Quantitative analysis by reversed-phase high-performance liquid chromatography and retinal neuroprotection after topical administration of moxonidine.

AIM: To determine moxonidine in aqueous humor and iris-ciliary body by reversed-phase high performance liquid chromatography (RP-HPLC), and to evaluate the retinal neuroprotective effect after topical administration with moxonidine in a high intraocular pressure (IOP) model. METHODS: The eyes of albino rabbits were administered topically and ipsilaterally with 0.2% moxonidine. A RP-HPLC method was employed for the identification and quantification of moxonidine between 2 and 480min, which presented in the aqueous humor and iris-ciliary body. Flash electroretinography (F-ERG) amplitude and superoxide dismutase (SOD) level were measured between day 1 and day 15 after topical administration with moxonidine in a rabbit model of high IOP. Histological and ultrastructural observation underwent to analyze the changes of retinal morphology, the inner retinal layers (IRL) thickness, and retinal ganglion cell (RGC) counting. RESULTS: Moxonidine was detectable between 2 and 480min after administration, and the peak concentration developed both in the two tissues at 30min, 0.51 µg/mL in aqueous humor and 1.03 µg/g in iris-ciliary body. In comparison to control, F-ERG b-wave amplitude in moxonidine eyes were significantly differences between day 3 and day 15 (P<0.01) in the high IOP model; SOD levels were significantly higher at all time-points (P<0.01) with a maximum level of 20.29 U/mgprot at day 15; and RGCs were significantly higher (P<0.05). CONCLUSION: Moxonidine is a viable neuroprotective agent with application to high IOP model. All layers of retina, including RGC layer, retinal nerve fiber layer and INL, are more preserved after moxonidine administration. SOD plays a neuroprotective role in ocular hypertension-mediated RGC death.

Age-related Changes in the Global DNA Methylation Profile of Oligodendrocyte Progenitor Cells Derived from Rat Spinal Cords.

Demyelination of axons plays an important role in the pathology of many spinal cord diseases and injuries. Remyelination in demyelinated lesions is primarily performed by oligodendrocyte progenitor cells (OPCs), which generate oligodendrocytes in the developing and mature central nervous system. The efficiency of remyelination decreases with age. Many reports suggest that this decline in remyelination results from impaired OPC recruitment and differentiation during aging. Of the various molecular mechanisms involved in aging, changes in epigenetic modifications have received particular attention. Global DNA methylation is a major epigenetic modification that plays important roles in cellular senescence and organismal aging. Thus, we aimed to evaluate the dynamic changes in the global DNA methylation profiles of OPCs derived from rat spinal cords during the aging process. We separated and cultured OPCs from the spinal cords of neonatal, 4-month-old, and 16-month-old rats and investigated the age-related alterations of genomic DNA methylation levels by using quantitative colorimetric analysis. To determine the potential cause of dynamic changes in global DNA methylation, we further analyzed the activity of DNA methyltransferases (DNMTs) and the expression of DNMT1, DNMT3a, DNMT3b, TET1, TET2, TET3, MBD2, and MeCP2 in the OPCs from each group. Our results showed the genomic DNA methylation level and the activity of DNMTs from OPCs derived from rat spinal cords decreased gradually during aging, and OPCs from 16-month-old rats were characterized by global hypomethylation. During OPC aging, the mRNA and protein expression levels of DNMT3a, DNMT3b, and MeCP2 were significantly elevated; those of DNMT1 were significantly down-regulated; and no significant changes were observed in those for TET1, TET2, TET3, or MBD2. Our results indicated that global DNA hypomethylation in aged OPCs is correlated with DNMT1 downregulation. Together, these data provide important evidence for partly elucidating the mechanism of age-related impaired OPC recruitment and differentiation and assist in the development of new treatments for promoting efficient remyelination.

Controlled chondrogenesis from adipose-derived stem cells by recombinant transforming growth factor-ß3 fusion protein in peptide scaffolds.

Adipose-derived stem cells (ADSCs) are promising for cartilage repair due to their easy accessibility and chondrogenic potential. Although chondrogenesis of transforming growth factor-ß (TGF-ß) mediated mesenchymal stem cells (MSCs) is well established in vitro, clinical tissue engineering requires effective and controlled delivery of TGF-ß in vivo. In this work, a self-assembled peptide scaffold was employed to construct cartilages in vivo through the chondrogenesis from ADSCs controlled by recombinant fusion protein LAP-MMP-mTGF-ß3 that was transfected by lentiviral vectors. During this course, the addition of matrix metalloproteinases (MMPs) can trigger the release of mTGF-ß3 from the recombinant fusion protein of LAP-MMP-mTGF-ß3 in the combined scaffolds, thus stimulating the differentiation of ADSCs into chondrogenesis. The specific expression of cartilage genes was analyzed by real-time polymerase chain reaction and Western blot. The expression of chondrocytic markers was obviously upregulated to a higher level compared to the one by commonly used TGF-ß3 alone. After 3 weeks of in vitro culturing, the hybrids with differentiated chondrogenesis were then injected subcutaneously into nude mice and retrieved after 4 weeks of culturing in vivo. Histological analysis also confirmed that the recombinant fusion protein was more effective for the formation of cartilage matrix than the cases either with TGF-ß3 alone or without LAP-MMP-mTGF-ß3 (P<0.05). This study demonstrates that controlled local delivery of the LAP-MMP-mTGF-ß3 constructs can accelerate differentiation of ADSCs into the cartilage in vivo, which indicates the great potential of this hybrid in rapid therapy of osteoarthritis.

Decreased in the number and function of circulation endothelial progenitor cells in patients with avascular necrosis of the femoral head.

INTRODUCTION: Once non-traumatic avascular necrosis of the femoral head (ANFH) happened, vascular impairment and feeble collateral circulation are followed by poor outcomes. Circulating endothelial progenitor cells (EPCs) may substantially contribute to vascular homeostasis such as vascular repair and new blood vessel growth. We investigated whether abnormalities in EPCs levels and functions are present in ANFH patients. METHODS: 54 ANFH patients were enrolled, including steroid-induced (n=21), alcohol-induced (n=15) and idiopathic ANFH (n=18), and 30 healthy subjects as control (HC). The numbers of circulation EPCs were determined by fluorescence-activated cell-sorting (FACS) analysis. EPCs cultured from peripheral blood mononuclear cells on fibronectin to induce the expression of receptors for acetylated low-density lipoprotein and ulex-lectin. EPCs colony-forming units (CFUs) were observed from 54 patients and 30 healthy controls. Migratory capacity to chemo-attractants (vascular endothelial growth factor) cellular senescence levels and in vitro angiogenesis ability were assessed in age-matched subjects (n=10 per groups). RESULTS: Mean numbers of circulating EPC were 1460+/-265 cells/ml in HC, 545+/-177 in ANFH, (P<0.001). Mean numbers of CFUs were 26.2+/-6.2 in HC, 19.6+/-7.7 in ANFH,(P<0.001). Although there were not significant differences in circulating EPC and CFUs among the steroid-induced, alcohol-induced or idiopathic three groups, all these risk factors contributed to the decreased circulating EPCs numbers and CFUs. In addition, EPCs from ANFH patients showed reduced migratory capacity and increased cellular senescence compared with EPCs from normal subjects, furthermore the ability of angiogenesis in vitro was also impaired. CONCLUSION: Circulating endothelial progenitor cells (EPCs) numbers and functions are reduced in ANFH patients, suggesting that risk factors of ANFH may alter EPCs biology in angiogenesis and vascular repair.

Effects of TGF beta1 autocrine blockage on osteosarcoma cells.

OBJECTIVE: To evaluate the effects of transforming growth factor beta1 (TGFbeta1) autocrine blockage on proliferation activity and drug sensitivity of osteosarcoma. METHODS; Northern blot, MTT determination, and 3H thymidine incorporation were used to investigate the effects of antisense TGF beta1 gene on osteosarcoma. RESULTS: The proliferation of osteosarcoma cells transfected by antisense TGF beta1 gene was suppressed markedly, and adriamycin sensitivity was significantly increased. CONCLUSION: Blockage of osteosarcoma cells TGF beta1 autocrine loop inhibits cell proliferation and enhances chemotherapy sensitivity.