Post-loading of proangiogenic growth factors in PLGA microspheres.

Active self-encapsulation (ASE) is a recently developed post-loading method based on absorption of (positively charged) proteins in microporous PLGA microspheres loaded with negatively charged polysaccharides (trapping agents). The aim of this study was to investigate ASE for simultaneous loading and controlled release of multiple growth factors. For this purpose, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and insulin-like growth factor (IGF) were loaded in microspheres containing high molecular weight dextran sulfate (HDS) as trapping agent; loading was performed in a concentrated growth factor solution of low ionic strength and of pH 5 under conditions at which the proteins are positively charged. Subsequent pore closure was induced by incubation of the growth factor-loaded microspheres at 42.5 °C, i.e. above the Tg of (hydrated) PLGA (~30 °C). A 1:1:1 combination of VEGF, FGF and IGF was loaded with high loading (4.3%) and loading efficiency (91%). The in vitro release kinetics and bioactivity of loaded growth factors were studied for 4 weeks using ELISA and an endothelial cell proliferation assay, respectively. While IGF was released quickly, VEGF and FGF were continuously released for 4 weeks in their bioactive form, whereby a growth factor combination had a synergistic angiogenic effect. Therefore, ASE is a suitable method for co-loading growth factors which can provide sustained release profiles of bioactive growth factors, which is attractive for vascularization of biomaterial implants.

The effect of local application of insulin-like growth factor for prevention of inner-ear damage caused by electrode trauma.

BACKGROUND: Electrode insertion during cochlear implantation causes cochlear damage and apoptosis. Insulin-like growth factor applied locally was investigated in 21 rats. METHODS: In the sham group, an intracochlear dummy electrode was inserted through the round window. In the control group, after the same insertion procedure, saline-soaked porcine skin gelatine was placed on the round window. In the study group, insulin-like growth factor 1 soaked gelatine was placed on the round window. Auditory brainstem response thresholds were measured and histopathological examination was performed. RESULTS: In the study group, at 2-4 kHz, one rat had deterioration, one showed improvement and the rest had stable thresholds 14 days after intervention. At 6 kHz, four rats showed improvement and the rest remained stable. At 8 kHz, four showed improvement, one had deterioration and two remained stable. In the other groups, hearing loss deteriorated in about half of the rats and remained stable in the rest. The mean post-operative 6 kHz threshold was significantly lower than that immediately after the intervention in the study group, contrary to the other groups. The study group had significantly better mean histopathological grading than the other groups. CONCLUSION: Local insulin-like growth factor 1 application may protect hearing after cochlear implantation.

Gene Therapy Strategies for Alzheimer's Disease: An Overview.

Key neuropathological hallmarks of Alzheimer's disease (AD) are extracellular amyloid plaques and intracellular accumulation of hyperphosphorylated Tau protein. The mechanisms underlying these neuropathological changes remain unclear. So far, research on AD therapy has had limited success in terms of symptomatic treatments although it has also had several failures for disease-modifying drugs. Gene transfer strategies to the brain have contributed to evaluate in animal models many interesting tracks, some of which should deserve clinical applications in AD patients in the future.

Study of modified electroconvulsive therapy combined with risperidone oral solution in the treatment of agitation in the acute stage of epilepsy and expression level changes of insulin-like growth factor-1.

BACKGROUND: The aim of this paper was to explore the efficacy of modified electroconvulsive therapy (MECT) combined with risperidone oral solution in the treatment of agitation in the acute stage of epilepsy, and the effects of insulin-like growth factor-1 mRNA and protein expression. METHODS: Forty-six cases with seizures and agitation in the acute stage were included from February 2012 to February 2014. This study was approved by the ethics committee in our hospital. Patients were divided randomly into the experimental (23 cases) and control (23 cases) groups. The patients in the experimental group were treated with MECT combined with risperidone oral solution. The patients in the control group were treated with risperidone oral solution. The Positive and Negative Symptom Scale (PANSS) score and Treatment-Emergent Symptom Scale (TESS) score were compared before and after treatment. The insulin-like growth factor-1 (IGF-1) protein and mRNA expression level were detected with enzyme-linked immunosorbent assay (ELISA) and qRT-PCR, respectively. RESULTS: There were no significant differences on positive and negative symptom scores or total score in the two groups before treatment (P>0.05). After treatment, the positive symptom score, negative symptom score, and total score all decreased in both groups, and the decrease was more obvious in the experimental group (P<0.05). After treatment, TESS scores of the experimental group were significantly lower than those of the control group (P<0.05). In the experimental group, the total efficiency was significantly higher than that in the control group (P<0.05). Before treatment, there were no significant differences in the expression levels of IGF-1 protein or mRNA in the two groups (P>0.05), while after treatment the expression levels of IGF-1 protein and mRNA decreased in both groups. However, the decrease was more obvious in the experimental group. The differences were all significant for scores (P<0.05). CONCLUSIONS: The combined application of MECT and risperidone oral solution in the treatment of agitation can improve the clinical efficacy to some extent.

Theranostic mesoporous silica nanoparticles biodegrade after pro-survival drug delivery and ultrasound/magnetic resonance imaging of stem cells.

Increasing cell survival in stem cell therapy is an important challenge for the field of regenerative medicine. Here, we report theranostic mesoporous silica nanoparticles that can increase cell survival through both diagnostic and therapeutic approaches. First, the nanoparticle offers ultrasound and MRI signal to guide implantation into the peri-infarct zone and away from the most necrotic tissue. Second, the nanoparticle serves as a slow release reservoir of insulin-like growth factor (IGF)-a protein shown to increase cell survival. Mesenchymal stem cells labeled with these nanoparticles had detection limits near 9000 cells with no cytotoxicity at the 250 µg/mL concentration required for labeling. We also studied the degradation of the nanoparticles and showed that they clear from cells in approximately 3 weeks. The presence of IGF increased cell survival up to 40% (p<0.05) versus unlabeled cells under in vitro serum-free culture conditions.

The role of phosphoinositide 3-kinase in adhesion of oral epithelial cells to titanium.

BACKGROUND: Oral epithelial cells (OECs) adhesion to titanium may improve the success rate of implant restoration. PURPOSE: We investigated the mechanism by which OECs adhere to titanium dental implants. MATERIALS AND METHODS: (1) After culturing rat OECs on titanium plates (Ti) or culture dishes in the presence or absence of a phosphoinositide 3-kinase (PI3K) activator or inhibitors and/or growth factors, and OEC morphology under these conditions were analyzed. (2) Right maxillary first molars were extracted and replaced with experimental implants. The rats were treated with or without growth factors. RESULTS: (1) Cell adherence was lower of OECs on Ti than in those on culture dishes, as were the levels of integrin ß4 and the continuity of F-actin structures. After PI3K inhibition, markedly reducing adherence to both substrates. In contrast, PI3K activation with activator or insulin-like growth factor restored the OEC adherence and the expression of adhesion molecules on Ti to the levels seen in OECs cultured on dishes. Cell migration was inhibited by PI3K activation. (2) High expression of integrin ß4 was observed in the peri-implant epithelia of PI3K-activated rats. CONCLUSION: These findings suggest that PI3K plays an important role in the adhesion of OECs to Ti.

Insulin-like growth factors in the brain and their potential clinical implications.

Insulin and insulin-like growth factors (IGFs) including IGF1 and IGF2 are members of the insulin-like peptide superfamily and have an important role in development, cell differentiation, plasticity, and survival of the nervous system. These insulin-like peptides act at several receptors that initiate downstream phosphorylation cascades that in turn regulate transcription, synaptic maturation, and apoptosis. In the adult brain, insulin and IGF1 act as circulating signals that reach the CNS by crossing the blood-brain barrier (BBB) or the blood-CSF barrier; IGF1 and IGF2 also act as paracrine signals released from all neural cells. The bioavailability of IGF1 and IGF2 is regulated by their binding to IGF binding proteins (IGFBPs). Insulin-like peptides participate in neuroprotection and may have an important role in the pathophysiology of several neurologic disorders and as potential therapeutic targets for these conditions. The insulin-like peptides, their receptors, effects in the nervous system, and potential clinical correlations have been the subject of several recent reviews.(1-6).

Doping control analysis of selected peptide hormones using LC-MS(/MS).

With the constantly increasing sensitivity and robustness of liquid chromatography-mass spectrometry-based instruments combined with enhanced reproducibility as well as mass accuracy and resolution, LC-MS(/MS) has become an integral part of sports drug testing programs particularly concerning the detection of peptide hormones. Although several of the relevant peptidic drugs such as insulins (Humalog LisPro, Novolog Aspart, etc.), growth hormone releasing peptides (GHRPs, e.g., GHRP-2, GHRP-6, Hexarelin, etc.), and insulin-like growth factors (e.g., IGF-1, IGF-2, long-R(3)-IGF-1) are currently analyzed using dedicated top-down analytical procedures, i.e. employing specifically tailored sample preparation procedures followed by targeted LC-MS(/MS) measurements focusing on intact analytes, first approaches towards multi-analyte methods have been established. These allow the determination of the prohibited substances in blood and urine doping control specimens following therapeutic applications. In addition, the use of new complementary devices such as ion mobility analyzers, e.g., in hybrid mass spectrometers yielded promising data for the differentiation of isobaric insulins, which outlines the potential to further accelerate and multiplex doping control analytical assays to meet the continuously increasing demands of rapid and unambiguous test methods. Moreover, the potential of LC-MS/MS to target recombinant peptide hormones such as human growth hormone using bottom-up approaches has been demonstrated by targeting proteotypic peptides that unambiguously differentiate the recombinant molecule from the naturally occurring and endogenously produced analog.

Viability and growth of buffalo preantral follicles and their corresponding oocytes in vitro: effect of growth factors and beta mercaptoethanol.

The present study was undertaken to isolate buffalo preantral follicles (PFs), to test the viability and sizes of buffalo PFs and to examine the effect of various growth factors (insulin-like growth factor, fibroblast growth factor) and an antioxidant (beta mercaptoethanol) on the in vitro growth, survival and antrum formation rates of buffalo PFs and growth rates of oocytes in cultured PFs. Preantral follicles from slaughtered buffalo ovaries were recovered by a combined mechanical and enzymatic method. The recovery rates of >40-100, 101-200, 201-300, 301-400 and 401-500 microm PFs were 5.1, 3.2, 3.1, 6.3 and 5.1 per ovary, respectively. The corresponding viability rates were 76.1%, 78.1%, 85.2%, 92.5% and 92.6%, respectively. There was a positive correlation (r = 0.73) between oocyte size and the follicular size. However, there was no significant correlation between the size of oocyte and its viability at the time of its retrieval from ovary. Insulin-like growth factor and fibroblast growth factor improved the survival of buffalo PFs and regulated their growth in culture. The growth factors and beta mercaptoethanol in association synergically improved the growth and survival of buffalo PFs.

Insulin-like growth factor-induced signals activate mitochondrial respiration.

From experiments with lower eukaryotes it is known that the metabolic rate and also the rate of aging are tightly controlled by the insulin-like growth factor (IGF)/insulin signal transduction pathway. The mitochondrial theory of aging implies that an increased metabolic rate leads to increased mitochondrial activity; increased production of reactive oxygen species due to these alterations would speed up the aging process. To address the question if mitochondrial activity is influenced by insulin/IGF signaling, we have established an experimental system to determine the influence of IGF-I-dependent signaling on mitochondrial function. We used DU145 prostate cancer cells, known for the intact IGF signal transduction pathway, to address the influence of IGF receptor activation on mitochondrial function by high-resolution respirometry. These experiments revealed that indeed mitochondrial function is regulated by IGF signaling, and up-regulation of respiration seems to require phosphoinositide 3-kinase/AKT signaling, but is independent of IGF effects on cell cycle progression. Collectively these data establish a regulatory cross-talk between insulin/IGF signal transduction and mitochondrial function, two major pathways implicated in controlling the rate of aging.

Insulin-like growth factor-I stimulates Na+-dependent glutamine absorption in piglet enterocytes.

Agents that enhance intestinal glutamine transport may be useful under conditions in which intestinal mucosal function is compromised. Here we examined whether oral administration of insulin-like growth factor (IGF-I) stimulates absorption of L-glutamine in piglet intestine. Colostrum-deprived piglets received human recombinant IGF-I (3.5 mg/kg/day) or vehicle orogastrically every 8 hr for four days after birth. Piglets were killed on day 5 and the proximal jejunum was removed. Basal electrical parameters and L-glutamine-stimulated changes in short-circuit current were measured in muscle-stripped tissues, and rates of L-glutamine uptake were measured in everted jejunal sleeves. Oral IGF-I had no effect on jejunal mucosal mass. Short-circuit current responses to mucosal addition of 10 mM L-glutamine were increased by oral IGF-I. Total and carrier-mediated uptakes of L-glutamine per milligram were greater in tissues from IGF-I-treated piglets due to a significantly greater maximal rate of uptake (Jmax). Thus, oral administration of IGF-I stimulates Na+-dependent glutamine absorption in piglet small intestine, an effect that is independent of changes in intestinal mucosal mass.

Nutritional regulation of circulating insulin-like growth factors (IGFs) and their binding proteins in the ovine fetus.

Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are important for fetal and postnatal development, but the regulation of circulating IGFs and IGFBPs has not been as thoroughly investigated in the maternal/fetal unit as in the adult animal where nutrition status plays a regulatory role. We used the chronically-catheterized, late-gestation ovine model and compared circulating IGFs and IGFBPs levels, and hepatic IGF-I mRNA levels. Following a five-day maternal fast, both IGF-I and IGF-II levels were decreased in the maternal and fetal circulation (P < 0.05), accompanied by a decrease in fetal hepatic IGF-I mRNA levels, but the IGFBP2 level was increased and the IGFBP3 level was decreased in maternal circulation, whereas the IGFBP1 level was increased in fetal circulation. In both fed and fasting states, the infusion of glucose (150% of baseline) did not alter IGFs or IGFBPs in either maternal or fetal circulation. To understand the regulation of the endogenous IGF system, rhIGF-I was infused (6.7 nmol/kg fetus/h) into the fetal circulation. While maternal IGFs or IGFBPs remained unchanged, IGF-I infusion into fetal circulation resulted in an increase in IGF-I, a decrease in IGF-II, and an overall increase in the IGFBPs (P < 0.05). Taken together, circulating IGFs and IGFBPs in the ovine fetus are more sensitive to prolonged nutrient deficit than to a brief glucose increase. The nutrition status therefore regulates the IGF system in maternal and fetal circulation which, in turn, may regulate the nutrient utilization for fetal growth.

Development of a microcomputer-based system to monitor healing from injury.

It is well documented that induction of electric current in bone not only prevents the bone loss of functional disuse, but also induces new bone formation. Moreover, the literature suggests that the skeletal response is optimal at a distinct frequency range 10-30 Hz. Indeed, even at peak strains, well below those typical of habitual physiological loading, applications of 30 Hz were shown to be osteogenic. This evidence supports the concept that inducing even very low strains may generate an effective osteogenic stimulus, provided that they are induced at optimal frequency (10 to 30 Hz). Bone appears to respond with greater selectivity and sensitivity to this frequency range of electrical stimulation. Inducing insulin-like growth factors, which are negatively charged, will provide the required electrical stimulus. Traditionally, the progression of the cellular events during trauma is normally followed by x-ray to determine a healing rate. Frequent use of this method can result in serious side effects to the vital and reproductive organs. The objective of this study is to develop a microcomputer-based system to monitor the cellular events associated with healing. The system is capable of transmitting an electrical signal directly to the site of injury to improve the healing process and to monitor the progress of osteogenesis. The system consists of a base unit and implanted units. One implanted unit will be inserted in the femur with induced trauma and the other implant will be in the control femur. The base unit will transmit low frequency electromagnetic waves to the implanted units as well as receive periodic information about the ion movement in both femurs.

Systemic administration of rhIGF-I or rhIGF-I/IGFBP-3 increases cortical bone and lean body mass in ovariectomized rats.

The purpose of this study was to compare dose-related effects on cortical bone and lean body mass following subcutaneous administration of rhIGF-I alone, or bound to an equimolar amount of rhIGFBP-3 to adult Ovx rats. At the age of 16 weeks, rats were ovariectomized or sham-operated and were allowed 8 weeks to develop osteopenia. After being divided into control (saline treated) or treatment groups, rats were injected daily during an 8-week period with 0.9 and 2.6 mg/kg of rhIGF-I, or with 0.9, 2.6, and 7.5 mg/kg of rhIGF-I bound to rhIGFBP-3. Fluorescent bone markers were given 9 and 2 days prior to necropsy. Body weights and lean body mass were monitored throughout the experiment. Cortical bone histomorphometry was performed on tibial cross-sections at the tibiofibular junction, and endochondral bone growth was measured at the distal femoral metaphysis. All rats treated with rhIGF-I or the rhIGF-I/IGFBP-3 complex had increased body weights, corresponding to a dose-dependent increase in lean body mass. Endochondral growth was slightly increased in all experimental groups, but was not dose-dependent. A dramatic increase in periosteal, modeling-dependent formation, coupled with decreased or unchanged resorption on the endocortical envelope resulted in a dose-dependent increase in cortical thickness and cross-sectional area in groups treated with the complex of rhIGF-I/IGFBP-3. This complex appeared to be more effective in promoting positive musculoskeletal changes than rhIGF-I alone.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of IGF-1 treatment after hypoxic-ischemic brain injury in adult rats.

Intraventricular injection of insulin-like growth factor 1 (IGF-1) 2 h after hypoxic-ischemic injury reduces neuronal loss. To clarify the mode of action, we compared histological outcome between treatment groups in the following three studies: 0, 0.5, 5, and 50 micrograms IGF-1 given 2 h after injury; 0 and 20 micrograms IGF-1 given 1 h before; and 20 micrograms IGF-1 and insulin or vehicle alone given 2 h after. Unilateral hypoxic-ischemic injury was induced in adult rats by ligation of the right carotid and exposure to 6% O2 for 10 min. Histological outcome was evaluated in the cortex, striatum, and hippocampus 5 days later. Five to 50 micrograms IGF-1 reduced the incidence of infarction and neuronal loss in a dose-dependent manner in all regions (p < 0.05), and 50 micrograms reduced the infarction rate from 87 to 26% (p < 0.01). Pretreatment did not alter outcome. IGF-1 improved outcome compared with equimolar doses of insulin (p < 0.05) and did not affect systemic glucose concentrations or cortical temperature. The results indicate that the neuronal protective effects of IGF-1 are specific and are not mediated via insulin receptors, hypothermia, or hypoglycemic mechanisms. Centrally administered IGF-1 appears to provide worthwhile trophic support to cells within most cerebral structures after transient hypoxic-ischemic injury.

Human osteoblast-derived insulin-like growth factor (IGF) binding protein-5 stimulates osteoblast mitogenesis and potentiates IGF action.

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) either inhibit or enhance IGF-stimulated cellular effects. While inhibition occurs by sequestration of IGF from cell-surface receptors, the exact mechanism of IGF-enhancement remains undefined. Human osteoblast-like bone cells in culture secrete several IGF-binding proteins, one of which we have previously identified as IGFBP-5. In this study we purified a 23-kDa IGFBP-5 from cultures of human osteoblast-like cells using ligand affinity chromatography and reversed-phase high performance liquid chromatography and tested its bioactivity in serum-free cultures of normal mouse osteoblast-like cells. Binding studies with radioiodinated IGF showed similar and relatively low affinities for IGF-I and IGF-II consistent with a carboxyl truncated IGF-binding protein. Mitogenic assays demonstrated that the binding protein, when coincubated with IGF-I or -II, enhanced mitogenesis. This enhancement was unique from other binding proteins in not requiring a preincubation period or serum co-factors. Furthermore, the osteoblast-derived IGFBP-5 stimulated mitogenesis in the absence of exogenous or endogenous IGF. Using radioiodinated IGFBP-5 we found that the binding protein could associate with the osteoblast surface, an effect which did not require IGF nor an interaction with IGF receptors. We suggest that osteoblast-derived IGFBP-5 may stimulate osteoblast mitogenesis in at least two ways, by association with IGF and by a second pathway that is independent of IGF receptor activation.

Does sulfation of gastrin influence gastric acid secretion in man?

To assess the physiologic significance of tyrosine o-sulfation of gastrin in humans, the gastric acid stimulatory potencies of sulfated and non-sulfated human gastrin-17 were compared in six normal young subjects. Sulfated and non-sulfated forms of synthetic human gastrin-17 were infused intravenously in doses from 12.7 to 478 pmol/kg/h. Similar acid secretory responses were observed. The calculated maximal acid response for sulfated gastrin-17 was 35.7 +/- 4.3 mmol/h, and that for non-sulfated gastrin-17 was 39.8 +/- 7.5 mmol/h (mean +/- SEM, NS). The 50% effective dose of sulfated gastrin-17 was 22.2 +/- 6.7 pmol/kg/h, whereas it was 29.3 +/- 5.8 pmol/kg/h for non-sulfated gastrin-17 (NS). Finally, the 50% effective concentration of gastrin in serum was 34.7 +/- 5.0 pmol sulfated gastrin-17/l and 42.5 +/- 11.8 pmol non-sulfated gastrin-17/l (NS). The results show that tyrosine o-sulfation is without significant influence on the gastric acid secretory potency of gastrin in man. Moreover, the results also suggest that sulfated and non-sulfated gastrin-17 in man have similar rates of metabolism.

Small stature and insulin-like growth factors: prolonged treatment of mini-poodles with recombinant human insulin-like growth factor I.

The short stature of mini-poodles is associated with low serum levels of IGF-I. Standard poodles are taller and have considerably higher serum levels of IGF-I. Low IGF-I serum levels may be a symptom or the cause of small stature. We, therefore, undertook a study in which serum IGF-I levels of mini-poodles were elevated over a prolonged period of time by a constant infusion of rhIGF-I and the growth rate of the mini-poodles was followed. We infused four mini-poodles from day 91 to day 221 of age with 6 mg/day of recombinant human insulin-like growth factor I (rhIGF-I). Serum levels of IGF-I rose from about 160 to about 500 micrograms/l. Blood glucose remained within normal limits. Stimulation tests with clonidine and with GHRH revealed suppression of endogenous GH secretion during the IGF-I infusion. Serum levels of IGF-II and of creatinine were lower in the IGF-I-infused animals. Radial length and body weight did not increase to a greater extent in the IGF-I infused dogs than in controls. However, 'adapted body mass index' (aBMI = gram body weight/(mm radial length)2) decreased in each of the IGF-I infused animals, whereas it increased in each of the control dogs (p less than 0.05). We conclude that long-term infusion of IGF-I does not stimulate growth in young mini-poodles, but may change body composition.