Wonhee Suh

Enzyme-catalyzed in situ forming gelatin hydrogels as bioactive wound dressings: effects of fibroblast delivery on wound healing efficacy

In this study, in situ forming gelatin hydrogels via horseradish peroxidase (HRP)-catalyzed cross-linking were developed to serve as bioactive wound dressings with suitable tissue adhesive properties to deliver dermal fibroblasts (DFBs). The DFB-encapsulated gelatin hydrogels with different stiffnesses, GH-soft (1.1 kPa) and GH-hard (6.2 kPa), were prepared by controlling the hydrogen peroxide (H2O2) concentrations. The GH-soft hydrogel was capable of facilitating the proliferation of DFBs and the synthesis of extracellular components, as compared to GH-hard hydrogels. In addition, the subcutaneously injected GH-soft hydrogel with bioluminescent reporter cells provided enhanced cell survival and local retention over 14 days. In vivo transplantation of DFB-encapsulated GH-soft hydrogels accelerated wound contraction, and promoted collagen deposition and neovascularization within the incisions performed on mice skin. Therefore, we expect that HRP-catalyzed in situ forming gelatin hydrogels can be useful for local delivery of cells with high viability in wounds, which holds great promise for advancing wound healing technologies and other tissue engineering applications.

Direct and differential effects of stem cell factor on the neovascularization activity of endothelial progenitor cells

AIMS Previous studies on the role of stem cell factor (SCF) in endothelial progenitor cell (EPC)-mediated neovascularization have focused on the EPC mobilization and homing process. However, the direct effects of SCF on neovascularization activity of EPCs have not been characterized. We sought to determine whether SCF regulates the neovascularization ability of EPCs by comparing its roles in mature endothelial cells. METHODS AND RESULTS In vitro and in vivo assays revealed that SCF substantially increased the neovascularization activity of human EPCs through the c-Kit receptor. Notably, the SCF-induced increase in neovascularization activity was substantially greater in EPCs than that in human umbilical vein endothelial cells (HUVECs). SCF-induced phosphorylation of c-Kit and downstream signalling molecules was consistently found to be more potent and longer-lasting in EPCs than in HUVECs. This high responsiveness of EPCs to SCF was explained by the finding that the cell-surface expression of c-Kit is far higher in EPCs than in HUVECs. A c-Kit promoter assay revealed that the increased expression of c-Kit in EPCs could be attributed to the greater expression of stem cell leukaemia, LIM-only 2, and GATA-binding protein 2. CONCLUSION In addition to its documented role in the mobilization and recruitment of EPCs, our findings show that SCF directly enhances the neovascularization activity of EPCs. Furthermore, the present study provides further evidence that EPCs exhibit differentially greater responsiveness to hypoxia-inducible cytokines, including SCF, than mature endothelial cells, suggesting that EPCs in ischaemic tissues function differently from mature endothelial cells, although they exhibit very similar phenotypes.

Tie1 regulates the Tie2 agonistic role of angiopoietin-2 in human lymphatic endothelial cells

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.

Establishment of isolation and expansion protocols for human cardiac C-kit-positive progenitor cells for stem cell therapy.

Although cardiac stem cells (CSCs) have emerged in regeneration research, the number of isolated CSCs is low, making a sufficient supply of functional elements an important consideration in cardiovascular research. In this study, we established an efficient method for CSC isolation. We directly compared cultures of single cells to human cardiac-derived c-kit-positive progenitor cells (hCPCs(c-kit+)). The two protocols employed enzymatically digested hCPCs(c-kit+) (ED-hCPCs) with tissue-expanded hCPC(c-kit+) (TE-hCPCs). Using fluorescence-activated cell sorting, we showed the concentration of c-kit in TE-hCPCs to be higher than in ED-hCPCs, although the total number of c-kit positive cells resulting from ED-hCPCs was similar to that resulting from TE-hCPCs. The cardiomyocyte-associated proteins, GATA4 and Nkx2-5, which were expressed during hCPCs expansion, did not differ between the isolation methods. Importantly, the expression of the CSC stem cell marker, c-kit, was more efficiently preserved using the ED-hCPCs versus the TE-hCPCs method. In a cell proliferation assay, the ED-hCPCs method produced a significantly greater number of cells. Finally, hCPCs derived using both protocols differentiated into endothelial, smooth muscle, and cardiomyocyte lineages. In conclusion, the single-cell culture protocol using an enzymatic digestion method may be more useful to isolate human cardiac-derived c-kit-positive elements compared with the tissue expansion method.

Amine-enriched surface modification facilitates expansion, attachment, and maintenance of human cardiac-derived c-kit positive progenitor cells

BACKGROUND Stem cells have a low expansion rate and are difficult to maintain in vitro. To overcome the problems of cardiovascular regeneration, we developed a novel method of stem cell cultivation in culture vessels with amine and carboxyl coatings. METHODS AND RESULTS We isolated cardiac stem/progenitor cells from infant-derived heart tissue by using c-kit antibody (human cardiac-derived c-kit positive progenitor cells; hCPC(c-kit+)); the cells differentiated into endothelial cells, smooth muscle cells, and cardiomyocytes. To characterize the effect of surface modification on hCPC(c-kit+) expansion, cellular attachment, c-kit expression maintenance, and cardiomyocyte differentiation, we tested hCPC(c-kit+) cultured on non-coated (control), amine-coated (amine), and carboxyl-coated (carboxyl) vessels. Ex vivo proliferation, c-kit maintenance, and cellular attachment were significantly enhanced in the amine group. The amine coating also increased procollagen type I (pro-COL1) expression and increased phosphorylation signals, such as focal adhesion kinase (FAK) and cytosolic Src, as well as enhanced ERK/CDK2 signaling. In addition, there was significant downregulation of the stress signal transducer, JNK, in the amine group. However, cardiomyogenesis remained unchanged in the control, amine, and carboxyl groups. CONCLUSIONS Although surface modifications had no effect on early induction cardiomyogenesis, amine-enriched surface modification may increase hCPC(c-kit+) expansion. The amine-enriched surface improved cellular proliferation and attachment during ex vivo hCPC(c-kit+) expansion, possibly by modulating intracellular signal transducers.

Angiopoietin-1 gene therapy attenuates hypertension and target organ damage in nitric oxide synthase inhibited spontaneously hypertensive rats.

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, Nw-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.

Reactive oxygen species regulate the quiescence of CD34-positive cells derived from human embryonic stem cells

AIMS Reactive oxygen species (ROS) are involved in a wide range of cellular processes. However, few studies have examined the generation and function of ROS in human embryonic vascular development. In this study, the sources of ROS and their roles in the vascular differentiation of human embryonic stem cells (hESCs) were investigated. METHODS AND RESULTS During vascular differentiation of hESCs, CD34(+) cells had quiescence-related gene expression profiles and a large fraction of these cells were in G0 phase. In addition, levels of ROS, which were primarily generated through NOX4, were substantially higher in hESC-derived CD34(+) cells than in hESC-derived CD34(-) cells. To determine whether excess levels of ROS induce quiescence of hESC-derived CD34(+) cells, ROS levels were moderately reduced using selenium to enhance antioxidant activities of thioredoxin reductase and glutathione peroxidase. In comparison to untreated CD34(+) cells, selenium-treated CD34(+) cells exhibited changes in gene expression that favoured cell cycle progression, and had a greater proliferation and a smaller fraction of cells in G0 phase. Thus, selenium treatment increased the number of hESC-derived CD34(+) cells, thereby enhancing the efficiency with which hESCs differentiated into vascular endothelial and smooth muscle cells. CONCLUSION This study reveals that NOX4 produces ROS in CD34(+) cells during vascular differentiation of hESCs, and shows that modulation of ROS levels using antioxidants such as selenium may be a novel approach to increase the vascular differentiation efficiency of hESCs.

Distinct transcriptional profiles of angioblasts derived from human embryonic stem cells.

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.

Direct comparison of distinct cardiomyogenic induction methodologies in human cardiac-derived c-kit positive progenitor cells

Cardiac stem/progenitor cells can be differentiated into cardiomyocytes in vitro using several differentiation methodologies. However, the methodology of cardiomyogenic induction in human c-kit positive progenitor cells (hCPCsc-kit+) was not fully demonstrated. Thus, the purpose of our study was to directly evaluate each cardiomyocyte induction system using hCPCsc-kit+. In this study, cardiomyocyte induction methodologies were divided into the following three groups; treatment with dexamethasone, 5-azacytidine, and co-treatment with 5-azacytidine and Transforming Growth Factor Beta 1 (TGF-β1), using different serum concentrations [2% or 10% fetal bovine serum (FBS)]. GATA4 and Nkx2-5, cardiac-specific transcription factors, were expressed in our hCPCsckit+. However, the GATA4 and Nkx2-5 expressions were significantly decreased in 10% FBS/cardiomyogenic induction system (p < 0.01), whereas the GATA4 and Nkx2-5 expressions were preserved in 2% FBS/cardiomyogenic induction system (p > 0.05). GATA4 and Nkx2-5 is crucial roles in cardiac development, thus we considered the low serum conditions more affected in our cardiomyogenic induction system. In addition, c-kit expression decreased significantly during cardiomyogenic differentiation. Importantly, we demonstrated that co-treated with 5-azacytidine and TGF-β1 led to an earlier expression pattern of alpha-sarcomeric actin (α-SA), implying that this cardiomyocyte induction system facilitates early cardiomyocyte differentiation of hCPCsc-kit+. Thus, the present study provides a pivotal cardiomyogenic differentiation methodology using hCPCsc-kit+for basic or clinical research.

Analysis of disease progression-associated gene expression profile in fibrillin-1 mutant mice: new insight into molecular pathogenesis of marfan syndrome.

Marfan syndrome (MFS) is a dominantly inherited connective tissue disorder caused by mutations in the gene encoding fibrillin-1 (FBN1) and is characterized by aortic dilatation and dissection, which is the primary cause of death in untreated MFS patients. However, disease progression-associated changes in gene expression in the aortic lesions of MFS patients remained unknown. Using a mouse model of MFS, FBN1 hypomorphic mouse (mgR/mgR), we characterized the aortic gene expression profiles during the progression of the MFS. Homozygous mgR mice exhibited MFS-like phenotypic features, such as fragmentation of elastic fibers throughout the vessel wall and were graded into mgR1–4 based on the pathological severity in aortic walls. Comparative gene expression profiling of WT and four mgR mice using microarrays revealed that the changes in the transcriptome were a direct reflection of the severity of aortic pathological features. Gene ontology analysis showed that genes related to oxidation/reduction, myofibril assembly, cytoskeleton organization, and cell adhesion were differentially expressed in the mgR mice. Further analysis of differentially expressed genes identified several candidate genes whose known roles were suggestive of their involvement in the progressive destruction of aorta during MFS. This study is the first genome-wide analysis of the aortic gene expression profiles associated with the progression of MFS. Our findings provide valuable information regarding the molecular pathogenesis during MFS progression and contribute to the development of new biomarkers as well as improved therapeutic strategies.