Revealing the impact of local access-site complications and upper extremity dysfunction post transradial percutaneous coronary procedures.

OBJECTIVES: Little is known about local access-site complications and upper extremity dysfunction after transradial percutaneous coronary procedures (TR-PCP). This systematic review study aimed to summarise the current knowledge on the incidences of access-site complications and upper extremity dysfunction after TR-PCP. METHODS: Two independent, trained investigators searched MEDLINE, EMBASE and CENTRAL for eligible studies published before 1 January 2015. Also, they hand-searched the conference proceedings of the annual scientific sessions of the American College of Cardiology, the American Heart Association, European Society of Cardiology, and the Trans-catheter Cardiovascular Therapeutics. Inclusion criteria were cohort studies and clinical trials discussing the incidence of access-site complications and upper extremity function after transradial percutaneous coronary intervention (TR-PCI) and/or transradial coronary angiography (TR-CAG) as endpoints. RESULTS: 176 articles described access-site complications. The incidence is up to 9.6 %. Fourteen articles described upper extremity dysfunction, with an incidence of up to 1.7 %. Upper extremity dysfunction was rarely investigated, hardly ever as primary endpoint, and if investigated not thoroughly enough. CONCLUSION: Upper extremity dysfunction in TR-PCP has never been properly investigated and is therefore underestimated. Further studies are needed to investigate the magnitude, prevention and best treatment of upper extremity dysfunction. Optimising TR-PCP might be achieved by using slender techniques, detection of upper extremity dysfunction and early referral to a hand rehabilitation centre.

Lotus petal perforator flap for reconstruction of an extended chronic presacral defect after radiotherapy and rectal cancer resection.

AIM: As a result of their extent and complexity, pelvic wounds after surgery for anorectal malignancy often require a multidisciplinary approach to accomplish closure. This report describes a successful reconstruction using the lotus petal perforator flap. METHOD: This flap is based on perforators of the internal pudendal artery and was partially depithelialized for plugging the defect. RESULTS: Wound healing was achieved after 12 days. CONCLUSION: The lotus petal flap is a relatively simple and successful choice for reconstruction of an extended chronic presacral defect after radiotherapy and rectal cancer resection.

Evaluation of alternative sites for islet transplantation in the minipig: interest and limits of the gastric submucosa.

Since the introduction of glucocorticoid-free immunosuppressive regimens, islet transplantation offers a less invasive alternative to pancreas transplantation. However, complications associated with intraportal islet injection and the progressive functional decline of intrahepatic islets encourage the exploration of alternative sites. Herein we evaluated, in the minipig, the use of the gastric submucosa (GS; group 1, n = 5) for islet transplantation compared with the kidney capsule (KC; group 2, n = 5). Subsequently we attempted to improve the vascularization of the submucosal graft (group 3, n = 5) by the addition of an extracellular matrix rich in growth factors (Matrigel). One month after grafting, we evaluated transplanted islet function in vivo and in vitro. Our study showed better function of islets engrafted in the GS than in the KC (P < .05). Despite the growth factors, Matrigel did not offer a more suitable environment to further improve engraftment (group 3, P < .05). Thus, even if the liver remains the gold standard, the GS represents a potential islet engraftment site, confirming the data obtained in vitro and in the rodent. Offering easy access by endoscopy, this site could constitute an interesting alternative for experimental studies in large mammals and, eventually, for clinical application.

Can transcutaneous oxygen tension measurement determine re-amputation levels?

OBJECTIVE: To review the preoperative transcutaneous oxygen tension (TcPO2) measurements in patients having major lower leg amputation, and also consider the re-amputation rate, wound infection and the definitive level of amputation. METHOD: A case-control study was performed in a consecutive cohort of 170 patients (1999-2003). Fifty-two patients underwent preoperative TcPO2 measurements (cases) and 118 patients did not (control). Multiple logistic regression analysis was performed to analyse independent risk factors associated with re-amputations. RESULTS: Primary and definitive (in case of a re-amputation) amputation levels were lower in the TcPO2 group, although this did not reach statistical significance. The number of re-amputations in the TcPO2 group was significantly higher: 15 versus 18 patients (p=0.039). Selection of an amputation level with aTcPO2 of 30mmHg resulted in a positive predictive value of re-amputation of 41% and a negative predictive value of 90%. A cut off value of 20mmHg resulted in 41% and 77% respectively. CONCLUSION: The use of TcPO2 measurements for major amputation level selection resulted in an increased rate of re-amputation. However, there was a trend in gaining a more distal definitive amputation level. Selection of an amputation level solely based on a TcPO2 value is unreliable.

1,25-Dihydroxyvitamin D3 protects human pancreatic islets against cytokine-induced apoptosis via down-regulation of the Fas receptor.

Beta cell loss occurs at the onset of type 1 diabetes and after islet graft. It results from the dysfunction and destruction of beta cells mainly achieved by apoptosis. One of the mediators believed to be involved in beta cell apoptosis is Fas, a transmembrane cell surface receptor transducing an apoptotic death signal and contributing to the pathogenesis of several autoimmune diseases. Fas expression is particularly induced in beta cells by inflammatory cytokines secreted by islet-infiltrating mononuclear cells and makes cells susceptible to apoptosis by interaction with Fas-ligand expressing cells. We have previously demonstrated that 1,25(OH)2D3, the active metabolite of vitamin D, known to exhibit immunomodulatory properties and prevent the development of type 1 diabetes in NOD mice, is efficient against apoptosis induced by cytokines in human pancreatic islets in vitro. The effects were mainly mediated by the inactivation of NF-kappa-B. In this study we demonstrated that 1,25(OH)2D3 was also able to counteract cytokine-induced Fas expression in human islets both at the mRNA and protein levels. These results were reinforced by our microarray analysis highlighting the beneficial effects of 1,25(OH)2D3 on death signals induced by Fas activation. Our results provides additional evidence that 1,25(OH)2D3 may be an interesting tool to help prevent the onset of type 1 diabetes and improve islet graft survival.

Hepatocyte nuclear factor 4 alpha isoforms originated from the P1 promoter are expressed in human pancreatic beta-cells and exhibit stronger transcriptional potentials than P2 promoter-driven isoforms.

The nuclear receptor hepatocyte nuclear factor (HNF) 4 alpha is involved in a transcriptional network and plays an important role in pancreatic beta-cells. Mutations in the HNF4 alpha gene are correlated with maturity-onset diabetes of the young 1. HNF4 alpha isoforms result from both alternative splicing and alternate usage of promoters P1 and P2. It has recently been reported that HNF4 alpha transcription is driven almost exclusively by the P2 promoter in pancreatic islets. We observed that transcripts from both P1 and P2 promoters were expressed in human pancreatic beta-cells and in the pancreatic beta-cell lines RIN m5F and HIT-T15. Expression of HNF4 alpha proteins originating from the P1 promoter was confirmed by immunodetection. Due to the presence of the activation function module AF-1, HNF4 alpha isoforms originating from the P1 promoter exhibit stronger transcriptional activities and recruit coactivators more efficiently than isoforms driven by the P2 promoter. Conversely, activities of isoforms produced by both promoters were similarly repressed by the corepressor small heterodimer partner. These behaviors were observed on the promoter of HNF1 alpha that is required for beta-cell function. Our results highlight that expression of P1 promoter-driven isoforms is important in the control of pancreatic beta-cell function.

The temporary use of allograft for complicated wounds in plastic surgery.

A retrospective analysis was performed on the use of glycerol-preserved allografts (GPA) in the preparation of complicated wounds for secondary wound closure. All files from the plastic surgery department in the period 1992-1998 were screened. Thirty-three patients within a total 85 GPA treatments were selected and screened for indication of use of GPA, frequency of GPA changes, duration of treatment and whether or not subsequent autografting was possible. GPA was used as a biological cover for the following indications: problematic wound healing, 13 cases; non-healing burns, 12 cases; carcinoma, 4 cases; unstable scar, 2 cases; shortage of skin, 2 cases. The average frequency of GPA application was 2.6 times, with a mean duration of 5 days per application. In 84 cases (32 patients) the wound was successfully covered with autograft. In conclusion, GPA was used with good results as a temporary cover for complicated wounds. We postulate that angiogenic effects of this biological dressing may have contributed to the improved healing conditions and successful secondary wound closure.

Formulation and delivery mode affect disposition and activity of tyrphostin-loaded nanoparticles in the rat carotid model.

Poor drug residence in the arterial wall hinders clinical implementation of local drug delivery strategies for the treatment of restenosis. A rat carotid model of vascular injury and intraluminal delivery of tyrphostin-containing polylactic acid (PLA) nanoparticles (NPs) were used to determine the relationship between residence properties and biological activity of different formulations and administration modes. The effects of delivery modes (denudation and delivery time) and formulation variables (adsorbed vs encapsulated drug, and NP size) on arterial drug/NP retention were examined. Antirestenotic effects of large (160 nm) and small (90 nm) tyrphostin-containing NPs, surface-absorbed tyrphostin, and systemic treatment were compared. Fluorescent NPs were used to study the spatial distribution of the carrier in the arterial wall. The decrease in arterial tyrphostin level over time fitted a biexponential model. Delivery time and pressure, endothelium integrity, particle size, and drug-polymer association affected local pharmacokinetics and the antirestenotic results after 14 days. The PLA-based tyrphostin NP formulation ensured a prolonged drug residence at the angioplasty site after single intraluminal application. Several readily adjustable formulation and procedural factors considerably modified arterial ingress of the drug-loaded NPs and governed their subsequent redistribution, tissue binding, elimination, and ensuing antirestenotic effect.

Inhibition of Osf2/Cbfa1 expression and terminal osteoblast differentiation by PPARgamma2.

Cells of the bone marrow stroma can reversibly convert among different phenotypes. Based on this and on evidence for a reciprocal relationship between osteoblastogenesis and adipogenesis, we have isolated several murine bone marrow-derived clonal cell lines with phenotypic characteristics of osteoblasts or adipocytes, or both. Consistent with a state of plasticity, cell lines with a mixed phenotype synthesized osteoblast markers like type I collagen, alkaline phosphatase, osteocalcin, as well as the adipocyte marker lipoprotein lipase, under basal conditions. In the presence of ascorbic acid and beta-glycerophosphate-agents that promote osteoblast differentiation-they formed a mineralized matrix. In the presence of isobutylmethylxanthine, hydrocortisone, and indomethacin-agents that promote adipocyte differentiation-they accumulated fat droplets, but failed to express adipsin and aP2, markers of terminally differentiated adipocytes. Furthermore, they were converted back to matrix mineralizing cells when the adipogenic stimuli were replaced with the osteoblastogenic ones. A prototypic cell line with mixed phenotype (UAMS-33) expressed Osf2/Cbfa1-a transcription factor required for osteoblast differentiation, but not PPARgamma2-a transcription factor required for terminal adipocyte differentiation. Stable transfection with a PPARgamma2 expression construct and activation with the thiazolidinedione BRL49653 stimulated aP2 and adipsin synthesis and fat accumulation, and simultaneously suppressed Osf2/Cbfa1, alpha1(I) procollagen, and osteocalcin synthesis. Moreover, it rendered the cells incapable of forming a mineralized matrix. These results strongly suggest that PPARgamma2 negatively regulates stromal cell plasticity by suppressing Osf2/Cbfa1 and osteoblast-like biosynthetic activity, while promoting terminal differentiation to adipocytes.

Cellular and molecular biomarkers indicate precocious in vitro senescence in fibroblasts from SAMP6 mice. Evidence supporting a murine model of premature senescence and osteopenia.

A variety of short-lived mouse strains (SAMP strains) and control strains of less abbreviated life span (SAMR strains) have been proposed as murine models of accelerated senescence. Each SAMP strain, in addition to displaying "progeroid" traits of accelerated aging, exhibits a singular age-related pathology. The application of this animal model to the study of normal aging processes has been and remains controversial. Therefore, we have undertaken a study of dermal fibroblasts derived from the short-lived SAMP6 strain, which shows early-onset and progressive osteopenia. We have investigated cellular and molecular characteristics that are associated with in vitro aging of normal human fibroblasts, and which are exacerbated in fibroblasts from patients with Werner syndrome, a human model of premature senescence. We found that SAMP6 dermal fibroblasts, relative to SAMR1 and C57BL/6 controls, exhibit characteristics of premature or accelerated cellular senescence with regard to in vitro life span, initial growth rate, and patterns of gene expression.

Characterization of IGFBP-3, PAI-1 and SPARC mRNA expression in senescent fibroblasts.

The RNA species encoded by IGFBP-3 (insulin-like growth factor binding protein-3), PAI-1 (plasminogen activator inhibitor-1) and SPARC (secreted protein-acidic and rich in cysteine; a.k.a. osteonectin) are overexpressed in senescent human diploid fibroblasts (HDF). Their extracellular products have the ability to modulate cell growth in culture and have been shown to have inhibitory effects on DNA synthesis and/or cell growth. This overproduction may contribute to a number of features of aging, including osteoporosis, atherosclerosis and diabetes mellitus type II. Based on analysis of steady-state mRNA levels, which showed similar patterns for all three along with overexpression in senescent cells, we further investigated their transcription rates and stability to determine reasons for their overexpression and to determine if coordinate gene regulation was involved. Characterization of the rates of transcription and the levels of message stability of these genes in early passage (young) versus late passage (old) HDF revealed that IGFBP-3, PAI-1 and SPARC are coordinately overexpressed but not regulated by a unique or simple mechanism encompassing all three transcripts. Only PAI-1 shows an increase in the rate of transcription, while all three show evidence that their overexpression is due to an increase in the stability of RNA. Thus, the overexpression of these genes in senescent fibroblasts involves interactions not only at the transcriptional level but also with protein factors involved in determining the stability and the degradation of RNA.

Altered expression and regulation of the alpha 5beta1 integrin-fibronectin receptor lead to reduced amounts of functional alpha5beta1 heterodimer on the plasma membrane of senescent human diploid fibroblasts.

Previously, we reported that fibronectin (FN) mRNA was overexpressed in normal late-passage (old) and pre- maturely senescent Werner syndrome (WS) fibroblasts when compared to normal early-passage (young) cells (Murano et al. Mol. Cell. Biol. 11, 3905-3914, 1991). Therefore, we investigated the expression and function of the alpha5 beta1 FN receptor (FNR), a member of the integrin family, in young and senescent normal and WS cells. Levels of the alpha5 polypeptide, a unique subunit of the alpha5 beta1 FNR, were reduced in old cells, so that old cells produced fewer alpha5 beta1 heterodimers on the plasma membrane. The reduced levels of alpha5 polypeptide might be due to deficient translation and/or nonfunctional alpha5 mRNA since increased mRNA levels and unchanged polypeptide turnover were found in these cells. Moreover, the alpha5 polypeptides on the senescent cell surface were less accessible to monoclonal antibody, suggesting sequestration of this subunit, which might affect receptor-ligand binding. In contrast, beta1 subunit, a common subunit for the beta1 integrin subfamily, showed relatively stable levels during cellular aging, but underwent slower intracellular processing. Old cells exhibited reduced attachment to FN, which might be in part mediated by the alpha5 beta1 FNR. More importantly, old cells were deficient in response to FN-induced DNA synthesis and cell proliferation. This induction was pronounced in young cells, however, and could be completely inhibited by alpha5-specific monoclonal antibody, indicating mediation by alpha5 beta1 FNR. WS cells behaved like normal old cells in the above assays. Our results indicate that reduction of alpha5 beta1 FNR expression and its mediated effects are associated with the senescent phenotype of fibroblasts. These findings provide new insight into the mechanism(s) of replicative senescence in human fibroblasts.

Senescent fibroblasts as feeder cells for lymphoid cell cloning.

Senescent primary human skin fibroblasts were used as feeder layers for cloning lymphoid cells by limiting dilution. B-cells including mouse hybridoma cells, human multiple myeloma cells C1R and DIG, as well as an immortalized T-cell line (Jurkat cells) were cloned using this approach. From heterogenous populations, homogeneous (clonal) populations were obtained and further analyzed. The major advantages of senescent fibroblasts as feeder cells are (i) the need to establish primary cultures from experimental animals for preparing feeder cells is obviated; (ii) the risk of contamination with infectious agents is diminished; (iii) primary skin fibroblasts are easily maintained in culture, can be kept at confluence for extended periods of time, and do not undergo spontaneous transformation; and (iv) lymphoid cells are readily separated from fibroblast monolayers. The use of senescent fibroblasts overcomes limitations inherent with primary mouse cell cultures and irradiated cells commonly used as feeder cells in cloning techniques.

Expression of senescence-induced protein WS3-10 in vivo and in vitro.

In our efforts to characterize cellular senescence we have shown that the mRNA encoding WS3-10 protein is overexpressed in senescent human diploid fibroblasts (HDF) when compared with their younger counterparts, and that forced expression of the WS3-10 cDNA in young HDF results in suppression of calcium-dependent membrane currents, presumably due in part to the presence of a calcium binding domain within the WS3-10 protein. We have now expressed this protein in E. coli and have obtained affinity purified antibodies. Western blot analysis utilizing these antibodies showed that WS3-10 protein is also overexpressed in senescent HDF when compared to young HDF, and in normal fetal lung HDF when compared to SV40-transformed fetal lung HDF. HeLa cells do not express WS3-10 protein. In addition, we looked for WS3-10-related species in a variety of rat tissues. Analysis of WS3-10 immunologically related proteins in rat tissue extracts revealed two WS3-10 homologs, sized 22 kDa and 20 kDa. The latter presumably result from proteolytic removal of the C-terminal end of the 22 kDa polypeptide. The ratio between these polypeptides varies in a tissue-specific manner. Two proteins immunologically related to WS3-10 with sizes of 39 kDa and 91 kDa were present in rat spleen and skeletal muscle, respectively.

Identification of gene sequences overexpressed in senescent and Werner syndrome human fibroblasts.

The phenotype of replicative senescence is a dominant trait in human diploid fibroblasts (HDF). Therefore, we have sought to identify overexpressed and/or newly expressed causal genes by constructing and screening a subtracted cDNA library derived from polyA+RNA of prematurely senescent Werner syndrome (WS) HDF. We have identified 15 cDNA clones that are overexpressed in senescent and WS HDF. Among them are six known sequences coding for: acid sphingomyelinase, fibronectin, SPARC, nm23-metastasis suppressor protein, and two translation factors, eIF-2 beta and EF-1 alpha. Among the 10 unknown clones are: S1-5, which encodes a secreted protein containing EGF-like domains and paradoxically stimulates DNA synthesis of young HDF in an autocrine and paracrine manner, S1-3, which encodes a protein containing "zinc finger" domains, suggesting nucleic acid binding properties; S1-15, which shows sequence similarities to human alpha 2-chimerin; and S2-6, which represents a new member of the LIM family of proteins. The other five clones do not have any significant homology to known sequences. Steady-state mRNA levels of all gene sequences thus far studied are elevated in both WS and senescent normal HDF when compared to young HDF, which suggests that senescent and WS HDF enter a final common pathway where multiple gene overexpression may generate diverse antiproliferative mechanisms and pathogenic sequelae.

Overexpression of insulin-like growth factor binding protein-3 by senescent human fibroblasts: attenuation of the mitogenic response to IGF-I.

We have previously demonstrated that senescent human diploid fibroblasts (HDF) produce large amounts of IGF-binding protein-3 (IGFBP-3) in comparison to early-passage vigorously proliferative HDF. In order to determine whether this excess IGFBP-3 accumulation plays a role in the observed attenuation of DNA synthesis in senescent HDF, we examined the response of these cells to IGF-I and two IGF-I functional analogs, [QAYL]IGF-I and insulin, both of which have extremely low binding affinity for IGFBP-3 but which exert their mitogenic effect via the IGF-I plasma membrane receptor. Senescent HDF showed an increased sensitivity of DNA synthetic response to [QAYL]IGF-I and insulin compared to IGF-I. IGF binding activity was significantly higher in conditioned medium of senescent HDF than the medium of young HDF, and virtually all of this enhanced binding capacity could be accounted for by IGFBP-3. Addition of recombinant IGFBP-3 to young cells at a constant molar ratio of 1:1 with respect to IGF-I significantly attenuated the response to IGF-I and abolished the response at a 2:1 molar ratio. These data indicate that IGFBP-3 accumulated in medium of senescent HDF can bind and sequester IGFs when present in molar excess and thereby account for a significant part of the attenuated response of senescent HDF to IGF-I.

Overexpression of plasminogen activator inhibitor type-1 in senescent fibroblasts from normal subjects and those with Werner syndrome.

We previously reported that plasminogen activator inhibitor type-1 (PAI-1) mRNA was present at higher steady-state levels in prematurely senescent fibroblasts derived from a subject with Werner syndrome (WS) compared to early passage (EP) fibroblasts from an age-matched normal subject (Murano et al., 1991, Mol. Cell. Biol. 11:3905-3914). To explore the generally of this phenomenon with respect to chronological age of donor (in vivo aging) and the late-passage (LP) or senescent phase of the fibroblast replicative lifespan, we assayed PAI-1 mRNA in cells and PAI-1 antigen in medium conditioned by 20 normal fibroblast strains at EP and LP and six WS strains during their curtailed replicative lifespans. The lowest accumulations of PAI-1 were found in medium conditioned by fetal and newborn cells with a shallow but progressive rise seen in postnatal cells from normal donors of increasing chronological age. With few exceptions, normal LP fibroblasts showed increased PAI-1 accumulations in medium compared to their EP counterparts. Conditioned medium from four of the six WS strains showed PAI-1 accumulations that were significantly higher than the media of any normal controls at EP and LP. PAI-1 mRNa levels were generally commensurate with the cumulative amount of PAI-1 in the medium but the frequent exceptions indicate that translational and post-translational mechanisms also regulate PAI-1 output. The augmentation in PAI-1 output of fibroblasts as a direct function of chronological age and during in vitro senescence suggests that PAI-1 may play an important role in the reduced capacity for wound healing and the increasing tendency to thrombogenesis and atherogenesis seen during biological aging and in particular in persons with Werner syndrome.

Senescence and cell density of human diploid fibroblasts influence metabolism of insulin-like growth factor binding proteins.

In order to analyze changes in metabolism of insulin-like growth factor binding proteins (IGFBPs) related to cell senescence and cell density, we compared human diploid fibroblasts (HDF) in the proliferatively vigorous first half (young cells) and senescent HDF in the last 10% (old cells) of the replicative lifespan after seeding cells over an eightfold range and proliferation to high density. Increasing the seeding cell density of both young and old HDF led to elevated rates of IGFBP-3 secretion, an increasing ratio of the 42/38 kDa species of IGFBP-3, and degradation of all species of IGFBPs derived from both the fetal bovine serum component of the culture medium and from HDF. At a given seeding density old HDF produced more IGFBP-3 and degraded more IGFBPs than young HDF. IGFBP-4 was degraded by a protease that appeared to be different from the protease(s) involved in degradation of the other IGFBPs. Young HDF at all seeding densities contained a cell-associated 29 kDa IGFBP, whereas this protein could not be detected in old cells. Thus, although certain changes in IGFBP metabolism are similar in young HDF seeded at high densities and in old HDF, young and old phenotypes can be distinguished by characteristic qualitative and quantitative changes in IGFBPs derived from fetal bovine serum and from HDF.

Accumulation of insulin-like growth factor binding protein-3 in conditioned medium of human fibroblasts increases with chronologic age of donor and senescence in vitro.

We have found that insulin-like growth factor binding protein-3 (IGFBP-3) accumulates to higher levels in medium conditioned by a strain of normal fibroblasts at late passage (LP) and a strain derived from subjects with Werner syndrome (WS) of premature aging, compared to medium conditioned by the same normal cells at early passage (EP) (Goldstein et al., Proc. Natl. Acad. Sci. USA, 88:9680-9684, 1991). To explore the generality of this phenomenon with respect to chronological age of donor (in vivo aging) and LP (in vitro senescence) we assayed IGFBP-3 in medium conditioned by 18 normal fibroblast strains at EP and LP and two WS strains at the midpoint of their curtailed replicative lifespans and assessed IGFBP-3 mRNA levels in cells by Northern analysis. The lowest accumulations of IGFBP-3 were found in medium conditioned by fetal cells with progressively increasing amounts postnatally; direct correlations between IGFBP-3 levels and donor age were seen in EP cells 3 days after subculture (during logarithmic growth) r = 0.80, P < 0.001, and 7 days after subculture (at confluence) r = 0.77, P < 0.001. With two exceptions, conditioned medium of cell strains accumulated more IGFBP-3 at LP; IGFBP-3 levels correlated with chronological age after 3 days, r = 0.50, P = 0.05, and after 7 days, r = 0.75, P < 0.001. IGFBP-3 content of WS culture medium fell within the range of LP normal cells. Cumulative IGFBP-3 levels were inversely proportional to the thymidine labeling index, a measure of proliferative vigor. With some exceptions IGFBP-3 mRNA levels were commensurate with the amount of IGFBP-3 accumulated in the medium, suggesting that distal translational and posttranslational mechanisms also regulate IGFBP-3 production in some strains. The trend toward augmented IGFBP-3 output of fibroblasts as a direct function of chronological age and in vitro senescence and as an inverse function of proliferative vigor is consistent with the known inhibitory effect of excess IGFBP-3 on IGF-mediated DNA synthesis and the reduced regenerative potential that is evident during biological aging in vivo.

Insulin-like growth factor binding protein-3 is overexpressed in senescent and quiescent human fibroblasts.

Cellular insulin-like growth factor binding protein-3 (IGFBP-3) mRNA and IGFBP-3 levels in conditioned medium were consistently higher in cultures of late passage normal (old) fibroblasts and prematurely senescent fibroblasts derived from Werner syndrome (WS) during quiescence induced by serum depletion and during the renewed growth ensuing after serum repletion, compared to cultures of early passage normal (young) fibroblasts. Molar ratios of IGFBP-3/IGF-II were always higher in senescent cultures and maintained a hierarchy of old > WS > young human diploid fibroblasts. Transfection into fibroblasts of the normal full-length IGFBP-3 cDNA in an expression vector resulted in a significant reduction in colony formation compared to cells transfected with an empty expression vector (no cDNA) or with IGFBP-3 cDNA altered by a 273 base pair (bp) deletion. Addition to old and young cultures of recombinant human IGFBP-3 and IGF-I at 1:1 or 5:1 molar ratios inhibited IGF-I-mediated DNA synthesis by approximately 70-80%. These data indicate that IGFBP-3 may play an important role in the quiescent and senescent growth arrest of HDF.