Insulin Growth Factor Binding Protein Research Articles

Reference change values for insulin and insulin-like growth factor binding protein-1 (IGFBP-1) in individuals with varying degrees of glucose tolerance

Background. The reference change value (RCV) is an important parameter of biological variation used to assess the significance of differences between consecutive results obtained in a single individual. This study evaluated the RCV for insulin-like growth factor binding protein-1 (IGFBP-1) and fasting serum insulin (FSI) in individuals with different degrees of glucose tolerance. Methods. IGFBP-1 and FSI concentrations were measured in 33 fasting subjects who had two blood samples taken 10 days apart. Subjects were distributed in the following categories: Normal glucose tolerance (NGT), n = 15, impaired fasting glucose (IFG), n = 9 and impaired glucose tolerance (IGT), n = 9. Results. The RCV values for IGFBP-1 were 59.9%, 83.2% and 93.0% and for FSI were 68.5%, 79.0% and 93.4% for subjects with NGT, IFG and IGT respectively. Conclusions. The RCVs for IGFBP-1 and FSI increase with deteriorating glucose tolerance. When monitoring IGFBP-1 and FSI, changes in concentrations should be interpreted using glycaemic category-specific RCV values.

Insulin Growth Factor 1 and Cardiac Allograft Vasculopathy

Purpose Cardiac Allograft Vasculopathy (CAV) is major comorbidity after heart transplantation (OHT) with no definitive therapy besides re-transplantation. Its pathophysiology is not well defined and non-invasive markers of early disease are lacking. Low Insulin Growth Factor 1 (IGF1) and high Insulin Growth Factor Binding protein 3(IGFBP3) have been implicated in the development of atherosclerosis and native coronary artery disease (CAD). The purpose of this study is to evaluate the role of IGF1 and IGFBP3 in CAV. Methods and Materials The Intermountain Heart Collaborative Study Registry was queried for stored blood samples for heart transplant recipients (1995-2010). IGF1 and IGFBP3 plasma levels were done by ELISA and measured at baseline before the diagnosis of CAV in the CAV group and at similar time points after transplantation in the control group (±2 years) which was matched by gender and age. Demographic, clinical and laboratory data were collected at the time of the sample collections. CAV was defined CAV1-3 per International Society for Heart and Lung Transplantation. A non-parametric analysis for comparing the IGF1 and IGFBP3 for the CAV and non-CAV matched patients was done. Results 41 matched CAV and control pairs were analyzed. Average age was 53 ±12 years, 83% were male, and 96% were Caucasian. There was no difference in IGF1 and IGFBP3 serum levels between the two groups at baseline (see Table). Conclusions Although low serum IGF1 is a risk factor of development of CAD, this association was not found in patients with CAV post-transplant. This observation likely reflects differences in pathophysiology between the two diseases as immunologic process plays a major role in the CAV compare to native atherosclerosis. Larger studies are needed to further explore the role of IGF1 in CAV. CAV(n=41) Control (n=41) P value DM % 7.8 (9.8 0.69 HTN % 48.8 36.6 0.37 Average IGF1 (ng/ml)±std 116.66 ±55.73 105±39.02 0.61 Average IGFBP3 (ng/ml))±std 1670.14 ±513.28 1623.75 ±1026.93 0.82 DM: Diabetes Mellitus, HTN: Hypertension, std: standard deviation

Copper induces cellular senescence in human glioblastoma multiforme cells through downregulation of Bmi-1

Most human tumor cells, including glioblastoma multiforme (GBM) cells, have aberrant control of cell aging and apoptosis. Subcytotoxic concentrations of oxidative or stress‑causing agents, such as hydrogen peroxide, may induce human cell senescence. Thus, induction of tumor cells into premature senescence may provide a useful in vitro model for developing novel therapeutic strategy to combat tumors. In the present study, we assessed the molecular mechanism(s) underlying senescence in GBM cells induced by copper sulfate. Following pretreatment with subcytotoxic concentrations of copper sulfate, U87-MG tumor cells showed typical aging characteristics, including reduced cell proliferation, cell enlargement, increased level of senescence-associated β-galactosidase (SA β-gal) activity, and overexpression of several senescence-associated genes, p16, p21, transforming growth factor β-1 (TGF-β1), insulin growth factor binding protein 3 (IGFBP3) and apolipoproteinJ (ApoJ). We further demonstrated that the Bmi-1 pathway was downregulated in GBM cells in parallel with the induced senescence. The present study for the first time demonstrates the ability of copper to induce GBM cell senescence by downregulating Bmi-1.

Insulin/Insulin-Like Growth Factor-1 Pathway in Barrett's Carcinogenesis

OBJECTIVES:Obesity-associated carcinogenesis is postulated to be mediated through the proliferative actions of insulin and the insulin-like growth factor (IGF) family. The aim of this study was to determine whether the insulin/IGF-1 pathway is involved in the sequential progression from metaplastic Barrett's esophagus (BE) to dysplasia to esophageal adenocarcinoma (EAC).METHODS:Fasting serum levels of insulin, glucose, IGF-1, insulin growth factor binding protein-1 (IGFBP1), and IGFBP3 were measured in 44 non-dysplastic, 9 low-grade dysplasia (LGD), 12 high-grade dysplasia (HGD), and 10 EAC subjects. Immunohistochemistry was performed on paraffin-embedded tissue derived from BE cases using rabbit monoclonal antibodies to p-mammalian target of rapamycin (mTOR) and p-AKT, mouse monoclonal antibody to Ki-67, and rabbit polyclonal antibody to p-insulin receptor substrate 1 (IRS1).RESULTS:Nineteen of 44 (43.2%) BE, 5/9 (55%) LGD, 8/12 (66.7%) HGD and EAC 7/10 (70%) cases showed strong staining for p-IRS1. A significantly higher proportion of HGD/EAC subjects showed p-IRS1 staining when compared with BE/LGD subjects, 63.6% vs. 41.5%, P<0.05. p-IRS1 immunostaining was moderately correlated with strong immunostaining of the downstream mediators p-AKT and p-mTOR (Spearman correlation coefficient=0.167 and 0.27 for p-IRS1/p-AKT and for p-IRS1/p-mTOR, respectively) and the proliferation marker Ki-67 (Spearman correlation coefficient=0.20, P=0.09). However, systemic levels of insulin, IGF-1, or IGF-2 were not associated with tissue immunostaining of p-IRS1.CONCLUSIONS:Activation of the insulin/IGF-1 pathway in BE may be associated with cellular proliferation and appears to have a role in the progression from metaplasia to cancer. The activation of the insulin/IGF-1 pathway at the tissue level is likely complex and does not have a simple association with systemic measures of insulin or IGF-1.

Does three months genistein and daidzein in the form of soy protein supplementation have any effects on bone formation markers after menopause

Postmenopausal osteoporosis is caused by a sharp decrease in estrogen levels leading to an increased rate of bone remodeling. Dietary supplements are preferred as alternative therapeutic options for many women instead of estrogen therapy. These alternative therapies include the use of natural substances such as soy isoflavones due to their weak estrogenic activity and affinity for estrogen receptors.Present study was carried out as a before and after clinical trial on 25 postmenopausal women aged 45-64 years. Soy protein at 35g level containing 98.3mg isoflavones (containing 47.2 genistein and 37.8 daidzein) were given to subjects daily for 12 weeks. Blood and urine samplings were done in 3 stages, in the beginning and at the end of 6th and 12th week. Repeated measurement analysis was employed to analyze any possible changes in food intake and biochemical variables in 3 stages. The level of significance was set at below 5 percent (P<0.05). Mean body mass index and physical activity level had no change and mean daily intake of macronutrients and important micronutrients were not different at 6 and 12 weeks compared to the start of the study The results showed a total serum alkaline phosphatase (TALP) significant increase while the other bone formation indicators namely osteocalcin and insulin growth factor binding protein (IGFBP3) did not change significantly. These data suggest that soy protein or its isoflavones may increase bone formation by supplementation.

Experimental Pain and Opioid Analgesia in Volunteers at High Risk for Obstructive Sleep Apnea

BackgroundObstructive sleep apnea (OSA) is characterized by recurrent nocturnal hypoxia and sleep disruption. Sleep fragmentation caused hyperalgesia in volunteers, while nocturnal hypoxemia enhanced morphine analgesic potency in children with OSA. This evidence directly relates to surgical OSA patients who are at risk for airway compromise due to postoperative use of opioids. Using accepted experimental pain models, we characterized pain processing and opioid analgesia in male volunteers recruited based on their risk for OSA.MethodsAfter approval from the Intitutional Review Board and informed consent, we assessed heat and cold pain thresholds and tolerances in volunteers after overnight polysomnography (PSG). Three pro-inflammatory and 3 hypoxia markers were determined in the serum. Pain tests were performed at baseline, placebo, and two effect site concentrations of remifentanil (1 and 2 µg/ml), an μ-opioid agonist. Linear mixed effects regression models were employed to evaluate the association of 3 PSG descriptors [wake after sleep onset, number of sleep stage shifts, and lowest oxyhemoglobin saturation (SaO2) during sleep] and all serum markers with pain thresholds and tolerances at baseline, as well as their changes under remifentanil.ResultsForty-three volunteers (12 normal and 31 with a PSG-based diagnosis of OSA) were included in the analysis. The lower nadir SaO2 and higher insulin growth factor binding protein-1 (IGFBP-1) were associated with higher analgesic sensitivity to remifentanil (SaO2, P = 0.0440; IGFBP-1, P = 0.0013). Other pro-inflammatory mediators like interleukin-1β and tumor necrosis factor-α (TNF-α) were associated with an enhanced sensitivity to the opioid analgesic effect (IL-1β, P = 0.0218; TNF-α, P = 0.0276).ConclusionsNocturnal hypoxemia in subjects at high risk for OSA was associated with an increased potency of opioid analgesia. A serum hypoxia marker (IGFBP-1) was associated with hypoalgesia and increased potency to opioid analgesia; other pro-inflammatory mediators also predicted an enhanced opioid potency.Trial Registration: ClinicalTrials.gov NCT00672737.

MicroRNA-140 Expression During Chondrogenic Differentiation of Equine Cord Blood-Derived Mesenchymal Stromal Cells

MicroRNAs are a class of short noncoding RNAs that are involved in various biological processes, including differentiation. MicroRNA-140 (miR-140) has been identified as a cartilage-specific microRNA with several targets involved in cartilage development and homeostasis. The aim of this study was to investigate the expression of miR-140 during chondrogenic differentiation of equine cord blood-derived mesenchymal stromal cells (eCB-MSCs). We demonstrate both that miR-140 is highly expressed in normal equine articular cartilage and that eCB-MSCs express significantly higher levels of this microRNA after 14 days of chondrogenic differentiation. Furthermore, miR-140 expression closely paralleled that of the cartilage-specific transcription factor Sox9, suggesting that miR-140 may be under the transcriptional regulation of Sox9 in these cells. The expression patterns of miR-140 targets the chemokine (CXC motif) ligand 12 (CXCL12), A disintegrin and metalloproteinase with thrombosponin motifs (ADAMTS)-5 and insulin growth factor binding protein 5 (IGFBP5) were also determined; however, only CXCL12 and ADAMTS-5 were repressed while miR-140 expression was upregulated. Together, these studies suggest that miR-140 is an important regulator of cartilage development and homeostasis in eCB-MSCs that may act, in part, through the regulation of CXCL12 and ADAMTS-5.

MDA-9/Syntenin and IGFBP-2 Promote Angiogenesis in Human Melanoma

Melanoma differentiation-associated gene-9 (mda-9/syntenin) encodes an adapter scaffold protein whose expression correlates with and mediates melanoma progression and metastasis. Tumor angiogenesis represents an integral component of cancer metastasis prompting us to investigate a possible role of mda-9/syntenin in inducing angiogenesis. Genetic (gain-of-function and loss-of-function) and pharmacologic approaches were used to modify mda-9/syntenin expression in normal immortal melanocytes, early radial growth phase melanoma, and metastatic melanoma cells. The consequence of modifying mda-9/syntenin expression on angiogenesis was evaluated using both in vitro and in vivo assays, including tube formation assays using human vascular endothelial cells, chorioallantoic membrane (CAM) assays and xenograft tumor animal models. Gain-of-function and loss-of-function experiments confirm that MDA-9/syntenin induces angiogenesis by augmenting expression of several proangiogenic factors/genes. Experimental evidence is provided for a model of angiogenesis induction by MDA-9/syntenin in which MDA-9/syntenin interacts with the extracellular matrix (ECM), activating Src and FAK resulting in activation by phosphorylation of Akt, which induces hypoxia inducible factor 1-α (HIF-1α). The HIF-1α activates transcription of insulin growth factor-binding protein-2 (IGFBP-2), which is secreted thereby promoting angiogenesis and further induces endothelial cells to produce and secrete VEGF-A augmenting tumor angiogenesis. Our studies delineate an unanticipated cell nonautonomous function of MDA-9/syntenin in the context of angiogenesis, which may directly contribute to its metastasis-promoting properties. As a result, targeting MDA-9/syntenin or its downstream-regulated molecules may provide a means of simultaneously impeding metastasis by both directly inhibiting tumor cell transformed properties (autonomous) and indirectly by blocking angiogenesis (nonautonomous).

Molecular and Circulatory Expression of Insulin Growth Factors in Indian Females with Advanced Cervical Cancer

Recent studies have demonstrated an association between insulin growth factor (IGF) and insulin growth factor binding protein-3 (IGFBP-III) serum levels and increased risk for various cancers. However, little information is available on clinical implications of the IGF system in Indian patients with cervical cancer. This study explored associations by analyzing their expression profiles in cervical cancer cases. Totals of 50 patients with advanced cervical cancer and 40 healthy controls were enrolled. Human papillomavirus (HPV) and cervical biopsy sample were obtained from all participating women. Circulatory levels were estimated by ELISA and the tissue expression was assessed using RT-PCR and Western blotting. Levels of IGF-I and II showed significant increase whereas IGFBP-III showed significant decline in all patients as compared to controls. Spearman correlation analysis between IGFs and HPV status showed significant correlations. We demonstrated elevated circulating levels and tissue expression of IGF-I and IGF-II in advancer cancer cervix patients, as compared with controls, with a converse trend being apparent for IGFBP-III. In future, associations of the IGF system and clinical outcome of cervical cancer patients in post treatment samples might point to significance in disease mapping as a prognostic marker after validation with a larger patient series.

Systemic inflammation associated with severe intestinal injury in extremely low gestational age newborns

To define the role of systemic inflammation in infants with intestinal perforation (IP) and necrotizing enterocolitis (NEC), we measured 25 blood protein concentrations on days 1, 7 and 14 in 939 infants born before 28 weeks’ gestation. On days 7 and 14, infants with NEC had elevated levels of C-reactive protein (CRP), serum amyloid A (SAA), IL-6 and IL-8. Infants with IP had elevated levels of CRP and insulin growth factor binding protein-1 on day 7 and elevated CRP, SAA, TNF-receptor-2 and matrix metalloproteinase-9 levels on day 14. A better understanding of systemic inflammation might help prevent and treat these disorders.

The dietary flavonoids naringenin and quercetin acutely impair glucose metabolism in rodents possibly via inhibition of hypothalamic insulin signalling

Secondary metabolites of herbs and spices are widely used as an alternative strategy in the therapy of various diseases. The polyphenols naringenin, quercetin and curcumin have been characterised as anti-diabetic agents. Conversely, in vitro, naringenin and quercetin are described to inhibit phosphoinositide-3-kinase (PI3K), an enzyme that is essential for the neuronal control of whole body glucose homoeostasis. Using both in vitro and in vivo experiments, we tested whether the inhibitory effect on PI3K occurs in neurons and if it might affect whole body glucose homoeostasis. Quercetin was found to inhibit basal and insulin-induced phosphorylation of Akt (Ser473), a downstream target of PI3K, in HT-22 cells, whereas naringenin and curcumin had no effect. In Djungarian hamsters (Phodopus sungorus) naringenin and quercetin (10 mg/kg administered orally) diminished insulin-induced phosphorylation of Akt (Ser473) in the arcuate nucleus, indicating a reduction in hypothalamic PI3K activity. In agreement with this finding, glucose tolerance in naringenin-treated hamsters (oral) and mice (oral and intracerebroventricular) was reduced compared with controls. Dietary quercetin also impaired glucose tolerance, whereas curcumin was ineffective. Circulating levels of insulin and insulin-like growth factor-binding protein were not affected by the polyphenols. Oral quercetin reduced the respiratory quotient, suggesting that glucose utilisation was impaired after treatment. These data demonstrate that low doses of naringenin and quercetin acutely and potently impair glucose homoeostasis. This effect may be mediated by inhibition of hypothalamic PI3K signalling. Whether chronic impairments in glucose homoeostasis occur after long-term application remains to be identified.

Molecular Characterization and Expression Pattern of Gene IGFBP-5 in the Cashmere Goat (Capra hircus).

Insulin-like growth factor-binding protein-5 (IGFBP-5) is one of the six members of IGFBP family, important for cell growth, apoptosis and other IGF-stimulated signaling pathways. In order to explore the significance of IGFBP-5 in cells of the Inner Mongolian Cashmere goat (Capra hircus), IGFBP-5 gene complementary DNA (cDNA) was amplified by reverse transcription polymerase chain reaction (RT-PCR) from the animal’s fetal fibroblasts and tissue-specific expression analysis was performed by semi-quantitative RT-PCR. The gene is 816 base pairs (bp) in length and includes the complete open reading frame, encoding 271 amino acids (GenBank accession number JF720883). The full cDNA nucleotide sequence has a 99% identity with sheep, 98% with cattle and 95% with human. The amino acids sequence shares identity with 99%, 99% and 99%, respectively. The bioinformatics analysis showed that IGFBP-5 has an insulin growth factor-binding protein homologues (IB) domain and a thyroglobulin type-1 (TY) domain, four protein kinase C phosphorylation sites, five casein kinase II phosphorylation sites, three prenyl group binding sites (CaaX box). The IGFBP-5 gene was expressed in all the tested tissues including testis, brain, liver, lung, mammary gland, spleen, and kidney, suggesting that IGFBP-5 plays an important role in goat cells.

The conserved Cockayne syndrome B-piggyBac fusion protein (CSB-PGBD3) affects DNA repair and induces both interferon-like and innate antiviral responses in CSB-null cells

Cockayne syndrome is a segmental progeria most often caused by mutations in the CSB gene encoding a SWI/SNF-like ATPase required for transcription-coupled DNA repair (TCR). Over 43Mya before marmosets diverged from humans, a piggyBac3 (PGBD3) transposable element integrated into intron 5 of the CSB gene. As a result, primate CSB genes now generate both CSB protein and a conserved CSB-PGBD3 fusion protein in which the first 5 exons of CSB are alternatively spliced to the PGBD3 transposase. Using a host cell reactivation assay, we show that the fusion protein inhibits TCR of oxidative damage but facilitates TCR of UV damage. We also show by microarray analysis that expression of the fusion protein alone in CSB-null UV-sensitive syndrome (UVSS) cells induces an interferon-like response that resembles both the innate antiviral response and the prolonged interferon response normally maintained by unphosphorylated STAT1 (U-STAT1); moreover, as might be expected based on conservation of the fusion protein, this potentially cytotoxic interferon-like response is largely reversed by coexpression of functional CSB protein. Interestingly, expression of CSB and the CSB-PGBD3 fusion protein together, but neither alone, upregulates the insulin growth factor binding protein IGFBP5 and downregulates IGFBP7, suggesting that the fusion protein may also confer a metabolic advantage, perhaps in the presence of DNA damage. Finally, we show that the fusion protein binds in vitro to members of a dispersed family of 900 internally deleted piggyBac elements known as MER85s, providing a potential mechanism by which the fusion protein could exert widespread effects on gene expression. Our data suggest that the CSB-PGBD3 fusion protein is important in both health and disease, and could play a role in Cockayne syndrome.

Cotransplantation of Ex Vivo Expanded and Unexpanded Cord Blood Units in Immunodeficient Mice Using Insulin Growth Factor Binding Protein-2–Augmented Mesenchymal Cell Cocultures

Ex vivo expansion of cord blood (CB) hematopoietic stem cells and cotransplantation of 2 CB units (CBUs) could enhance the applicability of CB transplantation in adult patients. We report an immunodeficient mouse model for cotransplantation of ex vivo expanded and unexpanded human CB, showing enhanced CB engraftment and provide proof of concept for this transplantation strategy as a means of overcoming the limiting cell numbers in each CBU. CBUs were expanded in serum-free medium supplemented with stem cell factor, Flt-3 ligand, thrombopoietin, and insulin growth factor binding protein-2 together with mesenchymal stromal cell coculture. Unexpanded and expanded CB cells were cotransplanted by tail vein injection into 45 sublethally irradiated nonobese diabetic SCID-IL2γ(-/-) (NSG) mice. Submandibular bleeding was performed monthly, and mice were sacrificed 4 months after transplantation to analyze for human hematopoietic engraftment. Expansion of non-CD34(+) selected CB cells yielded 40-fold expansion of CD34(+) cells and 3.1-fold expansion of hematopoietic stem cells based on limiting dilution analysis of NSG engraftment. Mice receiving expanded grafts exhibited 4.30% human cell repopulation, compared with 0.92% in mice receiving only unexpanded grafts at equivalent starting cell doses, even though the unexpanded graft predominated in long-term hematopoiesis (P = .07). Ex vivo expanded grafts with lower initiating cell doses also showed equivalent engraftment to unexpanded grafts with higher cell dose (8.0% versus 7.9%; P = .93). In conclusion, ex vivo expansion resulted in enhanced CB engraftment despite eventual rejection by the unexpanded CBU.

Effect of zinc supplementation on growth Hormone Insulin growth factor axis in short Egyptian children with zinc deficiency

BackgroundThe relationship between zinc (Zn) and growth hormone-insulin growth factor (GH-IGF) system and how Zn therapy stimulates growth in children has not been clearly defined in humans. Thus, we aimed to assess GH-IGF axis in short children with Zn deficiency and to investigate the effect of Zn supplementation on these parameters.MethodsFifty pre-pubertal Egyptian children with short stature and Zn deficiency were compared to 50 age-, sex-, and pubertal stage- matched controls. All subjects were subjected to history, auxological assessment and measurement of serum Zn, IGF-1, insulin growth factor binding protein-3 (IGFBP-3); and basal and stimulated GH before and 3 months after Zn supplementation (50 mg/day).ResultsAfter 3 months of Zn supplementation in Zn-deficient patients, there were significant increases in height standard deviation score (SDS, P = 0.033), serum Zn (P < 0.001), IGF-1 (P < 0.01), IGF-1 standard deviation score (SDS,P < 0.01) and IGFBP-3 (P = 0.042). Zn rose in all patients but reached normal ranges in 64 %, IGF-1 levels rose in 60 % but reached normal ranges in 40 % and IGFBP-3 levels rose in 40 % but reached reference ranges in 22 %. Growth velocity (GV) SDS did not differ between cases and controls (p = 0.15) but was higher in GH-deficient patients than non-deficient ones, both having Zn deficiency (p = 0.03).ConclusionSerum IGF-1 and IGFBP-3 levels were low in short children with Zn deficiency, and increased after Zn supplementation for 3 months but their levels were still lower than the normal reference ranges in most children; therefore, Zn supplementation may be necessary for longer periods.

A hippocampal insulin-growth factor 2 pathway regulates the extinction of fear memories

Extinction learning refers to the phenomenon that a previously learned response to an environmental stimulus, for example, the expression of an aversive behaviour upon exposure to a specific context, is reduced when the stimulus is repeatedly presented in the absence of a previously paired aversive event. Extinction of fear memories has been implicated with the treatment of anxiety disease but the molecular processes that underlie fear extinction are only beginning to emerge. Here, we show that fear extinction initiates upregulation of hippocampal insulin-growth factor 2 (Igf2) and downregulation of insulin-growth factor binding protein 7 (Igfbp7). In line with this observation, we demonstrate that IGF2 facilitates fear extinction, while IGFBP7 impairs fear extinction in an IGF2-dependent manner. Furthermore, we identify one cellular substrate of altered IGF2 signalling during fear extinction. To this end, we show that fear extinction-induced IGF2/IGFBP7 signalling promotes the survival of 17-19-day-old newborn hippocampal neurons. In conclusion, our data suggest that therapeutic strategies that enhance IGF2 signalling and adult neurogenesis might be suitable to treat disease linked to excessive fear memory.

The requirement for fibroblasts in angiogenesis: fibroblast-derived matrix proteins are essential for endothelial cell lumen formation

A role for fibroblasts in physiological and pathological angiogenesis is now well recognized; however, the precise mechanisms underlying their action have not been determined. Using an in vitro angiogenesis model in combination with a candidate gene approach, column chromatography, and mass spectrometry, we identify two classes of fibroblast-derived factors--one that supports vessel sprouting but not lumen formation, and one that promotes lumen formation. In the absence of fibroblasts a combination of angiopoietin-1, angiogenin, hepatocyte growth factor, transforming growth factor-α, and tumor necrosis factor drives robust endothelial cell (EC) sprouting; however, lumens fail to form. Subsequent addition of fibroblast-conditioned medium restores lumenogenesis. Using small interfering RNA-mediated knockdown, we show that five genes expressed in fibroblasts--collagen I, procollagen C endopeptidase enhancer 1, secreted protein acidic and rich in cysteine, transforming growth factor-β-induced protein ig-h3, and insulin growth factor-binding protein 7--are necessary for lumen formation. Moreover, lumen formation can be rescued by addition of purified protein to knockdown cultures. Finally, using rheology, we demonstrate that the presence of these matricellular proteins results in significantly stiffer gels, which correlates with enhanced lumen formation. These findings highlight the critical role that fibroblast-derived extracellular matrix components play in EC lumen formation and provide potential insight into the role of fibroblasts in the tumor microenvironment.

Copper ability to induce premature senescence in human fibroblasts

Human diploid fibroblasts (HDFs) exposed to subcytotoxic concentrations of oxidative or stressful agents, such as hydrogen peroxide, tert-butylhydroperoxide, or ethanol, undergo stress-induced premature senescence (SIPS). This condition is characterized by the appearance of replicative senescence biomarkers such as irreversible growth arrest, increase in senescence-associated β-galactosidase (SA β-gal) activity, altered cell morphology, and overexpression of several senescence-associated genes. Copper is an essential trace element known to accumulate with ageing and to be involved in the pathogenesis of some age-related disorders. Past studies using either yeast or human cellular models of ageing provided evidence in favor of the role of intracellular copper as a longevity modulator. In the present study, copper ability to cause the appearance of senescent features in HDFs was assessed. WI-38 fibroblasts exposed to a subcytotoxic concentration of copper sulfate presented inhibition of cell proliferation, cell enlargement, increased SA β-gal activity, and mRNA overexpression of several senescence-associated genes such as p21, apolipoprotein J (ApoJ), fibronectin, transforming growth factor β-1 (TGF β1), insulin growth factor binding protein 3, and heme oxygenase 1. Western blotting results confirmed enhanced intracellular p21, ApoJ, and TGF β1 in copper-treated cells. Thus, similar to other SIPS-inducing agents, HDF exposure to subcytotoxic concentration of copper results in premature senescence. Further studies will unravel molecular mechanisms and the biological meaning of copper-associated senescence and lead to a better understanding of copper-related disorder establishment and progression.

Effects of a short‐term increase in the nutritional plane before the mating period on metabolic and endocrine parameters, hepatic gene expression and reproduction in primiparous beef cows on grazing conditions

Sixty-four spring-calved primiparous crossbred cows paired by calving date and body condition score (BCS) at calving were used to study the effect of a short-term increase in the nutritional plane before the mating period on cow and calf performance, changes in metabolic and endocrine parameters and hepatic gene expression. At 48 ± 10 days post-partum (onset of nutritional treatment = day 0), cows were assigned to two treatments during 23 days: control (grazing of native pastures; NP; n = 31) and increased nutritional plane (NP improved with Lotus subbiflorous cv Rincon; IP; n = 33). Cow body weight (BW), BCS and total protein and albumin concentrations increased while urea and non-esterified fatty acids levels decreased from the beginning of the nutritional treatment in both groups, indicating the animal positive energy balance as forage growth and availability of pastures increased during spring. In addition, cow BW and BCS, as well as calf average daily gain and BW, were greater in IP than in NP cows groups. Insulin concentrations were less in IP than in NP (1.37 vs. 2.25 ± 0.26 μU/ml) because insulin increased owing to nutritional treatment only in NP cows. Hepatic insulin receptor mRNA at day 23 tended to be 1.5-fold greater, while insulin growth factor binding protein-3 mRNA expression was 1.7-fold greater in NP than in IP cows. Reproductive responses were not affected by nutritional treatment, but days to initiation of ovarian ciclicity (108 ± 10 days) were positively correlated with insulin concentrations. Grazing of improved NP for 23 days before the mating period did not improve cow reproductive performance but modified metabolic, endocrine and gene expression parameters, in agreement with greater nutrient and energy partitioning towards milk production, reflected in better calf performance.

Identification of Cells with Colony-Forming Activity, Self-Renewal Capacity, and Multipotency in Ovarian Endometriosis

Endometriosis, the growth of endometrial tissue outside the uterine cavity, is a common gynecological disorder affecting 10% to 15% of women in their reproductive years. Retrograde menstrual shedding containing endometrial stem/progenitor cells has been postulated to be involved in its pathogenesis. In this study, we identified putative endometriotic stem/progenitor cells by their colony-forming potential, self-renewal capacity, and multipotency. Purified epithelial and stromal cells isolated from ovarian endometriotic cysts formed large and small colony-forming units (CFUs) in clonogenic assay. The colony-forming activity of epithelial and stromal cells was found to differ greatly between autologous endometrium and ovarian endometrioma samples. The large CFUs could propagate more than the small CFUs. The endometriotic epithelial small CFUs expressed epithelial markers (epithelial cell adhesion molecule, cytokeratin, and α6 integrin); only occasional large CFUs expressed α6 integrin. Aside from the expression of fibroblast markers, stromal CFUs also expressed three somatic stem cell markers: sal-like 4, CD133, and Musashi-1. Endometriotic stromal cells derived from large CFUs could differentiate into four mesenchymal lineages when cultured in the respective inducing-media, as determined by histochemical staining and RT-PCR of lineage specific markers. These findings demonstrate that ovarian endometrioma contains a subset of cells displaying somatic stem cell properties.