Combination therapy with intranasal NGF and electroacupuncture enhanced cell proliferation and survival in rats after stroke

Abstract

Objective: This work was designed to investigate the effects of the combination therapy with intranasal (IN) administration of nerve growth factor (NGF) and electroacupuncture (EA) on neural progenitors and neurological functional recovery in adult rats after focal ischemia.

Methods: Rats subjected to 2 hours of middle cerebral artery occlusion (MCAO) were randomly assigned to four groups: Group 1, IN administration of phosphate-buffered saline (PBS) for control; Group 2, IN administration of NGF alone; Group 3, EA with IN administration of PBS; Group 4, IN administration of NGF with EA. Treatments were initiated at 2 hours after MCAO and continued for three consecutive days. All animals received daily injections of 5-bromodeoxyuridine (BrdU) intraperitoneally for 7 days starting at 24 hours after reperfusion and were killed at 2 hours or 21 days after the last BrdU injection. Neurological function and infarct volume were evaluated. Immunohistochemistry for BrdU was performed to identify newborn cells in the ipsilateral subventricular zone and striatum.

Results: The combination treatment led to significant improvement in neurological function and reduction in infarct volume. Cell proliferation and survival of progenitors were enhanced in rats treated with the combination treatment.

Conclusion: These results suggest that IN administration of NGF and EA may have a synergistic effect in preventing ischemic injury and enhancing functional recovery after focal cerebral ischemia, which may be attributed to enhanced cell proliferation and survival.     

The MET oncogene transforms human primary bone-derived cells into osteosarcomas by targeting committed osteo-progenitors

The MET oncogene is aberrantly overexpressed in human osteosarcomas. We have previously converted primary cultures of human bone-derived cells into osteosarcoma cells by overexpressing MET. To determine whether MET transforms mesenchymal stem cells or committed progenitor cells, here we characterize distinct MET overexpressing osteosarcoma (MET-OS) clones using genome-wide expression profiling, cytometric analysis, and functional assays. All the MET-OS clones consistently display mesenchymal and stemness markers, but not most of the mesenchymal–stem cell-specific markers. Conversely, the MET-OS clones express genes characteristic of early osteoblastic differentiation phases, but not those of late phases. Profiling of mesenchymal stem cells induced to differentiate along osteoblast, adipocyte, and chondrocyte lineages confirms that MET-OS cells are similar to cells at an initial phase of osteoblastic differentiation. Accordingly, MET-OS cells cannot differentiate into adipocytes or chondrocytes, but can partially differentiate into osteogenic-matrix-producing cells. Moreover, in vitro MET-OS cells form self-renewing spheres enriched in cells that can initiate tumors in vivo. MET kinase inhibition abrogates the self-renewal capacity of MET-OS cells and allows them to progress toward osteoblastic differentiation. These data show that MET initiates the transformation of a cell population that has features of osteo-progenitors and suggest that MET regulates self-renewal and lineage differentiation of osteosarcoma cells.     

Contrasting roles of leukemia inhibitory factor in murine bone development and remodeling involve region-specific changes in vascularization

We describe here distinct functions of leukemia inhibitory factor (LIF) in bone development/growth and adult skeletal homeostasis. In the growth plate and developing neonate bones, LIF deficiency enhanced vascular endothelial growth factor (VEGF) levels, enlarged blood vessel formation, and increased the formation of "giant" osteoclasts/chondroclasts that rapidly destroyed the mineralized regions of the growth plate and developing neonatal bone. Below this region, osteoblasts formed large quantities of woven bone. In contrast, in adult bone undergoing remodeling osteoclast formation was unaffected by LIF deficiency, whereas osteoblast formation and function were both significantly impaired, resulting in osteopenia. Consistent with LIF promoting osteoblast commitment, enhanced marrow adipocyte formation was also observed in adult LIF null mice, and adipocytic differentiation of murine stromal cells was delayed by LIF treatment. LIF, therefore, controls vascular size and osteoclast differentiation during the transition of cartilage to bone, whereas an anatomically separate LIF-dependent pathway regulates osteoblast and adipocyte commitment in bone remodeling.     

Modification of gold surface by grafting of poly(ethylene glycol) for reduction in protein adsorption and platelet adhesion

Gold surfaces were first treated in an alkanethiol solution to form self-assembled monolayers (SAMs). The thiolated Au surface was then subjected to Ar plasma pretreatment, followed by air exposure and UV-induced graft polymerization of poly(ethylene glycol) methacrylate (PEGMA) macromonomer. In comparison with the 3-mercaptopropionic acid-2-ethylhexyl ester (MPAEE) SAM, the (3-mercaptoproply)trimethoxysilane (MPTMS) SAM on Au exhibited higher stability under the conditions of Ar plasma pretreatment. The graft concentration of the PEGMA polymer on SAMmodified Au surface increased with increasing PEGMA macromonomer concentration and UV-graft polymerization time. The modified-Au surfaces were characterized by X-ray spectroscopy (XPS), atomic force microscopy (AFM), and water contact angle measurement. The Au surface with a high concentration of grafted PEGMA polymer could completely repel protein adsorption and platelet adhesion.     

In vitro and in vivo studies of PEO-grafted blood-contacting cardiovascular prostheses

The initial step of thrombus formation on blood-contacting biomaterials is known to be adsorption of blood proteins followed by platelet adhesion. Poly(ethylene oxide) (PEO) has been frequently used to modify biomaterial surfaces to minimize or prevent protein adsorption and cell adhesion. PEO was grafted onto a number of biomaterials in our laboratory. Nitinol stents and glass tubes were grafted with PEO by priming the metal surface with trichlorovinylsilane (TCVS) followed by adsorption of Pluronic and γ-irradiation. Nitinol stents were also coated with Carbothane® for PEO grafting. Chemically inert polymeric biomaterials, such as Carbothane, polyethylene, silicone rubber, and expanded polytetrafluoroethylene (e-PTFE), were first adsorbed with PEO-polybutadiene-PEO (PEO-PB-PEO) triblock copolymers and then exposed to γ-irradiation for covalent grafting. For PEO grafting to Dacron® (polyethylene terephthalate), the surface was sequentially treated with PEO-PB-PEO and Pluronics® followed by γ-irradiation. In vitro studies showed substantial reduction in fibrinogen adsorption and platelet adhesion to the PEO-grafted surfaces compared with control surfaces. Fibrinogen adsorption was reduced by 70-95% by PEO grafting on all surfaces, except for e-PTFE. The platelet adhesion corresponded to the fibrinogen adsorption. When the PEO-grafted surfaces were tested ex vivo/in vivo, however, the expected beneficial effect of PEO grafting was inconsistent. The beneficial effect of the PEO grafting was most pronounced on the PEO-grafted nitinol stents. Thrombus formation was reduced by more than 85% by PEO grafting on metallic stents. Only moderate improvement (i.e. 35% decrease in platelet deposition) was observed with PEO-grafted tubes of polyethylene, silicone rubber, and glass. For PEO-grafted heart valves made of Dacron, however, no effect of PEO grafting was observed at all. It appears that the extent of thrombus formation on PEO-grafted biomaterials was directly related to the extent of tissue damage during implantation surgery. Platelets can be activated and form aggregates in the bulk blood, and the formed platelet aggregates may be able to deposit on the PEO monolayer overcoming its repulsive property. Our studies indicate that the testing of in vitro platelet adhesion should include adhesion of large platelet aggregates, in addition to adhesion of individual platelets. Furthermore, the surface modification methods should be improved over the current monolayer grafting concept so that the repulsive force by the grafted PEO layers is large enough to prevent adhesion of platelet aggregates formed in the bulk blood before arriving at the biomaterial surface.     

Developmental expression of fibroblast growth factor (FGF) receptors in neural stem cell progeny. Modulation of neuronal and glial lineages by basic FGF treatment

Abstract

Neural stem cells (NSCs) are self-renewable, multipotential cells capable of differentiating into the three major neural cell types, but the mechanisms which regulate their development are not fully understood. Both basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) promote the proliferation of NSCs. However, studies on the role of FGFs in the differentiation of EGF-expanded NSCs are still incomplete. We have studied the expression of distinct FGF receptors (FGFRs) in the progeny of EGFexpanded NSCs isolated from E15 rat striatum. In situ hybridization analysis and immunocytochemistry showed a developmentally related expression pattern and a cell lineage-specific distribution of these receptors. FGFR1 and FGFR2 were identified in many early precursors and in the oligodendrocyte lineage. The latter receptor was also present in a subpopulation of astrocytes. FGFR3 was detected in a restricted population of early precursors, in oligodendroglial progenitors, and in neurons and protoplasmic astrocytes of late-term cultures. Basic FGF treatment of the progeny of NSCs increased the proliferative rate of precursors and the number of oligodendrocytes generated, whereas the number of differentiating neurons was significantly reduced. Together these data provide evidence that FGFs modulate the development of EGF-expanded NSCs, and that this is at least partly determined by a cell lineage-specific expression of multiple FGFRs. [Neurol Res 2001; 23: 612-621]     

Elevation of MCP-1 and MCP-1/VEGF ratio in cerebrospinal fluid of amyotrophic lateral sclerosis patients

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with progressive cell death of upper and lower motor neurons. In this study, we measured monocyte chemotactic protein-1 (MCP-1) and vascular endothelial growth factor (VEGF) levels in cerebrospinal fluid (CSF) and serum by enzyme-linked immunosorbent assay (ELISA) in 42 ALS patients, and compared these levels with those of control subjects with other neurodegenerative disorders or with those of normal controls. MCP-1 levels in CSF were significantly higher in ALS patients than in the control group. VEGF levels in CSF tended to be lower in ALS patients than in the control group, but not significantly. A positive correlation was found between MCP-1 levels in CSF of ALS patients and the total Norris scale. The elevation of MCP-1/VEGF ratio in CSF was more specific to ALS patients compared to other neurological diseases such as Parkinson's disease (PD) and spinocerebellar ataxia (SCA) and to controls. Our data suggested that both MCP-1 levels and MCP-1/VEGF ratio in CSF may be useful markers for the clinical diagnosis of ALS.     

Human apolipoprotein E isoforms differentially affect bone mass and turnover in vivo

The primary role of apolipoprotein E (apoE) is to mediate the cellular uptake of lipoproteins. However, a new role for apoE as a regulator of bone metabolism in mice has recently been established. In contrast to mice, the human APOE gene is characterized by three common isoforms APOE ε2, ε3, and ε4 that result in different metabolic properties of the apoE isoforms, but it remains controversial whether the APOE polymorphism influences bone traits in humans. To clarify this, we investigated bone phenotypes of apoE knock‐in (k.i.) mice, which express one human isoform each (apoE2 k.i., apoE3 k.i., apoE4 k.i.) in place of the mouse apoE. Analysis of 12‐week‐old female k.i. mice revealed increased levels of biochemical bone formation and resorption markers in apoE2 k.i. animals as compared to apoE3 k.i. and apoE4 k.i., with a reduced osteoprotegerin (OPG)/receptor activator of NF‐κB ligand (RANKL) ratio in apoE2 k.i., indicating increased turnover with prevailing resorption in apoE2 k.i. Accordingly, histomorphometric and micro–computed tomography (µCT) analyses demonstrated significantly lower trabecular bone mass in apoE2 than in apoE3 and apoE4 k.i. animals, which was reflected by a significant reduction of lumbar vertebrae maximum force resistance. Unlike trabecular bone, femoral cortical thickness, and stability was not differentially affected by the apoE isoforms. To extend these observations to the human situation, plasma from middle‐aged healthy men homozygous for ε2/ε2, ε3/ε3, and ε4/ε4 (n = 21, n = 80, n = 55, respectively) was analyzed with regard to bone turnover markers. In analogy to apoE2 k.i. mice, a lower OPG/RANKL ratio was observed in the serum of ε2/ε2 carriers as compared to ε3/ε3 and ε4/ε4 individuals (p = 0.02 for ε2/ε2 versus ε4/ε4). In conclusion, the current data strongly underline the general importance of apoE as a regulator of bone metabolism and identifies the APOE ε2 allele as a potential genetic risk factor for low trabecular bone mass and vertebral fractures in humans. © 2013 American Society for Bone and Mineral Research