Release Of Insulin-like Growth Factor Research Articles

Abstract 107: IGF-1 Nanoparticles Improve Functional Recovery After Nerve Repair

PURPOSE: Insulin-like growth factor 1 (IGF-1) is a potent mitogen with well-described trophic and anti-apoptotic effects on neurons, myocytes, and Schwann cells (SCs). Local delivery of IGF-1 is limited by its short half-life. The aims of this study are to (1) encapsulate IGF-1 into biodegradable nanoparticles (NPs) that stabilize IGF-1 in its bioactive state and enable sustained release at target tissue sites that persist throughout the regenerative period; and (2) assess the efficacy of locally delivered IGF-1 NPs in augmenting axonal regeneration while also reducing denervation-induced muscle atrophy and SC senescence to thereby improve functional recovery following nerve injury. METHODS: (1) NP Fabrication: IGF-1 was first complexed with dextran sulfate to create hydrophobic ionic paired (HIP) complexes, which were then encapsulated in biodegradable NPs. Varying ratios of HIP:polymer were evaluated to maximize loading efficiency and release kinetics. In vitro NP release kinetics were evaluated and mitogenic activity of released IGF-1 was compared to native IGF-1. (2) The effects of locally-delivered IGF-1 NPs on denervated muscle and Schwann cells were assessed in a rat median nerve transection-without-repair model. The effects of IGF-1 NPs on axonal regeneration, muscle atrophy and reinnervation, and recovery of forepaw function were assessed in a model in which chronic denervation is induced prior to nerve repair; functional recovery was assessed weekly with stimulated grip strength testing prior to sacrifice at 15 weeks for histologic analyses. RESULTS: (1) Fabrication of uniform NPs with an encapsulation efficiency of 83.2% was achieved. NPs composed of 1:5 PEG5k-PCL40k yielded optimal release of IGF-1. Near-zero-order release of IGF-1 can be achieved for at least 70 days and released IGF-1 exhibits comparable bioactivity to native IGF-1. (2) IGF-1 treated animals recovered significantly more forceful grip strength compared to negative controls. IGF-1 NP treatment limits muscle atrophy during periods of denervation compared to negative controls (620 vs. 340μm2, respectively; p <0.05). CONCLUSION: Encapsulation of bioactive IGF-1 with sustained release for over 70 days was achieved. IGF-1 NP treatment in vivo limits muscle atrophy during denervation and improves functional recovery of forelimb grip strength.

Polymeric mesh and insulin-like growth factor 1 delivery enhance cell homing and graft-cartilage integration.

Cartilage injury, such as full-thickness lesions, predisposes patients to the premature development of osteoarthritis, a degenerative joint disease. While surgical management of cartilage lesions has improved, long-term clinical efficacy has stagnated, owing to the lack of hyaline cartilage regeneration and inadequate graft-host integration. This study tests the hypothesis that integration of cartilage grafts with native cartilage can be improved by enhancing the migration of chondrocytes across the graft-host interface via the release of chemotactic factor from a degradable polymeric mesh. To this end, a polylactide-co-glycolide/poly-ε-caprolactone mesh was designed to localize the delivery of insulin-like growth factor 1 (IGF-1), a well-established chondrocyte attractant. The release of IGF-1 (100ng/mg) enhanced cell migration from cartilage explants, and the mesh served as critical structural support for cell adhesion, growth, and production of a cartilaginous matrix in vitro, which resulted in increased integration strength compared with mesh-free repair. Further, this neocartilage matrix was structurally contiguous with native and grafted cartilage when tested in an osteochondral explant model in vivo. These results demonstrate that this combined approach of a cell homing factor and supportive matrix will promote cell-mediated integrative cartilage repair and improve clinical outcomes of cartilage grafts in the treatment of osteoarthritis.

STAT6, PBX2, and PBRM1 Emerge as Predicted Regulators of 452 Differentially Expressed Genes Associated With Puberty in Brahman Heifers.

The liver plays a central role in metabolism and produces important hormones. Hepatic estrogen receptors and the release of insulin-like growth factor 1 (IGF1) are critical links between liver function and the reproductive system. However, the role of liver in pubertal development is not fully understood. To explore this question, we applied transcriptomic analyses to liver samples of pre- and post-pubertal Brahman heifers and identified differentially expressed (DE) genes and genes encoding transcription factors (TFs). Differential expression of genes suggests potential biological mechanisms and pathways linking liver function to puberty. The analyses identified 452 DE genes and 82 TF with significant contribution to differential gene expression by using a regulatory impact factor metric. Brain-derived neurotrophic factor was observed as the most down-regulated gene (P = 0.003) in post-pubertal heifers and we propose this gene influences pubertal development in Brahman heifers. Additionally, co-expression network analysis provided evidence for three TF as key regulators of liver function during pubertal development: the signal transducer and activator of transcription 6, PBX homeobox 2, and polybromo 1. Pathway enrichment analysis identified transforming growth factor-beta and Wnt signaling pathways as significant annotation terms for the list of DE genes and TF in the co-expression network. Molecular information regarding genes and pathways described in this work are important to further our understanding of puberty onset in Brahman heifers.

Delivering Growth Factors through a Polymeric Scaffold to Cell Cultures Containing both Nucleus Pulposus and Annulus Fibrosus.

To design a novel, polyvinyl alcohol (PVA)-based polymeric scaffold that permits the controlled release of insulin-like growth factor 1 (IGF-1)/bone morphogenetic protein (BMP)-2 following intervertebral disc administration. The drug delivery system was composed of two different solutions that formed a scaffold within seconds of coming into contact with each other. Swelling, pH, and temperature tests and analysis of the controlled release of growth factors (GFs) from this system were performed. The release kinetics of the GFs were determined through enzyme-linked immunosorbent assay (ELISA). Cell proliferation and viability were monitored with microscopy and analyzed using an MTT assay and acridine orange/propidium iodide (AO/PI) staining. Chondroadherin (CHAD), hypoxia inducible factor-1 alpha (HIF-1?), and collagen type II (COL2A1) gene expressions were determined with quantitative real-time polymerase chain reaction (qRT-PCR) analysis to show the effects of IGF-1/BMP-2 administration on annulus fibrosus cell (AFC)/nucleus pulposus cell (NPC) cultures. For the statistical evaluation of the obtained data, experimental groups were compared with a post hoc Tukey's test following an analysis of variance. The scaffold allowed for the controlled release of IGF-1 and BMP-2 in different time intervals. It was observed that as the application time increased, the number of cells and the degree of extracellular matrix development increased in AFC/NPC cultures. AO/PI staining and an MTT analysis showed that cells retained their specific morphology and continued to proliferate. It was observed that HIF-1? and CHAD expression increased in a time-dependent manner, and no COL2A1 expression in the AFC/ NPC cultures was observed. The designed scaffold may be used as an alternative method for intervertebral disc administration of GFs after further in vivo studies. Such prototype scaffolds may be an innovative technology in targeted drug therapies after reconstructive neurosurgical interventions.

What Happened to the IGF Binding Proteins?

Insulinlike growth factor (IGF) binding proteins (IGFBPs) 1 to 6 are high-affinity regulators of IGF activity. They generally inhibit IGF actions by preventing binding to the IGF-I receptor but can also enhance their actions under some conditions. Posttranslational modifications such as glycosylation and phosphorylation modulate IGFBP properties, and IGFBP proteolysis results in IGF release. IGFBPs have more recently been shown to have IGF-independent actions. A number of mechanisms are involved, including modulation of other growth factor pathways, nuclear localization and transcriptional regulation, interaction with the sphingolipid pathway, and binding to non-IGF biomolecules in the extracellular space and matrix, on the cell surface and intracellularly. IGFBPs modulate important biological processes, including cell proliferation, survival, migration, senescence, autophagy, and angiogenesis. Their actions have been implicated in growth, metabolism, cancer, stem cell maintenance and differentiation, and immune regulation. Recent studies have shown that epigenetic mechanisms are involved in the regulation of IGFBP abundance. A more complete understanding of IGFBP biology is necessary to further define their cellular roles and determine their therapeutic potential.

Controlled release of insulin-like growth factor 1 enhances urethral sphincter function and histological structure in the treatment of female stress urinary incontinence in a rat model.

To determine the effects of controlled release of insulin-like growth factor 1 (IGF-1) from alginate-poly-L-ornithine-gelatine (A-PLO-G) microbeads on external urethral sphincter (EUS) tissue regeneration in a rat model of stress urinary incontinence (SUI), as SUI diminishes the quality of life of millions, particularly women who have delivered vaginally, which can injure the urethral sphincter. Despite several well-established treatments for SUI, growth factor therapy might provide an alternative to promote urethral sphincter repair. In all, 44 female Sprague-Dawley rats were randomised into four groups: vaginal distension (VD) followed by periurethral injection of IGF-1-A-PLO-G microbeads (VD + IGF-1 microbeads; 1 × 104 microbeads/1 mL normal saline); VD + empty microbeads; VD + saline; or sham-VD + saline (sham). Urethral function (leak-point pressure, LPP) was significantly lesser 1 week after VD + saline [mean (sem) 23.9 (1.3) cmH2 O] or VD + empty microbeads [mean (sem) 21.7 (0.8) cmH2 O) compared to the sham group [mean (sem) 44.4 (3.4) cmH2 O; P < 0.05), indicating that the microbeads themselves do not create a bulking or obstructive effect in the urethra. The LPP was significantly higher 1 week after VD + IGF-1 microbeads [mean (sem) 28.4 (1.2) cmH2 O] compared to VD + empty microbeads (P < 0.05), and was not significantly different from the LPP in sham rats, demonstrating an initiation of a reparative effect even at 1 week after VD. Histological analysis showed well-organised skeletal muscle fibres and vascular development in the EUS at 1 week after VD + IGF-1 microbeads, compared to substantial muscle fibre attenuation and disorganisation, and less vascular formation at 1 week after VD + saline or VD + empty microbeads. Periurethral administration of IGF-1-A-PLO-G microbeads facilitates recovery from SUI by promoting skeletal myogenesis and revascularisation. This therapy is promising, but detailed and longer term studies in animal models and humans are needed.

Can xylene and chia (&lt;i&gt;Salvia hispanica&lt;/i&gt; L.) seed extract directly affect basic bovine ovarian cell functions?

It was supposed, that xylene as environmental contaminant and chia (Salvia hispanica L.) as a medicinal plant may affect ovarian cell functions, and that chia seed extract could modify the potentially adverse effect of xylene. So, the aim of our investigation was to study the effect of xylene, extract of chia seed and their combination on bovine ovarian cell functions (proliferation, apoptosis and secretory activity) in vitro. Proliferation, apoptosis and the release of hormones (insulin-like growth factor 1 (IGF-1), progesterone and testosterone) were analysed with the use of either immunocytochemistry or EIA/RIA. It was observed, that xylene when given alone stimulated proliferation and did not influence the apoptosis. Furthermore, xylene inhibited release of progesterone and testosterone but did not change the release of IGF-1. Chia seed extract inhibited proliferation, apoptosis and the release of IGF-1, progesterone and testosterone. Moreover, chia seed extract suppressed the stimulatory effect of xylene on proliferation, and added together stimulated apoptosis. The simultaneous addition of chia seed extract and xylene stimulated IGF-1 release and inhibited the release of progesterone, but not testosterone. The obtained results prove a direct effect of both xylene and chia seed extract on bovine ovarian cell proliferation, apoptosis and secretory activity. Moreover, it is the first study in which chia seed extract was able to suppress xylene effect on ovarian cell proliferation. However, it prevented only from one xylene effect among five analysed suggesting that chia seed extract could not be potentially useful for natural prevention of all negative effects of xylene on reproduction.

Essential Role of Growth Hormone and IGF-1 in Therapeutic Effect of Ghrelin in the Course of Acetic Acid-Induced Colitis.

Previous studies have shown that ghrelin exhibits a protective and therapeutic effect in the gut. The aim of the present study was to examine whether administration of ghrelin affects the course of acetic acid-induced colitis and to determine what is the role of growth hormone (GH) and insulin-like growth factor-1 (IGF-1) in this effect. In sham-operated or hypophysectomized male Wistar rats, colitis was induced by enema with 1 mL of 3% solution of acetic acid. Saline or ghrelin (given at the dose of 8 nmol/kg/dose) was administered intraperitoneally twice a day. Seven days after colitis induction, rats were anesthetized and the severity of the colitis was assessed. Treatment with ghrelin reduced the area of colonic mucosa damage in pituitary-intact rat. This effect was associated with increase in serum levels of GH and IGF-1. Moreover, administration of ghrelin improved blood flow in colonic mucosa and mucosal cell proliferation, as well as reduced mucosal concentration of proinflammatory interleukin-1β (IL-1β) and activity of myeloperoxidase. Hypophysectomy reduced serum levels of GH and IGF-1 and increased the area of colonic damage in rats with colitis. These effects were associated with additional reduction in mucosal blood follow and DNA synthesis when compared to pituitary-intact rats. Mucosal concentration of IL-1β and mucosal activity of myeloperoxidase were maximally increased. Moreover, in hypophysectomized rats, administration of ghrelin failed to affect serum levels of GH or IGF-1, as well as the healing rate of colitis, mucosal cell proliferation, and mucosal concentration of IL-1β, or activity of myeloperoxidase. We conclude that administration of ghrelin accelerates the healing of the acetic acid-induced colitis. Therapeutic effect of ghrelin in experimental colitis is mainly mediated by the release of endogenous growth hormone and IGF-1.

Sex-based difference in Achilles peritendinous levels of matrix metalloproteinases and growth factors after acute resistance exercise.

Several recent investigations have demonstrated that the ability of various tendons to alter structural and functional properties in response to exercise are muted in women compared with men. We hypothesize that this disparity between men and women may be due to a reduced tendon production of key mediators of tendon extracellular matrix (ECM) remodeling in response to mechanical loading, e.g., exercise. Using microdialysis before and after an acute bout of resistance exercise, we evaluated Achilles peritendinous levels of insulin-like growth factor-1 (IGF-1) and interleukin-6 (IL-6), which have both been shown to increase tendon collagen synthesis. Additionally, the matrix remodeling enzymes matrix metalloproteinase-2 (MMP-2), MMP-9, and tissue inhibitor of metalloproteinase-1 (TIMP-1) were also evaluated. IGF-1 levels were elevated (P < 0.05) to a similar extent in men and women after 3 h of exercise but remained elevated at 4 h in only women. IL-6 levels were ~4-fold greater after exercise in both men and women (P < 0.05). MMP-2 levels increased to a similar extent (~2-3-fold) in men and women (P < 0.05). In contrast, MMP-9 increased with exercise but only in men (P < 0.05). Last, TIMP-1 levels also increased (P < 0.05) with exercise in men and women but the increase was more prolonged in women. In conclusion, we observed modest sex differences in tendon release of MMP-9, TIMP-1, and IGF-1 after acute resistance exercise. If such differences persist throughout a chronic exercise training, they may contribute to the reduced ability of women to adapt to exercise compared with men.NEW & NOTEWORTHY In this investigation we utilized microdialysis of the peritendinous Achilles to evaluate potential differences between men and women in tendon production of key regulators of extracellular matrix remodeling. We demonstrate that a modest sex-specific difference exists in peritendinous levels of several key extracellular matrix modulators after an acute bout of resistance exercise.

Pancreatic solitary fibrous tumor causing ectopic adrenocorticotropic hormone syndrome

Solitary fibrous tumors occasionally present with hypoglycemia because of the excessive release of insulin-like growth factor II. We report the first case of pancreatic solitary fibrous tumor causing ectopic adrenocorticotropic hormone syndrome. An 82-year-old Japanese man presented with lower limb edema, uncontrolled hypertension, hypokalemia, and baseline hypercortisolism. Distal pancreatectomy was performed after the clinical diagnosis of a neuroendocrine tumor with ectopic secretion of adrenocorticotropic hormone. On histological examination, the tumor showed spindle cells in a fascicular arrangement. The diagnosis of the solitary fibrous tumor was confirmed by the identification of the NAB2-STAT6 fusion gene and positive immuno-histochemical staining for STAT6 and CD34. Using quantitative real-time polymerase chain reaction, mRNA that encoded proopiomelanocortin, precursor of adrenocorticotropic hormone, was detected. Proopiomelanocortin production through the demethylation of the promoter region Domain IV was detected. Pancreatic solitary fibrous tumors represent a new cause of ectopic adrenocorticotropic hormone syndrome.

Toll-like receptor 7 affects the pathogenesis of non-alcoholic fatty liver disease

Recently, a possible link between toll-like receptor 7 (TLR7) and liver disease was suggested, although it was limited to fibrosis. Based on this report, we investigated whether TLR7 has a pivotal role in non-alcoholic fatty liver disease (NAFLD). The TLR7 signaling pathway, which is activated by imiquimod (TLR7 ligand) naturally, induced autophagy and released insulin-like growth factor 1 (IGF-1) into medium from hepatocytes. Lipid accumulation induced by unsaturated fatty acid (UFA; arachidonic acid:oleic acid = 1:1) in hepatocytes, was attenuated in TLR7 and autophagy activation. Interestingly, TLR7 activation attenuated UFA-induced lipid peroxidation products, such as malondialdehyde (MDA) and 4-Hydroxy-2-Nonenal (4-HNE). To clarify a possible pathway between TLR7 and lipid peroxidation, we treated hepatocytes with MDA and 4-HNE. MDA and 4-HNE induced 2-folds lipid accumulation in UFA-treated hepatocytes via blockade of the TLR7 signaling pathway’s IGF-1 release compared to only UFA-treated hepatocytes. In vivo experiments carried out with TLR7 knockout mice produced results consistent with in vitro experiments. In conclusion, TLR7 prevents progression of NAFLD via induced autophagy and released IGF-1 from liver. These findings suggest a new therapeutic strategy for the treatment of NAFLD.

Insulin-like growth factor-1 release regulates chondrocyte migration and cartilage repair

Insulin-like growth factor-1 release regulates chondrocyte migration and cartilage repair

Converted marine coral hydroxyapatite implants with growth factors: in vivo bone regeneration.

Herein we report rabbit model in vivo bone regeneration of hydrothermally converted coralline hydroxyapatite (HCCHAp) scaffolds without (group I) and with growth factors namely insulin like growth factor-1 (IGF-1) (group II) and bone morphogenetic protein-2 (BMP-2) (group III). All HCCHAp scaffolds have been characterized for phase purity and morphology before implantation. Calcined marine coral was hydrothermally converted using a mineralizer/catalyst to phase pure HAp retaining original pore structure and geometry. After sintering at 1250°C, the HCCHAp found to have ~87% crystallinity, 70-75% porosity and 2±0.5MPa compressive strength. In vitro growth factor release study at day 28 revealed 77 and 98% release for IGF-1 and BMP-2, respectively. The IGF-1 release was more sustained than BMP-2. In vivo bone healing of different groups was compared using chronological radiology, histological evaluations, scanning electron microscopy and fluorochrome labeling up to 90days of implantation. In vivo studies showed substantial reduction in radiolucent zone and decreased radiodensity of implants in group II followed by group III and group I. These observations clearly suggest in-growth of osseous tissue, initiation of bone healing and complete union between implants and natural bone in group II implants. A statistical score sheet based on histological observations showed an excellent osseous tissue formation in group II and group III scaffolds and moderate bone regeneration in group I scaffolds.

Bioactive IGF-1 release from collagen-GAG scaffold to enhance cartilage repair in vitro.

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.

Abstract 17202: Synthetic Modified RNA Driven Delivery of Insulin-Like Growth Factor-1 Promotes Early Cardiomyocyte Survival Post-Acute Myocardial Infarction

To extend the temporal window for cytoprotection in cardiomyocytes undergoing apoptosis after hypoxia and myocardial infarction (MI), a synthetic modified RNA (modRNA) was used to drive delivery of insulin-like growth factor-1 (IGF1) within the area at risk in a murine model of MI. Transient transfection of synthetic GFP modRNA as an tracking indicator, with polyethylenimine (PEI)-based nanoparticle, showed efficient delivery of modRNA derived protein and minimum cytotoxicity in HL-1 cardiomyocytes (CM; 44±5%). ModRNA-IGF1 protein expression and secreted levels increased 3.5 fold ( p &lt;0.05) at 24 hours and peaked at 48 hours post-transfection. The expression efficiency of modRNA was further enhanced (~2 fold at 24 hours post-transfection; p &lt;0.05) under hypoxia-induced apoptosis conditions. ModRNA augmented secreted and cell associated IGF1 promoting CM survival and abrogating cell apoptosis (71±5% in controls to 37±7%, p &lt;0.05). Translation of modRNA-IGF1 was sufficient to induce downstream increases in Akt and Erk phosphorylation (3 fold and 2 fold, respectively; p &lt;0.01). Downregulation of IGF1 specific miRNA-1 and -133 (52% and 56%, respectively; p &lt;0.01) but not miR-145 expression, was also confirmed. As proof of concept, intramyocardial delivery of modRNA-IGF1 but not control modRNA-GFP significantly decreased in TUNEL-positive cells within the infarct border zone (BZ) at 24 hours (22±3% versus 53±8% in controls, p &lt;0.01). Akt phosphorylation was augmented while caspase-9 activity and cleavage was downregulated in the infarct BZ compared to controls ( p &lt;0.05). These findings demonstrate extended in vivo cytoprotective effect of IGF1 24 hours post-MI driven by synthetic modRNA delivery. This provides a novel strategy to reduce ischemic injury by controlled release of IGF1 using a controllable bioactive nanoparticle for ‘short burst’ IGF1 cytoprotective therapy.

Minimally Invasive Approach to the Repair of Injured Skeletal Muscle With a Shape-memory Scaffold

Repair of injured skeletal muscle by cell therapies has been limited by poor survival of injected cells. Use of a carrier scaffold delivering cells locally, may enhance in vivo cell survival, and promote skeletal muscle regeneration. Biomaterial scaffolds are often implanted into muscle tissue through invasive surgeries, which can result in trauma that delays healing. Minimally invasive approaches to scaffold implantation are thought to minimize these adverse effects. This hypothesis was addressed in the context of a severe mouse skeletal muscle injury model. A degradable, shape-memory alginate scaffold that was highly porous and compressible was delivered by minimally invasive surgical techniques to injured tibialis anterior muscle. The scaffold controlled was quickly rehydrated in situ with autologous myoblasts and growth factors (either insulin-like growth factor-1 (IGF-1) alone or IGF-1 with vascular endothelial growth factor (VEGF)). The implanted scaffolds delivering myoblasts and IGF-1 significantly reduced scar formation, enhanced cell engraftment, and improved muscle contractile function. The addition of VEGF to the scaffold further improved functional recovery likely through increased angiogenesis. Thus, the delivery of myoblasts and dual local release of VEGF and IGF-1 from degradable scaffolds implanted through a minimally invasive procedure effectively promoted the functional regeneration of injured skeletal muscle.

Abstract 137: Pharmacologically Controlled IGF-1 Release For Paracrine Support Of Engineered Heart Muscle

Tissue engineered heart repair is developing rapidly, but needs refinement before clinical translation. We tested the hypothesis that force generating human engineered heart muscle (EHM) can be enhanced by integration of insulin-like growth factor-1 (IGF-1) secreting fibroblasts. Methods: TetOn lentiviral particles encoding for IGF-1 and the Tet-transactivator (tTA) were cloned and used to stably transduce human foreskin fibroblasts (HFF). Baseline and doxycycline induced IGF-1 release from HFFTetOn+IGF1 was quantified by ELISA. HFFwt and HFFTetOn+IGF1 conditioned medium was layered over human embryonic stem cell (HES2) derived cardiomyocytes followed by an analysis of AKT-phosphorylation. EHMs were assembled from HES2-derived cardiomyocytes and HFF (HFFwt or HFFTetOn+IGF1) at 70:30 ratio. Transgene activation was induced by addition of doxycycline (10 ng/ml) for 7 days. Twitch forces and response to pharmacological stimuli were measured to assess the functional consequences of IGF-1 release. EHMs were subsequently subjected to morphological analysis or dissociated into single cells to assess cellular composition of EHMs. Results: HFFTetOn+IGF1 released IGF-1 upon doxycycline stimulation (3.3x10-6 vs 8.3x10-8 [HFFwt] ng/ml/cell/day. Secreted IGF-1 from HFFTetOn+IGF1 induced Akt phosphorylation in HES2-derived cardiomyocytes (2.4±0.6 fold increase of [HFFwt]; n=3). EHMs with HFFTetOn+IGF1 developed significantly higher twitch forces than EHMs with HFFwt (0.24±0.03 vs 0.15±0.02 mN; n=10) under baseline conditions (IGF-1 leak). Doxycycline induced IGF-1 release further enhanced (P&lt;0.05) EHM twitch force (0.26±0.03 mN; n=10). Single cell analysis from EHMs demonstrated cardiomyocyte hypertrophy in response to paracrine IGF-1 release (154±11% of [HFFwt] control; n=4). Histological analyses demonstrated that HFFTetOn+IGF1 supplemented EHMs contained thicker muscle bundles and enlarged cardiomyocytes. Conclusion: EHM can be functionally enhanced by integration of drug-controllable IGF-1 release. Drug controllable, cell based paracrine release of protective factors may not only be exploited to enhance tissue engineered myocardium in vitro but also to achieve better survival and integration of EHM grafts in vivo.

GATA-4 protects against hypoxia-induced cardiomyocyte injury: effects on mitochondrial membrane potential.

Our previous studies have suggested that GATA-4 increases the differentiation of bone-marrow-derived mesenchymal stem cells (MSCs) into cardiac phenotypes. This study further investigated whether GATA-4 enhances MSC-mediated cardioprotection following hypoxia. MSCs were harvested from rat bone marrow and transduced with GATA-4 (MSC(GATA-4)). To mimic ischemic injury, cultured cardiomyocytes (CMs) isolated from neonatal rat ventricles were exposed to hypoxia or were pretreated with concentrated conditioned medium (CdM) from MSC(GATA-4) or transduced control MSC (MSC(Null)) for 16 h before exposure to hypoxic culture conditions (low glucose and low oxygen). Myocyte damage was estimated by annexin-V-PE and TUNEL technique and by lactate dehydrogenase (LDH) release. Cell survival was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium (MTT) uptake. Mitochondrial membrane potential was determined using confocal microscopy. ELISA studies indicated that insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF) were significantly increased in MSC(GATA-4) compared with MSC(Null). Hypoxia-induced apoptosis/cell death was significantly reduced when CMs were co-cultured with MSC(GATA-4) in a dual-chamber system. Cell protection mediated by MSC(GATA-4) was mimicked by treating CMs with CdM from MSC(GATA-4) and abrogated with IGF-1- and VEGF-neutralizing antibodies. MSC(GATA-4) protects CMs under hypoxic conditions. The release of IGF-1 and VEGF from MSC(GATA-4) is likely to be responsible for protection of CMs.

IGF-1 release kinetics from chitosan microparticles fabricated using environmentally benign conditions

The main objective of this study is to maximize growth factor encapsulation efficiency into microparticles. The novelty of this study is to maximize the encapsulated growth factors into microparticles by minimizing the use of organic solvents and using relatively low temperatures. The microparticles were fabricated using chitosan biopolymer as a base polymer and cross-linked with tripolyphosphate (TPP). Insulin like-growth factor-1 (IGF-1) was encapsulated into microparticles to study release kinetics and bioactivity. In order to authenticate the harms of using organic solvents like hexane and acetone during microparticle preparation, IGF-1 encapsulated microparticles prepared by the emulsification and coacervation methods were compared. The microparticles fabricated by emulsification method have shown a significant decrease (p<0.05) in IGF-1 encapsulation efficiency, and cumulative release during the two-week period. The biocompatibility of chitosan microparticles and the bioactivity of the released IGF-1 were determined in vitro by live/dead viability assay. The mineralization data observed with von Kossa assay, was supported by mRNA expression levels of osterix and runx2, which are transcription factors necessary for osteoblasts differentiation. Real time RT-PCR data showed an increased expression of runx2 and a decreased expression of osterix over time, indicating differentiating osteoblasts. Chitosan microparticles prepared in optimum environmental conditions are a promising controlled delivery system for cells to attach, proliferate, differentiate and mineralize, thereby acting as a suitable bone repairing material.

Diurnal alterations in circadian genes and peptides in major depressive disorder before and after escitalopram treatment

Strong links exist between circadian disturbances and some of the most characteristic symptoms of clinical major depressive disorder (MDD). However, changes in the expression of clock genes or neuropeptides related to the regulation of circadian rhythm that may influence the susceptibility to recurrence after antidepressant treatment in MDD have not been investigated. Blood samples were collected at 4h intervals for 24h from 12 male healthy controls and 12 male MDD patients before and after treatment with escitalopram for 8 weeks. The outcome measures included the relative expression of clock gene mRNA (PERIOD1, PERIOD2, PERIOD3, CRY1, BMAL1, NPAS2, and GSK-3β), and the levels of serum melatonin, vasoactive intestinal polypeptide (VIP), cortisol, adrenocorticotropic hormone (ACTH), insulin-like growth factor-1 (IGF-1), and growth hormone (GH). Compared with healthy controls, MDD patients showed disruptions in the diurnal rhythms of the expression of PERIOD1, PERIOD2, CRY1, BMAL1, NPAS2, and GSK-3β and disruptions in the diurnal rhythms of the release of melatonin, VIP, cortisol, ACTH, IGF-1, and GH. Several of these disruptions (i.e., PER1, CRY1, melatonin, VIP, cortisol, ACTH, and IGF-1) persisted 8 weeks after escitalopram treatment, similar to the increase in the 24h levels of VIP and decreases in the 24h levels of cortisol and ACTH. These persistent neurobiological changes may play a role in MDD symptoms that are thought to contribute to the vulnerability to recurrence and long-term maintenance therapy.