Human recombinant insulin-like growth factor I. I. Development of a serum-free medium for clonal density assay of growth factors using balb/c 3T3 mouse embryo fibroblasts

Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin, 100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED₅₀), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin, ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED₅₀, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures. This work was supported by a contract from IMCERA Bioproducts, Inc.     

Ras signaling in NGF reduction and TNF-α-related pancreatic β cell apoptosis in hyperglycemic rats

Recent evidence suggested that tumor necrosis factor-alpha (TNF-α) and nerve growth factor (NGF) withdrawal activated a common apoptotic pathway. Here, we aimed to investigate the possible role of apoptotic Ras effectors RASSF1 and NORE1 in NGF reduction and TNF-α-related β cell apoptosis in streptozotocin (STZ)-induced hyperglycemic rats. Rats were divided into four groups: the first group was given saline and citrate buffer, the second group was injected 4-methylcatechol (4-MC), an inducer of NGF synthesis, the third group received STZ, and the fourth group was given both 4-MC and STZ. 4-MC (10 μg/kg) was administered by daily intraperitoneal injection for 10 days before the animals were rendered hyperglycemic by administration of single dose STZ (75 mg/kg). With 4-MC pretreatment to hyperglycemic rats the following results were noted: (i) Decrease in pancreatic NGF level was blocked, (ii) Increase in pancreatic TNF-α level and the number of TNF-α⁺ beta cell in the islets were prevented, (iii) Increase in the number of β cell synthhesized apoptotic Ras effectors that RASSF1 and NORE1 was blocked, (iv) While pancreatic lipid peroxidation level decreased, antioxidant molecule glutathione and antioxidant enzymes glutathione peroxidase, catalase and superoxide dismutase activities increased, (v) Pancreatic caspase-3 activity and the number of cleaved caspase-3⁺ β cells were decreased. These results strengthen the idea that TNF-α and reduction in NGF can activate a common apoptotic pathway. Moreover, these data display that new apoptotic Ras effector molecules RASSF1 and NORE1 play important role with oxidative stress in NGF reduction and TNF-α-related pancreatic β cell apoptosis in hyperglycemic rats. Furthermore, these findings suggest that 4-MC can prevent β cell apoptosis possibly through increasing NGF synthesis in hyperglycemic rats.     

Thymosin-β4 Attenuates Ethanol-induced Neurotoxicity in Cultured Cerebral Cortical Astrocytes by Inhibiting Apoptosis

Thymosin-β4 (Tβ4) is a major actin monomer-binding peptide in mammalian tissues and plays a crucial role in the nervous system in synaptogenesis, neuronal survival and migration, axonal growth, and plastic changes of dendritic spines. However, it is unknown whether Tβ4 is also involved in challenges with external stress such as ethanol-induced neurotoxicity. In the present study, we investigated the effects of Tβ4 on ethanol-induced neurotoxicity in cultured cerebral cortical astrocytes and the underlying mechanisms. Primarily cultured astrocytes were treated with 1 μg/ml Tβ4 2 h prior to administration of 100 mM ethanol for 0.5, 1, 3 and 6 days, respectively. The results showed that ethanol caused neurotoxicity in cultured astrocytes, as shown by declined cell viability, distinct astroglial apoptosis and increased intracellular peroxidation. Tβ4 markedly promoted cell viability, ameliorated the injury of intracellular glial fibrillary acidic protein-immunopositive cytoskeletal structures, reduced the percentage of apoptotic astrocyte and cellular DNA fragmentation, suppressed caspase-3 activity and upregulated Bcl-2 expression, inhibited the accumulation of reactive oxygen species and production of malondialdehyde in ethanol-treated astrocytes in a time-dependent manner. These data indicated that Tβ4 attenuates ethanol-induced neurotoxicity in cultured cortical astrocytes through inhibition of apoptosis signaling, and one of the mechanisms underlying the capacity of Tβ4 to suppress apoptosis may in part be due to its effect of anti-peroxidation.     

Effects of steroid hormones in fetal bovine serum on plating and cloning of human cells in vitro

Summary Fetal bovine sera from each of three different commercial sources were tested for their ability to support cloning of human fibroblastoid cells in vitro. Cloning efficiencies varied according to serum source. Serum (10 samples) from company A did not support growth, while sera (10 samples) from companies B and C provided adequate to excellent conditions for cloning and growth. Cells from neonatal foreskin or embryonic lung responded to each serum similarly. Bovine serum albumin type H7 from company C supported cell growth in media without serum. Sera containing 1.0 ng per ml or more of progesterone inhibited growth, whereas sera containing less than 1.0 ng per ml supported cloning and growth. In the low progesterone sera, the concentration of 17-β-estradiol exceeded 100 pg per ml. Growth supporting sera could be made non-supportive by adding 0.1 μg per ml of progesterone. The addition to non-supportive sera of 0.1 μg per ml of 17-β-estradiol or hydrocortisone made these sera supportive of cell growth. Addition of estrogen or hydrocortisone to a culture medium that inhibits growth, with subsequent reversal of the inhibitory effect, implies that these hormones competitively regulate growth of responsive cells in vitro. Supported in part by NIH-NCI-EC2074.     

Modification of glial-neuronal cell interactions prevents photoreceptor apoptosis during light-induced retinal degeneration

Prolonged or high-intensity exposure to visible light leads to photoreceptor cell death. In this study, we demonstrate a novel pathway of light-induced photoreceptor apoptosis involving the low-affinity neurotrophin receptor p75 (p75NTR). Retinal degeneration upregulated both p75NTR and the high-affinity neurotrophin receptor TrkC in different parts of Müller glial cells. Exogenous neurotrophin-3 (NT-3) increased, but nerve growth factor (NGF) decreased basic fibroblast growth factor (bFGF) production in Müller cells, which can directly rescue photoreceptor apoptosis. Blockade of p75NTR prevented bFGF reduction and resulted in both structural and functional photoreceptor survival in vivo. Furthermore, the absence of p75NTR significantly prevented light-induced photoreceptor apoptosis. These observations implicate glial cells in the determination of neural cell survival, and suggest functional glial-neuronal cell interactions as new therapeutic targets for neurodegeneration.     

Stimulation by GH of IGF1 proforms synthesized by rabbit chondrocytes cultured with bFGF in serum-free medium

The IR-IGF1 production by rabbit epiphyseal chondrocytes cultured in serum-free medium was analyzed. Cell proliferation was induced by the addition of 10 ng/ ml basic fibroblast growth factor (bFGF) without or with 100 ng/ml recombinant human growth hormone (hGH). GH alone induced no cell multiplication. Chondrocytes treated with bFGF alone secreted an IR-IGF1 activity proportional to the mitotic activity of the cells. A specific positive IGF1 immunostaining was localized in the Golgi of control and hGH-treated cells. The IR-IGF1 activity recovered into culture medium was mainly composed of three fractions of apparent MW 6-8 kDa, 9–14 kDa, and 16–18 kDa. [³⁵S]Methionine pulse-chase experiments indicated that the radiolabeled 16–18 kDa IR-IGF1 fraction was partly converted into the 9–14 kDa and 6–8 kDa fractions. At equilibrium, 70% of the chondrocyte IR-IGF1 activity was recovered as 9- to 18-kDa forms which contained high IR-proIGF1A activity. The 6–8 kDa fraction had biochemical characteristics similar to those of the mature IGF1 peptide. Similar results were observed when 4% fetal calf serum was added to the culture. The addition of 100 ng/ml of hGH significantly and specifically increased IGF1 precursor material, which thus represented 90% of total IR-IGF1 activity. On Day 16 of the culture, when cells stopped dividing, the amount of chondrocyte IR-IGF1 was significantly lower than during cell proliferation, and hGH had no effect on this production. These data indicate that cultured chondrocytes produce more IGF1 precursors than mature IGF1 and that GH specifically stimulates biosynthesis of IGF1 precursors but not IGF1 per se. A GH-dependent biological function of IGF 1 preforms in chondrocytes remains to be demonstrated.     

A new serum-free method of measuring growth factor activities for human breast cancer cells in culture

Summary Growth of the MCF-7, T47D, and ZR-75-1 human breast cancer cells was established in a serum-free defined medium (MOM-1) composed of a 1∶1 (vol/vol) mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium containing 15 mM HEPES (pH 7.2), 2 mM 1-glutamine, 20 μg/ml glutathione, 10 μg/ml insulin, 10 μg/ml transferrin (Tf), 10 ng/ml selenous acid, 0.3 nM triiodothyronine, 50 μg/ml ethanolamine, 20 ng/ml epidermal, growth factor, 2.0 nM 17β-estradiol, and 1.0 mg/ml bovine serum albumin (BSA). Proliferation in MOM-1 was 50 to 70% of the serum stimulated rate. Deletion of components from MOM-1 gave a medium (Tf-BSA) containing only HEPES, 10 μg/ml Tf, and 200 μg/ml BSA, which sustained MCF-7 and T47D cells in a slowly dividing and mitogen responsive state; ZR-75-1 cells required Tf plus 1.0 mg/ml BSA. In Tf-BSA, insulin and insulin-like growth factor I(IGF-I) were mitogenic with ED₅₀ values of 2 to 3 ng/ml and 30 to 150 pg/ml, respectively, with MCF-7 cells. The T47D cells were responsive to these factors in Tf-BSA but required 10-fold higher concentrations for ED₅₀. At saturating concentrations, insulin and IGF-I promoted 1.5 to 3.5 cell population doublings over controls in 8 d. At≤ng/ml concentrations, epidermal growth factor, insulin-like growth factor II, and basic fibroblast growth factor were mitogenic for human breast cancer cells in Tf-BSA. Mitogen activities in uterus and pituitary extracts were assayed readily in Tf-BSA. This new method offers a convenient means of comparing the potencies of growth-promoting factors on human breast cancer cells without interfering activities known to be present in serum. This work was supported by grants CA-38024 and CA-26617, from the National Cancer Institute, Bethesda, MD, and by American Cancer Society grant BC-255 and grant 2225 from the Council for Tobacco Research, USA, Inc.     

In vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor

When grown in the absence of astroglial cells, purified mouse cerebellar granule neurons survive less than 36 hr and do not extend neurites. Here we report that low concentrations of basic fibroblast growth factor (bFGF, 1-25 ng/ml) maintained the viability and promoted the differentiation of purified granule neurons. The effect of bFGF on granule cell neurite outgrowth was dose dependent. Neurite outgrowth was stimulated markedly in the presence of 1-25 ng/ml bFGF, but effects were not seen below 1 ng/ml or above 50 ng/ml. When affinity-purified antibodies against bFGF (1-5 micrograms/ml) were added either to purified granule cells or to co-cultures of neurons and astroglial cells, process extension by granule neurons was severely impaired. The inhibition of neurite outgrowth in the presence of anti-bFGF antibodies was reversed by the addition of 25 ng/ml of exogenous bFGF. In addition to neuronotrophic effects, bFGF influenced the rate of growth of the astroglial cells. This result depended on whether the astroglia were grown in isolation from neurons, where low doses of bFGF (10-25 ng) stimulated glial growth, or in coculture with neurons, where much higher doses of bFGF (100-250 ng/ml) were needed for glial mitogenesis. Immunoprecipitation of lysates from 35S-labeled cerebellar astroglial cells with anti-bFGF antibodies revealed a single band after SDS-PAGE at 18,000 Da, the molecular weight of bFGF. These results indicate that glial cells synthesize bFGF and are possibly an endogenous source of bFGF in cerebellar cultures. Thus, astroglial cells synthesize soluble factors needed for neuronal differentiation.     

Growth of MTW9/PL2 estrogen-responsive rat mammary tumor cells in hormonally defined serum-free media

Summary Growth of the estrogen-responsive MTW9/PL2 rat mammary tumor cells was demonstrated in serum-free defined medium (designated DDM-1) formulated with F12-DME (1:1 vol/vol) supplemented with 15 mM HEPES pH 7.4 insulin 10 μg/ml, transferrin 10 μg/ml, sodium selenite 10 ng/ml, triiodo-l-thyronine 0.3 nM, phosphoethanolamine 5 μM, epidermal growth factor (20 ng/ml), 17 β-estradiol 2 nM, and bovine serum albumin 20 μg/ml. In DDM-1, the growth rate was about one-half that seen in serum-containing medium. When ethanolamine (50 μM), glutathione (20 μg/ml), and linoleic acid/bovine serum albumin (150 μg/ml) were added (formulation DDM-2), the growth rate was 80% of serum-containing medium and not seed-density dependent. Deletion of estradiol from DDM-1 or DDM-2 had no effect on growth rate. Also, cells grown in steroid hormone deficient medium for 4 mo. continued to form estrogen-responsive tumors in rats as did cells cultured for the same period in 2 nM estradiol. To investigate autocrine growth factor secretion, a third medium (DDM-3) was prepared by deleting insulin, epidermal growth factor, phosphoethanolamine, estradiol, and both forms of bovine serum albumin from DDM-2. Growth in mitogen-free medium equaled 86% of the serum-stimulated rate and was seed-density dependent; phenol red deletion from DDM-3 had no effect on growth rate. Evidence presented suggests that autocrine factors stimulate growth of the MTW9/PL2 cells in DDM-3, and that this secretion may support the growth of estrogen-responsive cells in culture in the absence of steroid hormone. This work was supported by National Cancer Institute grants CA-38024 and CA-26617 and American Cancer Society grant BC255.     

Short- and Long-term Memory are Differentialy Modulated by Hippocampal Nerve Growth Factor and Fibroblast Growth Factor

Rats were implanted with cannulae in the CA1 area of the dorsal hippocampus and trained in one-trial step-down inhibitory avoidance. Two retention tests were carried out in each animal, one at 1.5 h to measure short-term memory (STM) and another at 24 h to measure long-term memory (LTM). The purpose of the present study was to evaluate the modulation on hippocampal nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) on short- and long-term memory. Immediately after training, animals received 5 l of NGF (0.05, 0.5 or 5.0 ng), bFGF (1.25, 12.5 or 125 ng) or saline per side. At the higher dose, NGF blocked STM. In contrast, NGF at dose of 0.5 and 5.0 ng improved LTM. The bFGF infusion at a dose of 125 ng enhanced LTM. However, bFGF did not alter STM. These findings indicate that hippocampal NGF and bFGF modulate STM and LTM in a different manner.     

How the concentration of insulin affects the development of preantral follicles in goats

This study investigated the effect of adding different insulin concentrations to the culture medium for goat preantral follicle development in vitro. The ovarian fragments were immediately fixed or cultured for 7 days in MEM with insulin (0, 5, 10 ng/ml and 5 or 10 μg/ml). The results showed that, after 7 days of culture, insulin at 10 ng/ml was the best concentration to preserve follicular viability and ultrastructure, resulting in the highest rates of normal follicles. After 7 days, only treatments with 10 ng/ml and 5 μg/ml of insulin increased follicular activation when compared to other concentrations. Regarding follicular and oocyte growth, the presence of 10 ng/ml of insulin promoted a larger diameter than other treatments. In conclusion, this study shows that addition of 10 ng/ml of insulin to the culture medium improved the survival and stimulated growth of goat preantral follicles.     

Epidermal growth factor rescues trophoblast apoptosis induced by reactive oxygen species

Pre-eclampsia and intrauterine growth restriction are associated with increased apoptosis of placental villous trophoblast. This may result from placental hypoperfusion, leading to the generation of reactive oxygen species (ROS). Apoptosis can be induced in villous trophoblast following exposure to oxidative stress. Epidermal growth factor (EGF) reduces trophoblast apoptosis resulting from exposure to hypoxia. We hypothesised that exposure to hydrogen peroxide, a potent generator of ROS, would induce apoptosis in term placental villous explants and that this could be reduced by treatment with EGF. Placental explants were taken from normal term pregnancies and exposed to increasing doses of hydrogen peroxide (0–1,000 μM) or to a combination of increasing doses of hydrogen peroxide and EGF (0–100 ng/ml) for either 6 or 48 h. Apoptosis was assessed by TUNEL, proliferation by Ki-67 immunostaining, necrosis by lactate dehydrogenase activity and trophoblast differentiation by human chorionic gonadotrophin (hCG) secretion in conditioned culture media. Immunoperoxidase staining was performed to identify phosphorylated-AKT (p-AKT) and phosphorylated-PI3 kinase (p-PI3k). Exposure to 1,000 μM hydrogen peroxide for 48 h induced apoptosis in placental explants. The increase in TUNEL positive nuclei predominantly localised to syncytiotrophoblast. The amount of apoptosis was reduced to control levels by treatment with 10 and 100 ng/ml EGF. Proliferation of cytotrophoblasts within villous explants was significantly reduced following exposure to 1,000 μM hydrogen peroxide, this was restored to control levels by simultaneous treatment with 10 or 100 ng/ml EGF. Neither exposure to hydrogen peroxide or EGF altered the amount of necrosis. There was increased immunostaining for pPI3K following treatment with EGF. This study shows that apoptosis may be induced in villous trophoblast following exposure to ROS, and demonstrates the anti-apoptotic effect of EGF in trophoblast, the maintenance of which is essential for normal pregnancy.     

Dose-related influence of sodium selenite on apoptosis in human thyroid follicles in vitro induced by iodine, EGF, TGF-β, and H2O2

Apoptosis of thyroid follicular cells is induced by high doses of iodide, epidermal growth factor (EGF), transforming growth factor-β (TGF-β), as well as H₂O₂ and might be attenuated by antioxidants. Therefore, we examined the apoptotic index induced by these substances in selenium-treated vs untreated human thyroid follicular cells. Reconstituted human thyroid follicles were incubated with sodium selenite (10 or 100 nM) for 72 h; controls received none. The follicles were then distributed to 24-well plates and incubated with potassium iodide (5, 10, or 20 nM), EGF (5 ng/mL), TGF-β (5 ng/mL), or H₂O₂ (100 μM). Apoptosis was determined by a mitochondrial potential assay and the number of apoptotic cells counted by two independent, experienced technicians and the glutathione peroxidase (GPx) activity was determined. A significant increase of apoptic cells was obtained in control thyroid follicles treated with iodine (5, 10, or 20 μM), thyroid-stimulating hormone (TSH) 1, or 10 mU/mL in combination with 5 and 10 μM iodine, EGF (5 ng/mL) and TGF-β (5 ng/mL), or H₂O₂ (100 μM) (p<0.001). In contrast, in thyroid follicles preincubated with 10 or 100 nM sodium selenite, the apoptototic index was identical to the basal rate. In H₂O₂-treated follicles, the apoptotic index was still significantly elevated but 50% lower compared to control cells. The GPx activity increased from 1.4±0.2 to 2.25±0.4 mU/μg DNA with 10 nM selenite and 2.6+0.4 mU/μg DNA with 100 nM selenite. Sodium selenite might increase the antioxidative potential in human thyroid follicles in vitro and therefore diminish the apoptosis induced by TGF-β, EGF, iodide, and even H₂O₂     

Interleukin-1β enhances the effect of serum deprivation on rat annular cell apoptosis

Excessive apoptosis of disc cells is believed to play an important role in intervertebral disc (IVD) degeneration. It has been shown that interleukin-1β (IL-1β) is involved in the failure of disc matrix by suppressing the synthesis of matrix components and stimulating the expression of matrix metalloproteinases. However, whether IL-1β induces disc cell apoptosis is still unclear. The objective of this study was to investigate the effect of IL-1β on the apoptosis of rat annular cells cultured with or without serum supplement. First-passage rat annular cells were cultured with 0% or 10% fetal bovine serum (FBS) supplement and stimulated with 0, 10, 20 or 50 ng/ml IL-1β for 12, 24 or 48 h. Apoptotic incidences were quantified by flow cytometry, morphologic changes in apoptotic cells were visualized by Hoechst 33258 staining and phase-contrast microscopy, and caspase-3 activity was also determined. When rat annular cells were cultured with 10% FBS supplement, no significant changes in apoptotic incidences, apoptotic morphology and caspase-3 activity were observed even when cells were stimulated with 50 ng/ml IL-1β for 48 h. In contrast, serum deprivation for 24 h led to an increase in apoptotic incidences, the number of apoptotic nuclei and caspase-3 activity, and IL-1β significantly increased the effects of serum deprivation in a dose-dependent manner. Our results indicate that IL-1β alone is not a sufficient stimulus to induce disc cell apoptosis and that in order to suppress disc cell apoptosis, improving the nutrient supply to the disc may be more effective than antagonizing the adverse effects of IL-1β.     

Growth of seminal vesicle epithelial cells in serum-free collagen gel culture

Summary Epithelial cells from mouse seminal vesicles were enzymatically dissociated enriched by gradient centrifugation, and maintained in collagen gel cultures with defined (serum-free) media. The epithelial origin of the cells was determined morhologically, immunocytochemically, and biochemically. Cells formed three-dimensional colonies with a lumen in collagen gels. Cell number was increased eight-fold within a 8 to 12-d culture period in a medium supplemented with epidermal growth factor (EGF) (10 ng/ml), insulin (10 μg/ml), transferrin (10 μg/ml), cholera toxin (10 ng/ml), and hydrocortisone (0.1 μg/ml). The cells required eGF and insulin; the growth-promoting effects of these two peptide hormones were optimized by transferrin, cholera toxin, and hydrocortisone. Fetal bovine serum did not support growth; rather, it suppressed the stimulated growth observed in serum-free media. A time-course study revealed that a lag period preceded rapi growth. The collagen gel, serum-free culture provides a powerful tool to study the effects of hormones on proliferation and differentiation of androgen sensitive cells.     

Neuroblast cell death in ovo and in culture: Interaction of ethanol and neurotrophic factors

We used two experimental paradigms to examine the influence of the neurotrophins, NGF, EGF, and bFGF on normal neuroblast survival and also after ethanol insult. In the first paradigm, chick embryos received in ovo at embryonic day 1 and 2 (E1 and E2) saline (control) ethanol (10mg/50 l/day), NGF (50 ng/50 l/day), or EGF (25 ng/50 l/day), or ethanol+NGF or EGF. At E3, cultures were prepared from whole embryos separately from each group. At C2, all cultures were labeled with [³H]thymidine and assessed for effects or neuronal survival. In the second paradigm, cultures were prepared from 3-day-old whole embryos and at CO, cultures were treated with either ethanol (50 mM) alone, NGF (50 ng/ml) alone, EGF (25 ng/ml) alone, bFGF (50 ng/ml) alone, or were treated concomitantly with ethanol plus one of the neurotrophins; control had only the culture medium, DMEM+5% FBS. We obtained the following findings. 1) Cultures derived from embryos treated with either of the three neurotrophins exhibited a higher neuronal survival as compared to controls (1st paradigm). 2) The survival-promoting effect was also observed when the neurotrophins were added directly to the cultures (2nd paradigm). 3) As reported previously, cultures derived from ethanol-treated embryos exhibited a marked decline in neuronal survival as compared to controls. 4) All three neurotrophins attenuated the decline in neuronal survival produced by ethanol. The rescuing effects of the neurotrophins support our early hypothesis that ethanol administration during early neurogenesis interferes with microenvironmental trophic signals essential for neuroblast survival and differentiation.     

BDNF-, IGF-1- and GDNF-Secreting Human Neural Progenitor Cells Rescue Amyloid β-Induced Toxicity in Cultured Rat Septal Neurons

Alzheimer’s disease (AD) is characterized by the depositions of amyloid-β (Aβ) proteins, resulting in a reduction of choline acetyltransferase (ChAT) activity of AD brain in the early stages of the disease. Several growth factors, including brain-derived neurotrophic factor (BDNF), insulin-like growth factor (IGF)-1 and glial cell-derived neurotrophic factor (GDNF) are known to protect neuronal cell death in several neurodegenerative both in vitro and in vivo models. In this study, septal neurons were prepared from septal nucleus of embryonic (day 16-17) rat brain and treated with monomeric, oligomeric or fibrillar Aβ_1-42 peptide. Oligomeric Aβ_1-42, (10 μM) was the most potent at sublethal dose. Septal neuron cultures treated with BDNF, IGF-1 or GDNF or co-cultured with genetically modified human neural progenitor cells (hNPCs) secreting these neurotrophic factors (but not allowing contact between the two cell types), were protected from oligomeric Aβ_1-42 peptide-induced cell death, and these trophic factors enhanced cholinergic functions by increasing ChAT expression level. These results indicate the potential of employing transplanted hNPCs for treatment of AD.     

An involvement of BDNF and PI3-K/Akt in the anti-apoptotic effect of memantine on staurosporine-evoked cell death in primary cortical neurons

Memantine, a clinically used NMDA receptor antagonist possesses neuroprotective properties, but the exact mechanisms of its beneficial action on neuronal survival are poorly recognized. In the present study, some intracellular mechanisms of memantine effects on staurosporine-evoked cell death were investigated in primary cortical neurons. Memantine (0.1–2 μM) suppressed neuronal apoptosis evoked by staurosporine in 7 DIV cortical neurons, whereas other antagonists of NMDA receptor, MK-801 (1 μM) and AP-5 (100 μM) were ineffective. The anti-apoptotic effects of memantine were not connected with any changes in cytoplasmic calcium concentration or reactive oxygen species level. The immunoblot analysis showed that the staurosporine induced a decrease in p-Akt protein kinase level and that this effect was reversed by memantine treatment. Moreover, the PI3-K inhibitors, wortmannin and LY 294002 attenuated the anti-apoptotic action of memantine on staurosporine-induced cell damage. Furthermore, the ELISA studies showed increased cellular and released BDNF protein level after combined treatment with memantine and staurosporine. There was no effect of memantine on the activation and expression of other protein kinases involved in the mechanism of cellular survival, i.e. ERK1/2, JNK and GSK3-β. The obtained data suggest an NMDAR-independent action of memantine in attenuation of neuronal apoptosis and point to the engagement of BDNF and PI3-K/Akt pathway in these processes.     

Modulation of calcification of vascular smooth muscle cells in culture by calcium antagonists, statins, and their combination

Background Vascular calcification is an organized process in which vascular smooth muscle cells (VSMCs) are implicated primarily. The purpose of the present study was to assess the effects of calcium antagonists and statins on VSMC calcification in vitro. Methods VSMC calcification was stimulated by incubation in growth medium supplemented with 10 mmol/l β-glycerophosphate, 8 mmol/l CaCl₂, 10 mmol/l sodium pyruvate, 1 μmol/l insulin, 50 μg/ml ascorbic acid, and 100 nmol/l dexamethasone (calcification medium). Calcification, proliferation, and apoptosis of VSMCs were quantified. Results Calcium deposition was stimulated dose-dependently by β-glycerophosphate, CaCl₂, and ascorbic acid (all P < 0.01). Addition of amlodipine (0.01–1 μmol/l) to the calcification medium did not affect VSMC calcification. However, atorvastatin (2–50 μmol/l) stimulated calcium deposition dose-dependently. Combining treatments stimulated calcification to a degree similar to that observed with atorvastatin alone. Both atorvastatin and amlodipine inhibited VSMC proliferation at the highest concentration used. Only atorvastatin (50 μmol/l) induced considerable apoptosis of VSMCs. Conclusion In vitro calcification of VSMCs is not affected by amlodipine, but is stimulated by atorvastatin at concentrations ≥10 μmol/l, which could contribute to the plaque-stabilizing effect reported for statins. J. W. Jukema is an Established Clinical Investigator of The Netherlands Heart Foundation 2001D032.     

Regulation of cerebroside sulfotransferase activity in cultured oligodendrocytes: effect of growth factors and insulin

Cerebroside sulfotransferase (EC 2.8.2.11, CST) specific activity has been determined in oligodendrocyte (OL)-enriched glial cell cultures from newborn rat brain grown in serum supplemented medium. This activity is detectable at 5 days in vitro (DIV) and reaches its maximum value at 12 DIV. This period corresponds to that of oligodendrocyte precursor proliferation in these cultures. The activity decreases thereafter and remains nearly constant after 24 DIV. The developmental curve of CST activity is parallel in pure oligodendrocyte subcultures but twice higher than in primary cultures. These data confirm that CST is highly enriched in OL. Basic fibroblast growth factor (bFGF) (15 ng/ml) and platelet derived growth factor (PDGF) (0.75 U/ml) both enhance CST activity by 90% and 72%, respectively. This increase is in the same range than that of DNA content in treated cultures, whereas protein increase is smaller (50% and 22%, respectively). In contrast, transforming growth factor beta 1 (TGF beta 1, 0.5 and 5 ng/ml) does not significantly enhance CST activity nor DNA content of OL cultures. Insulin at high concentrations (5 micrograms/ml) also enhances CST activity but has no effect at physiological concentrations (20 ng/ml). These results show that CST activity can be controlled by growth factors. They suggest that CST activity is more closely related to OL and OL precursor proliferation than to myelination itself since its maximal activity preceeds myelination in vitro.