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Stem cell factor (SCF) and its receptor, cKIT, are novel regulators of pathological neovascularization in the eye, which suggests that inhibition of SCF/cKIT signaling may be a novel pharmacological strategy for treating neovascular age-related macular degeneration (AMD). This study evaluated the therapeutic potential of a newly developed fully human monoclonal antibody targeting cKIT, NN2101, in a murine model of neovascular AMD. In hypoxic human endothelial cells, NN2101 substantially inhibited the SCF-induced increase in angiogenesis and activation of the cKIT signaling pathway. In a murine model of neovascular AMD, intravitreal injection of NN2101 substantially inhibited the SCF/cKIT-mediated choroidal neovascularization (CNV), with efficacy comparable to aflibercept, a vascular endothelial growth factor inhibitor. A combined intravitreal injection of NN2101 and aflibercept resulted in an additive therapeutic effect on CNV. NN2101 neither caused ocular toxicity nor interfered with the early retinal vascular development in mice. Ocular pharmacokinetic analysis in rabbits indicated that NN2101 demonstrated a pharmacokinetic profile suitable for intravitreal injection. These findings provide the first evidence of the potential use of the anti-cKIT blocking antibody, NN2101, as an alternative or additive therapeutic for the treatment of neovascular AMD.

Objective— Although stem cell factor (SCF) has been shown to play a critical role in hematopoiesis, gametogenesis, and melanogenesis, the function of SCF in the regulation of vascular integrity has not been studied. Approach and Results— We demonstrated that SCF binds to and activates the cKit receptor in endothelial cells, thereby increasing the internalization of vascular endothelial-cadherin and enhancing extravasation of dyes to a similar extent as vascular endothelial growth factor. SCF-mediated cKit activation in endothelial cells enhanced the phosphorylation of endothelial nitric oxide (NO) synthase via the phosphoinositide 3-kinase/Akt signaling pathway and subsequently increased the production of NO. Inhibition of endothelial NO synthase expression and NO synthesis using small interfering RNA knockdown and chemical inhibitors substantially diminished the ability of SCF to increase the internalization of vascular endothelial-cadherin and in vitro endothelial permeability. SCF-induced increase in extravasation of the dyes was abrogated in endothelial NO synthase knockout mice, which indicates that endothelial NO synthase–mediated NO production was responsible for the SCF-induced vascular leakage. Furthermore, we demonstrated that the expression of SCF and cKit was significantly higher in the retina of streptozotocin-injected diabetic mice than in the nondiabetic control animals. Depletion of SCF by intravitreous injection of anti-SCF–neutralizing immunoglobulin G significantly prevented vascular hyperpermeability in the retinas of streptozotocin-injected diabetic mice. Conclusions— Our data reveal that SCF disrupts the endothelial adherens junction and enhances vascular leakage, as well as suggest that anti-SCF/cKit therapy may hold promise as a potential therapy for the treatment of hyperpermeable vascular diseases.

Polycyclic aromatic hydrocarbons (PAHs) are a group of ubiquitous hazardous pollutants derived from fossil fuel, various combustion sources and pyrolysis of a wide range of plastics. Because PAHs can be uptake into crop plants, the inhibitory effects on rice and soybean plants were examined in greenhouse and growth chamber experiment. Soil-applied PAHs (phenanthrene of 0, 10, 30, 100 ppm) slightly reduced the plant height and dry weight both in transplanted rice and soybean plant. The inhibitory effect on growth was greater in soybean than rice. Plant height of soybean plants treated by 100 ppm was 58.9 cm and this value was 87.2% of untreated plant. In rice plant, the plant height was less inhibited (96.0% of untreated plant) by 100 ppm at 80 days after treatment (DAT). However, leaf chlorophyll content and chlorophyll fluorescence were less inhibited by PAHs at late growth stage (after heading) although the photosynthesis-related parameters were slightly inhibited from 20 DAT to 70 DAT. In agar medium experiment with infant seedlings, inhibition of seedling length and fresh weight by phenanthrene at 100 ppm were greater as compared to the experiment with adult plant in pot. Seedling length and fresh weight were reduced by 54.2% and 33.3% for rice and 27.9% and 13.2% for soybean, respectively. The results reflected that PAHs were more inhibitory during juvenile stage than adult stage and more inhibitory to rice plant than soybean for juvenile stage.

The objective of this study was to evaluate the effect of cutting types on microclimate and growth characteristics of afforested tree in Quercus acutissima stand after different types of cutting. The difference in temperature reaching 5.2℃ was shown in between clear cutting and selective cutting treatments. On July and August days with temperatures more than 35℃often appeared in clear cutting stand. The values of VPD in July and August were higher than those in other months. Maximum VPD of 3.99 kPa was shown in clear cutting stand on May 23 as a prolonged rainless days appeared. However, VPD in selective cutting stand always stayed under 3.0 kPa throughout growing season. A higher intensity was shown in clear cutting and strip clear cutting stands, reaching to more than 1,600 μmol m -2 s -1 at midday on early August, while that in selective cutting stand stayed about 1,500. In relative growth rate selective cutting stand showed a significantly higher relative growth rate in plant height than those in other cutting stands (p

Identification of differentially expressed genes in angioblasts derived from human embryonic stem cells (hESCs) is of great interest for elucidating the molecular mechanisms underlying human vasculogenesis. The aim of this study was to define hESC-derived angioblasts at the clonal level and to perform comparative transcriptional analysis to characterize their distinct gene expression profiles. In a clonal analysis performed in cell-specific differentiation media, hESC-derived CD34(+)CD31(+) cells were identified as angioblasts in that they exhibited a significantly higher ability to form endothelial cell (EC) and smooth muscle cell (SMC) colonies than CD34(+)CD31(-) and CD34(-) cell populations did. Microarray analysis showed that many genes involved in vascular development and signaling transduction were overexpressed in hESC-derived CD34(+)CD31(+) cells, whereas those related to mitosis, the DNA damage response, and translation were substantially downregulated. In addition, comparative gene expression profiling of hESC-derived CD34(+)CD31(+) cells and human somatic primary vascular cells demonstrated that hESC-derived CD34(+)CD31(+) cells expressed key genes involved in the EC and SMC differentiation processes, which supports the result that hESC-derived CD34(+)CD31(+) cells are bipotent angioblasts. Our results may provide insights into the identity and function of hESC-derived angioblasts and may also facilitate further investigation of the molecular mechanisms regulating human embryonic vasculogenesis.

Background Our goal was to investigate the correlation between the dysregulation of transforming growth factor-β1 (TGF-β1) and cystic medial degeneration in the aortic aneurysmal tissues of in Marfan syndrome (MFS) patients. Although aortic aneurysm in animal models of MFS is related to the dysregulation of TGF-β, it has yet to be determined whether TGF-β dysregulation correlates with pathogenic aneurysmal characteristics in MFS patients. Methods and results Compared with aortic tissue from normal individuals, the medial layers of aortic tissue from MFS patients exhibited profound cystic medial degeneration and cellular apoptosis. These histopathologic changes positively correlated with the extent of TGF-β1 signaling activation (Smad2 phosphorylation) in aneurysmal aortic tissue. In addition, the level of TGF-β1 expression in peripheral blood and aneurysmal aortic tissues was significantly elevated in MFS patients. A significant positive correlation was observed between the plasma level of active TGF-β1 in MFS patients and the severity of cystic medial degeneration and Smad2 phosphorylation in aneurysmal aortic medial layers. Conclusions We found a strong association between the dysregulation of TGF-β1 and aortic pathogenesis in human MFS patients. This suggests that the plasma concentration of TGF-β1 in MFS patients might be a useful biomarker of the progression of aortic aneurysms.

Objective— Vascular smooth muscle cells (VSMCs) modulate their phenotype between synthetic and contractile states in response to environmental changes; this modulation plays a crucial role in the pathogenesis of restenosis and atherosclerosis. Here, we identified fibroblast growth factor 12 (FGF12) as a novel key regulator of the VSMC phenotype switch. Approach and Results— Using murine models and human specimens, we found that FGF12 was highly expressed in contractile VSMCs of normal vessel walls but was downregulated in synthetic VSMCs from injured and atherosclerotic vessels. In human VSMCs, FGF12 expression was inhibited at the transcriptional level by platelet-derived growth factor-BB. Gain- and loss-of-function experiments showed that FGF12 was both necessary and sufficient for inducing and maintaining the quiescent and contractile phenotypes of VSMCs. FGF12 inhibited cell proliferation through the p53 pathway and upregulated the key factors involved in VSMC lineage differentiation, such as myocardin and serum response factor. Such FGF12-induced phenotypic change was mediated by the p38 MAPK (mitogen-activated protein kinase) pathway. Moreover, FGF12 promoted the differentiation of mouse embryonic stem cells and the transdifferentiation of human dermal fibroblasts into SMC-like cells. Furthermore, adenoviral infection of FGF12 substantially decreased neointima hyperplasia in a rat carotid artery injury model. Conclusions— In general, FGF family members induce a synthetic VSMC phenotype. Interestingly, the present study showed the unanticipated finding that FGF12 belonging to FGF family, strongly induced the quiescent and contractile VSMC phenotypes and directly promoted VSMC lineage differentiation. These novel findings suggested that FGF12 could be a new therapeutic target for treating restenosis and atherosclerosis.

BACKGROUND AND OBJECTIVES In our previous study, we found that the gene transfer of a potent derivative of cartilage oligomeric matrix protein Angiopoietin-1 (COMP-Ang-1) substantially prevented hypertension, microvascular rarefaction, and target organ damage in spontaneously hypertensive rats (SHRs). The purpose of the present study was to examine the role of nitric oxide (NO) in the therapeutic effects observed after COMP-Ang-1 gene transfer. MATERIALS AND METHODS To exclude the NO-mediated effects in COMP-Ang-1 gene therapy, the SHRs were treated with an NO synthase (NOS) inhibitor, N(w)-nitro-L-arginine methyl ester (L-NAME) before the electrophoretic gene transfer. RESULTS The pretreatment with L-NAME induced a severe and sustained increase in systolic blood pressure (BP) in a LacZ plasmid transferred control SHR. However, the electrophoretic transfer of a COMP-Ang-1 plasmid instead of LacZ plasmid in L-NAME-pretreated SHRs substantially blocked the development of hypertension without any significant difference in comparison with L-NAME-untreated COMP-Ang-1 plasmid transferred groups. In addition, the COMP-Ang-1 plasmid transfer substantially attenuated microvascular rarefaction and arteriole remodeling in the heart and kidney, which might account for the mild histological alterations observed in the COMP-Ang-1 plasmid transferred group, in contrast to the severe fibrosis and necrosis seen in the LacZ plasmid controls. CONCLUSION These therapeutic outcomes of COMP-Ang-1 gene transfer even in NOS inhibited SHRs suggested that the antihypertensive effect of COMP-Ang-1 was not merely secondary to NO-mediated vasorelaxation, but it may be associated with its ability to protect the vascular endothelium probably via an NO-independent mechanism which serves to attenuate microvascular rarefaction and target organ damage, and also to prevent hypertension by reducing peripheral vascular resistance.

Endothelial progenitor cells (EPCs), endothelial precursors that promote neovascularization in ischemic tissues, have shown the limited vascular regeneration efficacy due to their poor homing into injured sites and low survival, so that a variety of biosynthetic scaffolds have been employed as cell delivery vehicles to overcome the current cell transplantation methods. However, few paralleled studies that directly compare the efficacy of EPCs seeded within synthetic scaffolds to that of EPCs delivered by the conventional transplantation techniques used for EPC therapies have been performed. To address these issues, RGD-g-PLLA biosynthetic scaffold was developed for the targeted EPC delivery and was found to successfully support the in vitro growth and endothelial functions of EPCs. This scaffold also appeared to be good as in vivo targeted delivery carriers of EPCs as it promoted vascular regeneration in a murine dermal wound models. Furthermore, direct comparison with the intradermal EPC injection revealed that the targeted delivery of EPCs by using the RGD-g-PLLA scaffold was superior to their conventional local injection method in terms of the localization and survival/retention of the transplanted EPCs, and their vascular repairing potential. These results suggest that the development of an effective stem cell delivery system may help to maximize the tissue-repairing efficacy with a limited number of stem cells, thereby resolving the limited clinical success of current stem cell therapies that have utilized simple cell injections or infusions.

Bokbunja (wild raspberry) seed waste from the Bokbunja wine industry is a serious environmental problem due to the increasing popularity of Bokbunja wines in recent years. In the present study, Bokbunja seeds were carbonized and used as an adsorbent for the removal of Procion Red MX-5B. A batch of carbon was further treated with n-hexane to remove excess seed oil and used separately as an adsorbent for comparison. There was a slight variation in the adsorption capacities of n-hexane untreated (HUTC) and n-hexane treated carbon (HTC). The adsorption capacities predicted by the Langmuir isotherm were 29.37 and 30.65 mg/g for HUTC and HTC, respectively. The adsorption was found to be dependant on initial pH, contact time and initial dye concentration. The dye removal rates were higher at pH 2, while equilibrium time was achieved at 120 min. Kinetic studies showed a pseudo-first-order rate of adsorption. The results show that Bokbunja carbon could be used as a potential adsorbent for dye removal from wastewaters.

Aims The endothelium has emerged recently as a therapeutic target in the treatment of hypertension because endothelial dysfunction and subsequent vascular rarefaction cause target organ damage and further elevate blood pressure (BP). It led us to hypothesize that one of the endothelial survival factors, a potent derivative of angiopoietin-1 (cartilage oligomeric matrix protein, COMP-Ang-1), could be a novel class of antihypertensive agents that maintain endothelial integrity and function, thereby preventing the development of hypertension and target organ damage. Methods and results To study the role of COMP-Ang-1 in preventing hypertension and target organ damage, a COMP-Ang-1 plasmid was electroporated into adductor muscles of 6 weeks old, pre-hypertensive, spontaneously hypertensive rats (SHRs), and the secretion of its expressed protein into the bloodstream was confirmed by western blotting. In comparison with sham and reporter gene transfer, COMP-Ang-1 gene transfer significantly prevented increases in systolic BP and reduced microvascular rarefaction and tissue damage in the heart and kidney. However, overexpression of soluble Tie2 receptor completely abolished these beneficial effects of COMP-Ang-1 gene transfer on SHRs, indicating that expressed COMP-Ang-1 protein has antihypertensive effects in SHRs by binding Tie2 receptors on the vascular endothelium. In particular, COMP-Ang-1 gene-transferred SHRs had significantly higher plasma levels of nitrite than other controls, which was found to be due to that expressed COMP-Ang-1 protein promoted nitrite synthesis by activating endothelial nitric oxide synthase, one of the Tie2 downstream-signalling molecules. Conclusion The present study suggests a new potential of endothelial survival factor, COMP-Ang-1, as an antihypertensive agent that effectively reduces the hypertension-associated cardiovascular and renal damage, as well as prevents the further elevation of BP.

Human induced pluripotent stem cells (hiPSCs) are generated through the reprogramming of somatic cells into an embryonic stem cell-like state, such that vascular cells differentiated from hiPSCs might be a suitable autologous cell source for vascular regeneration. The goal of this study was to assess whether cotransplantation of endothelial cells (ECs) and smooth muscle cells (SMCs) differentiated from hiPSCs could promote neovascularization and tissue repair in a murine dermal wound model. hiPSCs were differentiated into ECs and SMCs; the differentiated cells displayed cell-specific surface markers. Compared to primary somatic cells, ECs and SMCs, which were differentiated from hiPSCs, strongly cooperated to enhance in vitro tubular network formation. In vivo gel assays in athymic nude mice showed that the coimplantation of differentiated ECs and SMCs significantly increased vascularization, unlike that observed in the case of implantation of differentiated ECs alone. In a murine full-thickness wound model, when compared with the transplantation of primary somatic cells or phosphate-buffered saline, cotransplantation of differentiated ECs and SMCs markedly enhanced neovascularization in injured tissues and accelerated wound healing. These results demonstrate that cotransplantation of hiPSC-derived ECs and SMCs may be feasible as a new autologous cell therapy for neovascularization and tissue repair.

It is well known that the expression of α B-crystallin (aBC) is increased in neurons and glia under pathologic conditions. However, the expression of aBC during the normal development of the central nervous system has not been reported. This study aimed to clarify the cell type in the chick retina in which aBC is expressed and timing of aBC expression in this cell type during development. Double immunofluorescence with cell-specific markers demonstrated that aBC was selectively expressed in oligodendrocytes (OLs) in the embryonic day 20 (E20) chick retina. A small number of aBC-expressing OLs first appeared in the nerve fiber layer of the central and peripheral retina at E16. Faint aBC expression was also observed in myelin sheaths near cell bodies in the central retina. The number of aBC-expressing OLs and intensity of aBC expression in myelin sheaths were increased in the periphery as well as in the center of the E19 retina. aBC signals in the post-hatching day 120 retina were observed in the entire nerve fiber layer. The spatiotemporal expression pattern of aBC was identical to that of myelin basic protein. These data indicate that aBC-expressing OLs are myelinating OLs among OL-lineage cells. Besides, intrayolk injection of tocopherol, an antioxidant, provoked a decrease in the levels of aBC expression in myelinating OLs. These data suggest that aBC expression in myelinating OLs responds to the change of physiological oxidative stress.

Although Angiopoietin (Ang) 2 has been shown to function as a Tie2 antagonist in vascular endothelial cells, several recent studies on Ang2-deficient mice have reported that, like Ang1, Ang2 acts as a Tie2 agonist during in vivo lymphangiogenesis. However, the mechanism governing the Tie2 agonistic activity of Ang2 in lymphatic endothelial cells has not been investigated. We found that both Ang1 and Ang2 enhanced the in vitro angiogenic and anti-apoptotic activities of human lymphatic endothelial cells (HLECs) through the Tie2/Akt signaling pathway, while only Ang1 elicited such effects in human umbilical vein vascular endothelial cells (HUVECs). This Tie2-agonistic effect of Ang2 in HLECs resulted from low levels of physical association between Tie2 and Tie1 receptors due to a reduced level of Tie1 expression in HLECs compared to HUVECs. Overexpression of Tie1 and the resulting increase in formation of Tie1/Tie2 heterocomplexes in HLECs completely abolished Ang2-mediated Tie2 activation and the subsequent cellular responses, but did not alter the Ang1 function. This inhibitory role of Tie1 in Ang2-induced Tie2 activation was also confirmed in non-endothelial cells with adenovirus-mediated ectopic expression of Tie1 and/or Tie2. To our knowledge, this study is the first to describe how Ang2 acts as a Tie2 agonist in HLECs. Our results suggest that the expression level of Tie1 and its physical interaction with Tie2 defines whether Ang2 functions as a Tie2 agonist or antagonist, thereby determining the context-dependent differential endothelial sensitivity to Ang2.

Rationale : Generation of induced pluripotent stem (iPS) cells has been intensively studied by a variety of reprogramming methods, but the molecular and functional properties of the cells differentiated from iPS cells have not been well characterized. Objective : To address this issue, we generated iPS cells from human aortic vascular smooth muscle cells (HASMCs) using lentiviral transduction of defined transcription factors and differentiated these iPS cells back into smooth muscle cells (SMCs). Methods and Results : Established iPS cells were shown to possess properties equivalent to human embryonic stem cells, in terms of the cell surface markers, global mRNA and microRNA expression patterns, epigenetic status of OCT4, REX1, and NANOG promoters, and in vitro/in vivo pluripotency. The cells were differentiated into SMCs to enable a direct, comparative analysis with HASMCs, from which the iPS cells originated. We observed that iPS cell–derived SMCs were very similar to parental HASMCs in gene expression patterns, epigenetic modifications of pluripotency-related genes, and in vitro functional properties. However, the iPS cells still expressed a significant amount of lentiviral transgenes (OCT4 and LIN28) because of partial gene silencing. Conclusions : Our study reports, for the first time, the generation of iPS cells from HASMCs and their differentiation into SMCs. Moreover, a parallel comparative analysis of human iPS cell–derived SMCs and parental HASMCs revealed that iPS-derived cells possessed representative molecular and in vitro functional characteristics of parental HASMCs, suggesting that iPS cells hold great promise as an autologous cell source for patient-specific cell therapy.

We previously reported the successful establishment of embryonic stem cell (ESC)-like multipotent spermatogonial stem cells (mSSCs) from neonatal mouse testis. Here, we examined the ability of mSSCs to differentiate into vascular endothelial cells and smooth muscle cells, and compared to that of mouse ESCs. We used real-time reverse transcriptase polymerase chain reaction and immunohistochemistry to examine gene expression profiles of mSSCs and ESCs during in vitro vascular differentiation. Both mSSCs and ESCs exhibited substantial increase in the expression of mesodermal markers, such as Brachyury, Flk1, Mesp1, Nkx2.5, and Islet1, and a decrease in the expression of pluripotency markers, such as Oct3/4 and Nanog during the early stage of differentiation. The mRNA levels of vascular endothelial (VE)-cadherin and CD31 gradually increased in both differentiated mSSCs and ESCs. VE-cadherin- or CD31-positive cells formed sprouting branch-like structures, as observed during embryonic vascular development. At the same time, vascular smooth muscle cell-specific markers, such as myocardin and α-smooth muscle actin (SMA), were also highly expressed in differentiated mSSCs and ESCs. Immunocytochemical analysis revealed that the differentiated cells expressed both α-SMA and SM22-α proteins, and exhibited the intracellular fibril structure typical of smooth muscle cells. Overall, our findings showed that mSSCs have similar vascular differentiation abilities to those of ESCs, suggesting that mSSCs may be an alternative source of autologous pluripotent stem cells for vascular regeneration.