, and losing pro-proliferation gene expressions such as c-fos, c-myc, and a cadre of proliferation-dependent kinases. Here, we present evidence that replicatively senescent fibroblasts are resistant to apoptotic death, associated with a lack of key enzyme activities, caspase-3 being the chief executioner. This observation, coupled with our earlier report that senescent fibroblasts maintain persistently high levels of pro-survival factor Bcl-2, suggests that the molecular signaling program present in fibroblasts at the end of their in vitro life span may not only cater to the state of permanent exit from cell cycle traverse, but also dictate an inability to commit cellular suicide. Future experiments will reveal whether replicatively senescent fibroblasts that can neither proliferate nor die contribute to organismic aging, and whether their accumulation over time in tissue becomes detrimental to the normal aging process.     

Molecular genetic approaches to the study of cellular senescence

Normal cells in culture exhibit limited division potential, which is used as a model for cellular aging. In contrast, tumor-derived, carcinogen- or virus-transformed cells are capable of dividing indefinetely (immortal). Fusion of normal with immortal human cells yielded hybrids having limited life span, indicating that cellular senescence is a dominant phenotype and that immortality is recessive. Fusions of various immortal human cell lines with each other led to the identification of four complementation groups for indefinite division. In order to identify the chromosomes and genes involved in growth regulation, that had been modified in immortal cells, we used the technique of fusion to introduce either a normal human chromosome 11 or 4 into cell lines representative of the different complementation groups. Chromosome 11 had no effect on the in vitro life span of the different immortal human tumor lines. However, when a normal human chromosome 4 was introduced into cell lines assigned to complementation group B, the cells lost the immortal phenotype. No effect on the proliferation potential of cell lines representing of the other complementation groups was observed. These results suggest that a gene(s) on human chromosome 4 has been modified in immortal cell lines assigned to complementation group B, to allow escape from senescence. They also provide evidence for a genetic basis for cellular aging.     

Roles and mechanisms of MFG-E8 in vascular aging-related diseases

The aging of the vasculature plays a crucial role in the pathological progression of various vascular aging-related diseases. As endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) are essential parts in the inner and medial layers of vessel wall, respectively, the structural and functional alterations of ECs and VSMCs are the major causes of vascular aging. Milk fat globule-epidermal growth factor 8 (MFG-E8) is a multifunctional glycoprotein which exerts a regulatory role in the intercellular interactions involved in a variety of biological and pathological processes. Emerging evidence suggests that MFG-E8 is a novel and outstanding modulator for vascular aging via targeting at ECs and VSMCs. In this review, we will summarise the cumulative roles and mechanisms of MFG-E8 in vascular aging and vascular aging-related diseases with special emphasis on the functions of ECs and VSMCs. In addition, we also aim to focus on the promising diagnostic function as a biomarker and the potential therapeutic application of MFG-E8 in vascular aging and the clinical evaluation of vascular aging-related diseases.     

Strict relationship between dialyzed serum concentration and cellular life span in vitro

To determine the extent of the contribution of growth factor level on the life span of human diploid fibroblasts in vitro, the growth rate changes of IMR-90 cells under altered dialyzed serum (d-FBS) concentrations were investigated at different population doubling levels (PDL). As the PDL increased, the cells showed an accelerated requirement for d-FBS in order to maintain a constant growth rate, thus indicating a rapid loss of responsiveness of the cells to serum growth factors. A similar relationship was observed when the growth rate was extrapolated to zero and the cellular life span was predicted from this relationship. The cells cultured with 0.3% and 10% d-FBS ceased their growth at 54 and 76 PDL, respectively, while their predicted life span was 58 and 80–85 PDL, respectively. The cells cultured with 0.3% d-FBS responded poorly to an increase in the d-FBS concentration after entering phase III. These results suggest that the serum growth factor level is one of the determinants of cellular life span in vitro.     

Modulation of mesangial cell proliferation by endothelial cells in coculture.

The effects of direct cell contact between endothelial (ECs) and mesangial cells (MCs) on MCs proliferation were examined in a coculture system in vitro. Mitomycin C treated ECs (M-ECs) were plated on culture dishes and MCs were cocultured with these M-ECs. Cell number was measured at the end of days 1, 3, and 5. In the coculture system with direct contact, the growth of cocultured MCs was modulated as follows: 1) the growth of MCs was inhibited up to day 3, and 2) a high level of proliferation was observed between days 3 and 5. This biphasic pattern of growth could not be detected in coculture of fibroblasts with MCs. In coculture without direct contact, using intercup chambers, the kinetics in cell proliferation between cocultured MCs and MCs alone were essentially the same. Conditioned media derived from cocultures up to day 3 in a contact-dependent manner inhibited the 3H-thymidine uptake of MCs. From these results, it would thus appear that MCs proliferation is regulated by intercellular contact with ECs.     

Abrogation of TGF-beta by antisense oligonucleotides modulates expression of VEGF and increases angiogenic potential in isolated fibroblasts from radiated skin

The transforming growth factor-beta (TGF-beta) has been identified as an important component of wound healing. Recent developments in molecular therapy offer good prospects for the modulation of wound healing, specifically those targeting TGF-beta. The aim of this study was to analyze the effect of TGF-beta targeting on the expression of angiogenic vascular endothelial growth factor (VEGF), a key regulator of angiogenesis and in vitro angiogenic activity in fibroblasts isolated from radiation-induced chronic dermal wounds. The expression of angiogenic VEGF in tissue samples from radiation-induced chronic dermal wounds was investigated by immunohistochemistry and microarray technique. The effect of TGF-beta targeting using antisense oligonucleotides on the expression of VEGF in isolated fibroblasts was analyzed by ELISA and multiplex RT-PCR. Human endothelial cells (ECs) were grown in conditioned medium produced from the treated fibroblasts. EC migration was measured using a modified Boyden chamber; EC tube formation was analyzed under a light microscope. Immunohistochemical investigation and microarray analysis demonstrated a decreased expression of VEGF protein and mRNA in tissue samples from radiation-induced chronic dermal wounds compared to normal human skin. Antisense TGF-beta oligonucleotide treatment significantly up-regulated VEGF secretion in vitro. Addition of conditioned medium from TGF-beta antisense-treated fibroblasts resulted in an increase in EC cell migration and tube formation. In conclusion, our results demonstrate that TGF-beta antisense oligonucleotide technology may be a potential therapeutic option for stimulation of angiogenesis in radiation-induced dermal wounds.     

Monocyte-Derived Influence on Growth of Normal Human Fibroblasts

The current study demonstrates that whereas high concentrations of recombinant tumour necrosis factor (rTNF, 0.05 microgram/ml) induced enhanced growth of normal human fibroblasts, supernatants from human monocytes with a similar TNF concentration induced cytostasis. This cytostasis was inhibited by an antiserum against rTNF. The TNF activity, measured as cytotoxicity against TNF-sensitive WEHI 164 cells, cochromatographed with the fibroblast growth inhibitory activity upon ion-exchange chromatography of monocyte supernatants. This indicates that TNF contributes to the fibroblast growth inhibition mediated by monocyte supernatants. Alpha interferon (IFN-alpha) abolished the growth of fibroblasts induced by rTNF, whereas rTNF in combination with gamma interferon (IFN-gamma) inhibited growth of fibroblasts. The interferon activity in the supernatants was determined to find out whether the growth-inhibitory activity of natural TNF was due to interferons present in the monocyte supernatants, which might modulate the TNF activity. Cytostasis of fibroblasts was mediated by monocyte supernatants which did not contain IFN-gamma in significant amounts. All supernatants contained IFN-alpha. An antiserum against IFN-alpha partially reduced the cytostasis induced by monocyte supernatants. This cytostasis was totally abolished by rTNF antiserum, suggesting that IFN-alpha modulates the growth-inhibitory activity of TNF in the monocyte supernatants. It appeared that the different effects of recombinant and natural TNF on fibroblast growth can probably not be attributed to monocyte-derived TNF-modulating factors alone. Thus, recombinant and natural TNF may differ in a way that affects their capacity to induce cytostasis of normal fibroblasts.     

A physico-chemical comparison of the monocyte-derived fibroblast growth factor and the tumour necrosis factor

The chromatographic characteristics of the human monocyte-derived growth stimulatory activity towards diploid FS-4 human fibroblasts has been studied. The fibroblast growth stimulatory factor had an apparent isoelectric point of 5.8 as determined by chromatofocusing, and a molecular weight in the range 70,000-30,000 as determined by gel filtration. The growth stimulatory factor eluted together with the tumour necrosis factor (TNF) upon cation exchange chromatography, chromatofocusing and gel filtration. Moreover, the fibroblast growth stimulatory activity in crude monocyte supernatants as well as the activity in the gel filtration column fractions was neutralized by antiserum raised against recombinant TNF (rTNF). The results indicate that the monocyte-derived fibroblast growth stimulatory activity is largely due to TNF.     

Glucose inhibition of human fibroblast proliferation and response to growth factors is prevented by inhibitors of aldose reductase

Diabetes mellitus is associated with premature senescence of cultured dermal fibroblasts. The present study investigated the effect of elevated glucose concentrations on cultured human fibroblasts from normal donors. Mean population doubling times, population doublings until senescence, saturation density at confluence (cells/cm2), tritiated thymidine incorporation, and response to platelet-derived growth factor (PDGF) were inhibited with the increasing glucose concentrations (11.0, 22, 44, or 55 mM glucose) (P less than 0.05). Replicative life span was markedly diminished by multiple passages in high glucose medium (5.5 mM glucose: 62.4 +/- 7.9 population doublings; 22 mM glucose: 22.8 +/- 3.4 population doublings: P less than 0.05). Aldose reductase activity was present in the cultured fibroblasts (3.9 +/- 0.5 nmol/min per mg protein), and inhibitors of aldose reductase, including sorbinil (10(-4) M--10(-6) M) and tolrestat (10(-6) M--10(-8) M), completely prevented glucose-mediated inhibition of fibroblast proliferation, restored the response to PDGF, and allowed a normal replicative life span. Myo-inositol (11 microM--5.5 mM) also reversed the adverse effects of glucose. These in vitro data demonstrate that elevated concentrations of glucose inhibit cell growth and promote premature senescence, effects which can be prevented with inhibitors of aldose reductase or supplemental myo-inositol. These aldose reductase-related effects may explain the impaired growth and premature senescence of cultured connective tissue from diabetic patients.     

Construction of tissue-engineered homograft bioprosthetic heart valves in vitro

Human mesenchymal stem cells (MSCs) differentiate into multiple cell-lineages and may serve as an alternative source of seed cells for tissue engineering. We investigated whether MSCs could be induced to differentiate into endothelial cells (ECs) and function as seed cells for the in vitro construction of tissue-engineered heart valves (TEHVs). Aortic or pulmonary valve homografts were decellularized with 0.1% sodium dodecylsulphate and used as scaffolds for TEHVs. The MSCs were isolated from human bone marrow by Percoll gradient centrifugation (1.073 g/ml), differentiated into ECs with vascular endothelial growth factor (10 ng/ml), and seeded onto a decellularized scaffold (high-density seeding, >10(5) cells/cm2) and grown in static culture for 14 days. Over 90% of the differentiated cells from MSCs stained positively for von Willebrand factor and Tie-2-related antigen. Additionally, Weibel-Palade corpuscle was observed in the cytoplasm of these cells. Levels of reendothelialization in static culture on days 7, 14, and 20, were 73%, 85%, and 95%, respectively. These results show that MSCs from human bone marrow can differentiate, in vitro, into ECs that can then be used to construct TEHVs. Reendothelialization in static culture can be used to provide the basic material for pulsatile-flow cultivation.     

The establishment of telomerase-immortalized cell lines representing human chromosome instability syndromes

The limited life span of normal human cells represents a substantial obstacle for biochemical analysis, genetic manipulation and genetic screens. To overcome this technical barrier, immortal human cell lines are often derived from tumors or produced by transformation with viral oncogenes such as SV40 large T antigen. Cell lines produced by these approaches are invariably transformed, genomically unstable and display cellular properties that differ from their normal counterpart. It was recently shown that the ectopic expression of hTERT, encoding the catalytic subunit of human telomerase, can extend the life span of normal human cells without causing cellular transformation and genomic instability. In the present study, we have used hTERT to extend the life span of normal human skin fibroblasts derived from patients afflicted with syndromes of genomic instability and/or premature aging. Our results show that hTERT efficiently extends the life span without altering the characteristic phenotypic properties of the cells. Thus, the ectopic expression of telomerase represents a major improvement over the use of viral oncogenes for the establishment of human cell lines.     

Lymphotoxin-Induced Growth Stimulation of Diploid Human Fibroblasts in the Presence and Absence of Gamma Interferon

In the present study we investigated the effect of lymphotoxin (LT) on the growth of human diploid fibroblasts, in the presence and absence of gamma interferon (IFN-gamma). Recombinant LT (rLT) had a growth stimulatory effect on diploid human FS-4 fibroblasts. This growth-stimulatory effect was reduced in the presence of recombinant IFN-gamma (rIFN-gamma). LT thus has a similar effect on diploid fibroblasts as tumour necrosis factor (TNF).     

Effect of exogenous opioid peptides on TNF-alpha-induced human neutrophil apoptosis in vitro

Polymorphonuclear leukocyte (neutrophil) apoptosis is an important mechanism regulating the life span and some functions of neutrophils at inflamed sites. Opioid peptides are present in the peripheral circulation and their concentrations rapidly increase as a result of stress and inflammation. The effect of opioid peptides such as met-enkephalin (M-ENK) and beta-endorphin (beta-END) on tumor necrosis factor (TNF)-alpha-induced apoptosis in human neutrophils in vitro was investigated. Neutrophils isolated from peripheral blood were cultured in the absence or presence of 10(-6)-10(-10) M of opioid peptides for 8, 12 and 18 h. Features of apoptotic neutrophils were measured by a flow cytometric method based on analysis of the apoptotic nuclei (DNA content). We found that M-ENK and beta-END enhanced both uninduced and TNF-alpha-induced neutrophil apoptosis in vitro in a dose-dependent manner. The effect of opioid peptides on the modulation of neutrophil apoptosis was not reversed by the opioid-receptor antagonist naloxone. The results suggest that M-ENK and beta-END can regulate neutrophil life span via apoptosis and in this way may participate in the resolution of inflammation.     

The effect of in vitro aging on human lung fibroblast beta-adrenergic receptor density, coupling and response.

Age-related changes in regulation of receptor response have been observed in several tissues and include regulation of beta-adrenergic receptor (beta-receptor) responses. The role of cellular aging in age-related changes in receptor response is not clear. We have examined the effect of aging in vitro on human fibroblast beta-receptor function. MRC-5 (embryonic lung) fibroblasts were aged by replication to produce cells of early, middle and late stages corresponding to the following cumulative population doublings: 15-20, 35-45 and greater than 50, respectively. Fibroblast membrane beta-receptor responses to isoproterenol (ISO, 0.1 mM) did not differ between the three stages. Adenylate cyclase responses to prostaglandin E1 (PGE1, 1 microM), guanosine triphosphate (GTP, 0.1 mM) and 5'-guanylimidodiphosphate (Gpp(NH)p, 0.1 mM) were also similar between the stages. Beta-receptor density (Bmax) was unaffected by in vitro aging. Beta-receptor agonist affinity, an indication of the capacity for beta-receptor coupling to the nucleotide binding protein (NS), was also unaffected by cell aging. These findings suggest that cellular aging in fibroblasts alone is not accompanied by changes in beta-receptor function.