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Exhibit 1:
ATM regulates insulin-like growth factor 1-secretory clusterin (IGF-1-sCLU) expression that protects cells against senescence.
Abstract
Downstream factors that regulate the decision between senescence and cell death have not been elucidated. Cells undergo senescence through three pathways, replicative senescence (RS), stress-induced premature senescence (SIPS) and oncogene-induced senescence. Recent studies suggest that the ataxia telangiectasia mutant (ATM) kinase is not only a key protein mediating cellular responses to DNA damage, but also regulates cellular senescence induced by telomere end exposure (in RS) or persistent DNA damage (in SIPS). Here, we show that expression of secretory clusterin (sCLU), a known pro-survival extracellular chaperone, is transcriptionally up-regulated during both RS and SIPS, but not in oncogene-induced senescence, consistent with a DNA damage-inducible mechanism. We demonstrate that ATM plays an important role in insulin-like growth factor 1 (IGF-1) expression, that in turn, regulates downstream sCLU induction during senescence. Loss of ATM activity, either by genomic mutation (ATM-deficient fibroblasts from an ataxia telangiectasia patient) or by administration of a chemical inhibitor (AAI, an inhibitor of ATM and ATR), blocks IGF-1-sCLU expression in senescent cells. Downstream, sCLU induction during senescence is mediated by IGF-1R/MAPK/Egr-1 signaling, identical to its induction after DNA damage. In contrast, administration of an IGF-1 inhibitor caused apoptosis of senescent cells. Thus, IGF-1 signaling is required for survival, whereas sCLU appears to protect cells from premature senescence, as IMR-90 cells with sCLU knockdown undergo senescence faster than control cells. Thus, the ATM-IGF-1-sCLU pathway protects cells from lethality and suspends senescence.
Exhibit 2:
Insulin-like growth factor-1 regulates the SIRT1-p53 pathway in cellular senescence
Summary
Cellular senescence, which is known to halt proliferation of aged and stressed cells, plays a key role against cancer development and is also closely associated with organismal aging. While increased insulin-like growth factor (IGF) signaling induces cell proliferation, survival and cancer progression, disrupted IGF signaling is known to enhance longevity concomitantly with delay in aging processes. The molecular mechanisms involved in the regulation of aging by IGF signaling and whether IGF regulates cellular senescence are still poorly understood. In this study, we demonstrate that IGF-1 exerts a dual function in promoting cell proliferation as well as cellular senescence. While acute IGF-1 exposure promotes cell proliferation and is opposed by p53, prolonged IGF-1 treatment induces premature cellular senescence in a p53-dependent manner. We show that prolonged IGF-1 treatment inhibits SIRT1 deacetylase activity, resulting in increased p53 acetylation as well as p53 stabilization and activation, thus leading to premature cellular senescence. In addition, either expression of SIRT1 or inhibition of p53 prevented IGF-1-induced premature cellular senescence. Together, these findings suggest that p53 acts as a molecular switch in monitoring IGF-1-induced proliferation and premature senescence, and suggest a possible molecular connection involving IGF-1-SIRT1-p53 signaling in cellular senescence and aging.
Exhibit 3:
IGF-1 Enhances Expression of iPS Cell Reprogramming Factors and Telomerase activities in Human Dermal Fibroblasts: A potential Role for IGF-1 in Induction of Pluripotent Stem Cells and Antisenescence
Background: Reprogramming somatic cells into induced pluropotent stem cells (iPS) using transcription factors (Oct4, Sox2, Klf4 and c-myc) offers a novel approach for regenerative medicine. However, the iPS cell generation has several obstacles, including the poor efficiency of iPS reprogramming and frequent re-entry of iPS cells into the stage of senescence during passage. Insulin-like growth factor (IGF-1) and its downstream phosphoinositide 3-kinase (PI3K) signaling pathway may maintain the proliferative potential of stem cells. This study was to test the effects of IGF-1 on iPS cell generation and senescence.
Conclusion: IGF-1 may enhance expression of programming factors and telomerase activities in human dermal fibroblasts transduced into iPS cells, while inhibiting expression of the senescence-inducing protein p53. The effects of IGF-1 may be mediated by the PI3 kinase/Akt pathway. Our data demonstrate that the novel biological effects of IGF-1 on iPS generation, and may allow development of an enhanced therapeutic option by employing iPS for treating cardiovascular diseases.
Exhibit 4:
Abstract
The accumulation of senescent stromal cells in aging tissue changes the local microenvironment from normal to a state similar to chronic inflammation. This inflammatory microenvironment can stimulate the proliferation of epithelial cells containing DNA mutations which can ultimately lead to cancer. Using geriatric skin as a model, we demonstrated that senescent fibroblasts also alter how epithelial keratinocytes respond to genotoxic stress, due to the silencing of IGF-1 expression in geriatric fibroblasts. These data indicate that in addition to promoting epithelial tumor growth, senescent fibroblasts also can promote carcinogenic initiation. We hypothesized that commonly used therapeutic stromal wounding therapies can reduce the percentage of senescent fibroblasts and consequently prevent the formation of keratinocytes proliferating with DNA mutations following acute genotoxic (UVB) stress. Sun-protected skin on the lower back of geriatric human volunteers was wounded by dermabrasion and the skin was allowed to heal for three months. In geriatric skin, we found that dermabrasion wounding decreases the proportion of senescent fibroblasts found in geriatric dermis, increases the expression of IGF-1, and restores the appropriate UVB response to epidermal keratinocytes in geriatric skin. Therefore, dermal rejuvenation therapies may play a significant role in preventing the initiation of skin cancer in geriatric patients.
Exhibit 5:
Bioactive IGF-1 release from collagen–GAG scaffold to enhance cartilage repair in vitro
Abstract
Tissue engineering is a promising technique for cartilage repair. Toward this goal, a porous collagen–glycosaminoglycan (CG) scaffold was loaded with different concentrations of insulin-like growth factor-1 (IGF-1) and evaluated as a growth factor delivery device. The biological response was assessed by monitoring the amount of type II collagen and proteoglycan synthesised by the chondrocytes seeded within the scaffolds. IGF-1 release was dependent on the IGF-1 loading concentration used to adsorb IGF-1 onto the CG scaffolds and the amount of IGF-1 released into the media was highest at day 4. This initial IGF-1 release could be modelled using linear regression analysis. Osteoarthritic (OA) chondrocytes seeded within scaffolds containing adsorbed IGF-1 deposited decorin and type II collagen in a dose dependent manner and the highest type II collagen deposition was achieved via loading the scaffold with 50 μg/ml IGF-1. Cells seeded within the IGF-1 loaded scaffolds also deposited more extracellular matrix than the no growth factor control group thus the IGF-1 released from the scaffold remained bioactive and exerted an anabolic effect on OA chondrocytes. The effectiveness of adsorbing IGF-1 onto the scaffold may be due to protection of the molecule from proteolytic digestion allowing a more sustained release of IGF-1 over time compared to adding multiple doses of exogenous growth factor. Incorporating IGF-1 into the CG scaffold provided an initial therapeutic burst release of IGF-1 which is beneficial in initiating ECM deposition and repair in this in vitro model and shows potential for developing this delivery device in vivo.
Exhibit 6:
GW25-e4120 IGF-1 Inhibits Apoptosis of Vascular Smooth Muscle Cells Through PI3/Akt Pathway
In this report, we show that the signaling cascade involved in IGF-1 protectes VSMC against Apop-1-induced apoptosis, while PDGF has no effect. In addition, pretreatment of Apop-1 transfected VSMCs with phosphatidylinositol-3-kinase inhibitor wortmannin, or infection with an adenoviral construct expressing the dominant negative Akt gene (Adeno-dnAkt) blocked the cytoprotective effect of IGF-1, whereas the MEK inhibitor PD98059 had no effect. Conversely, infection with an adenoviral construct expressing the constitutively active Akt (Adeno-MyrAkt) gene, protected VSMC from apoptosis induced by Apop-1 even in the absence of IGF-1, suggesting that IGF-1 prevents VSMC apoptosis induced by Apop-1 through activation of the PI3K/Akt pathway. Furthermore, IGF-1 elevated phospho-Akt expression in Apop-1 transfected VSMCs and Apop-1 decreased phospho-Akt expression. Importantly, IGF-1 inhibited cytochrome c release from mitochondria and blocked activation of intrinsic initiator caspase-9 in Apop-1 transfected VSMCs
Exhibit 7:
Abstract
Apoptosis and cell proliferation are two important cellular processes that determine the accumulation of pulmonary artery smooth muscle cells (PASMC) during pulmonary arterial hypertension (PAH). Insulin-like growth factor 1 (IGF-1) is an endocrine and autocrine/paracrine growth factor that circulates at high levels in the plasma and is expressed in most cell types. IGF-1 has major effects on development, cell growth and differentiation, also tissue repair. Inducible nitric oxide synthase (iNOS) has been shown to serve many vasoprotective roles in vascular smooth muscle cells (VSMCs) including inhibition of VSMC proliferation and migration and stimulation of endothelial cell growth. In this study, we investigated the involvement of iNOS in the process of IGF-1-induced inhibition of PASMC apoptosis. We also examined the role of p38 mitogen-activated protein kinase (MAPK) in the IGF-1-induced iNOS activation. Our results show that exogenous IGF-1 induced the up-regulation of iNOS in PASMC. Immunofluorescence of IGF-1 and iNOS showed a decreased immunostaining of both IGF-1 and iNOS in the cytoplasm and the perinucleus under serum deprivation condition. iNOS inhibition in PASMC in vitro markedly induced IGF-1-mediated anti-apoptosis as assessed by the cell viability measurement, Western blot, mitochondrial potential analysis and nuclear morphology determination. A p38 MAPK inhibitor blocked all the effects of IGF-1 on iNOS. Our findings suggest that IGF-1 inhibits cells apoptosis in PASMC by activating the p38 MAPK–iNOS transduction pathway. This mechanism may contribute to the accumulation of PASMC in early human PAH.
Exhibit 9:
Myostatin, a TGF-β family member, is associated with inhibition of muscle growth and differentiation and might interact with the IGF-1 signaling pathway. Since IGF-1 is secreted at a bioactive level by adipose tissue-derived mesenchymal stem cells (ASCs), these cells (ASCs) provide a therapeutic option for Duchenne Muscular Dystrophy (DMD). But the protective effect of stem cell secreted IGF-1 on myoblast under high level of myostatin remains unclear. In the present study murine myoblasts were exposed to myostatin under presence of ASCs conditioned medium and investigated for proliferation and apoptosis. The protective effect of IGF-1 was further examined by using IGF-1 neutralizing and receptor antibodies as well as gene silencing RNAi technology. MyoD expression was detected to identify impact of IGF-1 on myoblasts differentiation when exposed to myostatin. IGF-1 was accountable for 43.6% of the antiapoptotic impact and 48.8% for the proliferative effect of ASCs conditioned medium. Furthermore, IGF-1 restored mRNA and protein MyoD expression of myoblasts under risk. Beside fusion and transdifferentiation the beneficial effect of ASCs is mediated by paracrine secreted cytokines, particularly IGF-1. The present study underlines the potential of ASCs as a therapeutic option for Duchenne muscular dystrophy and other dystrophic muscle diseases.
Exhibit 10:
Abstract
Objective
Insulin-like growth factor 1 (IGF-1) increases the growth of cultured hair follicles and plays a role in regulating hair migration during the development of hair follicles in transgenic mice. However, the exogenous effect of IGF-1 on hair growth in wild-type mice has not been reported. In the present study, we examined whether IGF-1 was an important regulator of hair follicle growth in wide-type mice in vivo.
Design
C57BL/6 mice were injected with different concentrations of IGF-1 on dorsal skin. The treated tissues were analyzed by immunoassay methods for TGF-β1 and BrdU.
Results
Local injection of IGF-1 increased hair follicle number and prolonged the growing phase during the transition from anagen to telogen. Meanwhile, immunology analyses revealed that IGF-1 also stimulated the proliferation of follicle cells in anagen of the matrix and down regulated TGF-β1 expression in hair follicles.
Conclusions
These observations suggest that IGF-1 is an effective stimulator of hair follicle development in wide-type mice in vivo and may be a promising drug candidate for baldness therapy.
Exhibit 11:
Abstract
Reactive oxygen species (ROS)-induced oxidative stress increases in skeletal muscle with aging and decreases the viability of implanted cells. Type 1 insulin-like growth factor (IGF-1) promotes the survival of skeletal muscle cells under oxidative stress. It is unknown whether IGF-1 protects muscle-derived stem cells (MDSCs) from oxidative stress. In this study, we genetically engineered rat MDSCs to overexpress IGF-1 and determined cell viability, apoptosis, and VEGF secretion under oxidative stress. Overexpression of IGF-1 prevented MDSCs from H2O2-induced caspase-dependent apoptotic cell death by upregulating the PI3K/AKT pathway, accompanied with an increase of NF-κB, p-NF-κB, Bcl-2, and VEGF, as well as a decrease of Bax. In contrast, pre-administration of picropodophyllinb, wortmannin, 1L-6-hydroxymethyl-chiro-inositol-2-((R)-2-O-methyl-3-O-octadecylcarbonate), or pyrrolidine-dithiocarbamate, specific inhibitors of IGF-1R, PI3K, AKT, and NF-κB, respectively, followed by treatment with H2O2, resulted in cell death of MDSCs. Our data indicated that IGF-1 suppresses apoptosis and enhances the paracrine function of MDSCs under oxidative stress via enhancing IGF-1R/PI3K/AKT signaling. Thus, IGF-1 gene-modified MDSCs present a potential application in the treatment of muscle wasting, such as urethra intrinsic sphincter deficiency.
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