Yoon Mi Jin

Uric acid attenuates nitric oxide production by decreasing the interaction between endothelial nitric oxide synthase and calmodulin in human umbilical vein endothelial cells: A mechanism for uric acid-induced cardiovascular disease development

The elevated level of uric acid in the body is associated with increased risk of cardiovascular diseases, which is mediated by endothelial dysfunction. However, its underlying mechanism is not fully understood, although dysregulation of endothelial nitric oxide (NO) production is likely to be involved. Using human umbilical vascular endothelial cells (HUVEC), we explored the molecular mechanism of uric acid on endothelial NO synthase (eNOS) activity and NO production. Although high dose of uric acid (12mg/dl for 24h treatment) significantly decreased eNOS activity and NO production, it did not alter eNOS expression and phosphorylations at eNOS-Ser(1177), eNOS-Thr(495) and eNOS-Ser(114). Under this condition, we also found no alterations in the dimerization and acetylation of eNOS, compared with the control. Furthermore, uric acid did not change the activity of arginase II, an enzyme degrading l-arginine, a substrate of eNOS, and intracellular level of calcium, a cofactor for eNOS activation. We also found that uric acid did not alter xanthine oxidase activity, suggesting no involvement of xanthine oxidase-derived O2(-) production in the observed inhibitory effects. In vitro and in cell coimmunoprecipitation studies, however, revealed that uric acid significantly decreased the interaction between eNOS and calmodulin (CaM), an eNOS activator, although it did not change the intracellular CaM level. Like in HUVEC, uric acid also decreased eNOS-CaM interaction in bovine aortic EC. Finally, uric acid attenuated ionomycin-induced increase in the interaction between eNOS and CaM. This study suggests firstly that uric acid decreased eNOS activity and NO production through reducing the binding between eNOS and CaM in EC. Our result may provide molecular mechanism by which uric acid induces endothelial dysfunction.

Characterization of long-term in vitro culture-related alterations of human tonsil-derived mesenchymal stem cells: role for CCN1 in replicative senescence-associated increase in osteogenic differentiation

Although mesenchymal stem cells (MSC) isolated from bone marrow and adipose tissues are known to be subjected to in vitro culture‐related alterations in their stem cell properties, such data have not been reported in human tonsil‐derived MSC (T‐MSC). Here, we investigated the culture‐related changes of phenotypes, the senescence, and the differentiation potential of T‐MSC. T‐MSC were serially passaged by a standard protocol, and their characteristics were assessed, including MSC‐specific surface antigen profiles, the senescence, and the differentiation potentials into adipocytes, chondrocytes and osteocytes. Up to at least passage 15, we found no alterations in either MSC‐specific surface marker, CD14, CD34, CD45, CD73 and CD90, or the mRNA expression of embryonic stem cell gene markers, Nanog, Oct4‐A and Sox‐2. However, the expression of CD146, recently identified another MSC marker, dramatically decreased with increasing passages from ~ 23% at passage 3 to ~ 1% at passage 15. The average doubling time increased significantly from ~ 38 h at passage 10 to ~ 46 h at passage 15. From passage 10, the cell size increased slightly and SA‐β‐gal staining was evident. Both Alizarin Red S staining and osteocalcin expression showed that the osteogenic differentiation potential increased up to passage 10 and decreased thereafter. However, the adipogenic and chondrogenic differentiation potential decreased passage‐dependently from the start, as evidenced by staining of Oil Red O and Alcian Blue, respectively. Consistent with a passage‐dependent osteogenic differentiation, the expression of CCN1, an angiogenic protein known to be related to both senescence and osteogenesis, also increased up to passage 10. Furthermore, ectopic expression of small interfering RNA against CCN1 at passage 10 significantly reversed Alizarin Red S staining and osteocalcin expression. Altogether, our study demonstrates the characterization of long‐term in vitro cultured T‐MSC and that CCN1 may be involved in mediating a passage‐dependent increase in osteogenic potential of T‐MSC.

CCN1 Secreted by Tonsil‐Derived Mesenchymal Stem Cells Promotes Endothelial Cell Angiogenesis via Integrin αvβ3 and AMPK

CCN1 is highly expressed in cancer cells and has been identified in the secretome of bone marrow‐derived mesenchymal stem cells (BM‐MSC). Although secreted CCN1 is known to promote angiogenesis, its underlying mechanism remains unclear. Here, we examined whether our recently‐established tonsil‐derived MSC (T‐MSC) secrete CCN1 and, if any, how CCN1 promotes the angiogenesis of human umbilical vein endothelial cells (HUVEC). Compared with untreated control T‐MSC, a higher level of CCN1 was secreted by T‐MSC treated with activin A and sonic hedgehog, drugs known to induce endodermal differentiation. Expectedly, conditioned medium collected from differentiated T‐MSC (DCM) significantly increased HUVEC migration and tube formation compared with that from control T‐MSC (CCM), and these stimulatory effects were reversed by neutralization with anti‐CCN1 antibody. Treatment with recombinant human CCN1 (rh‐CCN1) alone also mimicked the stimulatory effects of DCM. Furthermore, treatment with either DCM or rh‐CCN1 increased the phosphorylation of AMP kinase (AMPK), and ectopic expression of siRNA of the AMPK gene inhibited all observed effects of both DCM and rh‐CCN1. However, no alteration of intracellular ATP levels or phosphorylation of LKB1, a well‐known upstream factor of AMPK activation, was observed under our conditions. Finally, the neutralization of integrin αvβ3 with anti‐integrin αvβ3 antibody almost completely reversed the effects of CCN1 on AMPK phosphorylation, and EC migration and tube formation. Taken together, we demonstrated that T‐MSC increase the secretion of CCN1 in response to endodermal differentiation and that integrin αvβ3 and AMPK mediate CCN1‐induced EC migration and tube formation independent of intracellular ATP levels alteration. J. Cell. Physiol. 230: 140–149, 2015.

Selective osteogenesis by a synthetic mineral inducing peptide for the treatment of osteoporosis

Mineralization in mammalian cells is accomplished by concerted regulation of protein-based extracellular matrix (ECM) components, such as non-collagenous proteins and collagen fibrils. In this study, we investigated the ability of a collagen-binding motif (CBM) peptide derived from osteopontin to selectively affect osteogenic or adipogenic differentiation in vitro and in vivo. In particular, increased osteogenic differentiation and decreased adipogenic differentiation were observed in human mesenchymal stem cells (hMSCs). Osteocalcin (OCN) protein expression in MC3T3-E1 cells without osteogenic inducers was then investigated following treatment with the CBM peptide. In ovariectomized (OVX) mice, estrogen deficiency induced osteoporosis and increased fat tissue deposition. However, after the CBM peptide or estradiol was injected into the OVX mice for 2 months, the increased serum OCN concentration and alkaline phosphate (ALP) activity were decreased in the estradiol-treated group (OVX-E) and the high-concentration CBM peptide-treated group (OVX-HP). Significant bone loss was also observed in the ovariectomized mice (OVX-PBS). In particular, the bone volume per total volume (BV/TV) and bone mineral density (BMD) were significantly decreased in the OVX mice; however, both of these markers were restored in the OVX-HP group, which also had significantly well-developed bone structure and bone formation. In contrast to the bone structural change, adipose tissue was increased in the OVX-PBS. However, a significant decrease in total fat and subcutaneous fat was observed in the low-concentration CBM peptide-treated group (OVX-LP) and the estradiol-treated group (OVX-E). Taken together, these results suggest that the CBM peptide could be an effective therapeutic agent for osteoporosis due to its selective stimulation of osteogenic differentiation, rather than adipogenesis.

Scaffold-free parathyroid tissue engineering using tonsil-derived mesenchymal stem cells

UNLABELLED To restore damaged parathyroid function, parathyroid tissue engineering is the best option. Previously, we reported that differentiated tonsil-derived mesenchymal stem cells (dTMSC) restore in vivo parathyroid function, but only if they are embedded in a scaffold. Because of the limited biocompatibility of Matrigel, however, here we developed a more clinically applicable, scaffold-free parathyroid regeneration system. Scaffold-free dTMSC spheroids were engineered in concave microwell plates made of polydimethylsiloxane in control culture medium for the first 7days and differentiation medium (containing activin A and sonic hedgehog) for next 7days. The size of dTMSC spheroids showed a gradual and significant decrease up to day 5, whereafter it decreased much less. Cells in dTMSC spheroids were highly viable (>80%). They expressed high levels of intact parathyroid hormone (iPTH), the parathyroid secretory protein 1, and cell adhesion molecule, N-cadherin. Furthermore, dTMSC spheroids-implanted parathyroidectomized (PTX) rats revealed higher survival rates (50%) over a 3-month period with physiological levels of both serum iPTH (57.7-128.2pg/mL) and ionized calcium (0.70-1.15mmol/L), compared with PTX rats treated with either vehicle or undifferentiated TMSC spheroids. This is the first report of a scaffold-free, human stem cell-based parathyroid tissue engineering and represents a more clinically feasible strategy for hypoparathyroidism treatment than those requiring scaffolds. STATEMENT OF SIGNIFICANCE Herein, we have for the first time developed a scaffold-free parathyroid tissue spheroids using differentiated tonsil-derived mesenchymal stem cells (dTMSC) to restore in vivo parathyroid cell functions. This new strategy is effective, even for long periods (3months), and is thus likely to be more feasible in clinic for hypoparathyroidism treatment. Development of TMSC spheroids may also provide a convenient and efficient scaffold-free platform for researchers investigating conditions involving abnormal calcium homeostasis, such as osteoporosis.

Sustained release of parathyroid hormone via in situ cross-linking gelatin hydrogels improves the therapeutic potential of tonsil-derived mesenchymal stem cells for hypoparathyroidism

Biomimetic parathyroid regeneration with sustained release of parathyroid hormone (PTH) into the blood stream is a considerable challenge in hypoparathyroidism treatment. We recently reported that tonsil‐derived mesenchymal stem cells (TMSCs), if these cells were both differentiated in vitro before implantation and incorporated into a scaffold Matrigel, are a good cell source for parathyroid regeneration in a parathyroidectomized (PTX) animal model. Here, we present a new strategy for improved clinical application that enhances the sustained release of PTH by controlling mechanical stiffness using in situ‐forming gelatin‐hydroxyphenyl propionic acid (GH) hydrogels (GHH). Differentiated TMSCs (dTMSCs) embedded in a GHH with a strength of 4.4 kPa exhibited the best sustained release of PTH and were the most effective in hypoparathyroidism treatment, showing improved blood calcium homeostasis compared with Matrigel‐embedded dTMSCs. Interestingly, undifferentiated control TMSCs (cTMSCs) also released PTH in a sustained manner if incorporated into GHH. Collectively, these findings may establish a new paradigm for parathyroid regeneration that could ultimately evolve into an improved clinical application. Copyright

Intracellular Remodeling and Accumulation of Aberrant Lysosomes in Differentiation of Tonsil-Derived Mesenchymal Stem Cells into Parathyroid-Like Cells

Differentiation of mesenchymal stem cells (MSC) into a variety of cell lineages such as adipocytes, osteocytes, and chondrocytes is often accompanied up-regulation of autophagy. In our study, we demonstrated that the expression of autophagy-associated proteins (p-Beclin 1, LC3A, LC3B, p-AMPK, p-mTOR and ATG3, ATG7, and ATG12-5) over a period of time was hardly distinguishable from control tonsil-derived MSC (TMSC). Despite the unnoticeable difference in autophagy activation between differentiated TMSC (dTMSC) and the control (cTMSC), we reported significant changes in intracellular compositions in differentiated TMSC into functional parathyroid-like cells secreting parathyroid hormone (PTH). By using transmission electron microscopy (TEM), we observed accumulation of multivesicular bodies (MVB) comprising small, degraded compartments densely accumulated as dark granular or amorphous clumps, multilamellar bodies and lipid droplets in dTMSC. However, no such structures were found in cTMSC. These results suggest that differentiation of TMSC into parathyroid-like cells producing PTH hormone is hardly dependent on autophagy activation in the beginning of our conditions. Furthermore, our results of intracellular remodeling and accumulated endo-lysosomal storage bodies in the later stages of TMSC differentiation present a possible role of the structures in PTH secretion.

Tonsil-derived mesenchymal stem cell-embedded in situ crosslinkable gelatin hydrogel therapy recovers postmenopausal osteoporosis through bone regeneration

We investigated therapeutic potential of human tonsil-derived mesenchymal stem cells (TMSC) subcutaneously delivered to ovariectomized (OVX) mice for developing more safe and effective therapy for osteoporosis. TMSC were isolated from tonsil tissues of children undergoing tonsillectomy, and TMSC-embedded in situ crosslinkable gelatin-hydroxyphenyl propionic acid hydrogel (TMSC-GHH) or TMSC alone were delivered subcutaneously to the dorsa of OVX mice. After 3 months, three-dimensionally reconstructed micro-computed tomographic images revealed better recovery of the femoral heads in OVX mice treated with TMSC-GHH. Serum osteocalcin and alkaline phosphatase were also recovered, indicating bone formation only in TMSC-GHH-treated mice, and absence in hypercalcemia or other severe macroscopic deformities showed biocompatibility of TMSC-GHH. Additionally, visceral fat reduction effects by TMSC-GHH further supported their therapeutic potential. TMSC provided therapeutic benefits toward osteoporosis only when embedded in GHH, and showed potential as a supplement or alternative to current therapies.

Toxicoproteomic analysis of bovine aortic endothelial cell under exposure to cigarette smoking extracts

The major health implications of exposure to cigarette smoke that leads to disease development are still being explored. The overall protein expression pattern of bovine aortic endothelial cell (BAEC) has been studied during the treatment with cigarette smoke extract (CSE) by using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Out of an average 950 spots in silver-stained gels, the synthetic levels of 4 proteins were significantly increased, whereas 7 proteins were decreased. The identified proteins have been found to be involved in anti-inflammation (Apolipoprotein A1), energy metabolism (ATP synthase subunit d), signaling pathway (Acetyl-CoA carboxylase; GTP-binding protein), oxidative stress response (Phosphatidylinositol transfer protein beta isoform; phosphatidylinositol transfer protein alpha isoform; Vascular endothelial growth factor), carcinogenesis (Insulin-like growth factor-binding protein 2; Visinin-like protein-1), exocytosis machinery (Synaptosomal-associated protein 25) and immune response (C4b-binding protein). A significantly different protein expression pattern between the control and the CSE-exposed BAEC has been observed and demonstrated on the synthesis level analysis. The systematic approach to the analysis of proteomic responses used as well as the detailed analysis results has been useful in understanding the novel molecular mechanisms for cigarette smoke induced pathological changes. This will help in designing the systemic strategies for the development of new therapeutic approaches to smokerelated diseases.

Double intratibial injection of human tonsil-derived mesenchymal stromal cells recovers postmenopausal osteoporotic bone mass

BACKGROUND AND AIMS Osteoporosis, which is a disease characterized by weakening of the bone, affects a large portion of the senior population. The current therapeutic options for osteoporosis have side effects, and there is no effective treatment for severe osteoporosis. Thus, we urgently need new treatment strategies, such as topical therapies and/or safe and effective stem cell therapies. METHODS We investigated the therapeutic potential of directly injecting human tonsil-derived mesenchymal stem cells (TMSC) into the right proximal tibias of ovariectomized postmenopausal osteoporosis model mice. Injections were given once (1×) or twice (2×) during the 3-month experimental period. At the end of the experiment, micro-computed tomographic images revealed some improvement in the proximal tibias and more significant improvement in the femoral heads of treated mice. RESULTS Osteogenic effect was qualitatively and quantitatively more pronounced in TMSC/2×-treated mice. Furthermore, TMSC/2× mice exhibited significant recovery of the serum osteocalcin level, which is pathologically elevated in osteoporosis, and increased serum alkaline phosphatase, which indicates bone formation. TMSC therapy was generally well tolerated and caused no apparent toxicity in the experimental mice. Moreover, TMSC therapy reduced visceral fat. CONCLUSION Our results demonstrate that double injection of TMSC directly into the proximal tibia triggers recovery of osteoporosis, and thus could be a potential therapeutic approach for severe bone loss.