A 70% Ethanol Neorhodomela munita Extract Attenuates RANKL-Induced Osteoclast Activation and H2O2-Induced Osteoblast Apoptosis In Vitro.

The rapid aging of the population worldwide presents a significant social and economic challenge, particularly due to osteoporotic fractures, primarily resulting from an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. While conventional therapies offer benefits, they also present limitations and a range of adverse effects. This study explores the protective impact of Neorhodomela munita ethanol extract (EN) on osteoporosis by modulating critical pathways in osteoclastogenesis and apoptosis. Raw264.7 cells and Saos-2 cells were used for in vitro osteoclast and osteoblast models, respectively. By utilizing various in vitro methods to detect osteoclast differentiation/activation and osteoblast death, it was demonstrated that the EN's potential to inhibit RANKL induced osteoclast formation and activation by targeting the MAPKs-NFATc1/c-Fos pathway and reducing H2O2-induced cell death through the downregulation of apoptotic signals. This study highlights the potential benefits of EN for osteoporosis and suggests that EN is a promising natural alternative to traditional treatments.

Enhanced Osteocyte Differentiation: Cathepsin D and L Secretion by Human Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (ASCs) have the potential to differentiate into bone, cartilage, fat, and neural cells and promote tissue regeneration and healing. It is known that they can have variable responses to hypoxic conditions. In the present study, we aimed to explore diverse changes in the cells and secretome of ASCs under a hypoxic environment over time and to present the possibility of ASCs as therapeutic agents from a different perspective. The expression differences of proteins between normoxic and hypoxic conditions (6, 12, or 24 h) were specifically investigated in human ASCs using 2-DE combined with MALDI-TOF MS analysis, and secreted proteins in ASC-derived conditioned media (ASC-derived CM) were examined by an adipokine array. In addition, genetic and/or proteomic interactions were assessed using a DAVID and miRNet functional annotation bioinformatics analysis. We found that 64 and 5 proteins were differentially expressed in hypoxic ASCs and in hypoxic ASC-derived CM, respectively. Moreover, 7 proteins among the 64 markedly changed spots in hypoxic ASCs were associated with bone-related diseases. We found that two proteins, cathepsin D (CTSD) and cathepsin L (CTSL), identified through an adipokine array independently exhibited significant efficacy in promoting osteocyte differentiation in bone-marrow-derived mesenchymal stem cells (BM-MSCs). This finding introduces a promising avenue for utilizing hypoxia-preconditioned ASC-derived CM as a potential therapeutic approach for bone-related diseases.

Structural and Biochemical Studies on Product Inhibition of S-Adenosylmethionine Synthetase from Corynebacterium glutamicum.

S-Adenosylmethionine (SAM) acts as a methyl donor in living organisms, and S-adenosylmethionine synthetase (MetK) is an essential enzyme for cells, as it synthesizes SAM from methionine and adenosine triphosphate (ATP). This study determined the crystal structures of the apo form and adenosine/triphosphate complex form of MetK from Corynebacterium glutamicum (CgMetK). Results showed that CgMetK has an allosteric inhibitor binding site for the SAM product in the vicinity of the active site and is inhibited by SAM both competitively and noncompetitively. Through structure-guided protein engineering, the CgMetKE68A variant was developed that exhibited an almost complete release of inhibition by SAM with rather enhanced enzyme activity. The crystal structure of the CgMetKE68A variant revealed that the formation of a new hydrogen bond between Tyr66 and Glu102 by the E68A mutation disrupted the allosteric SAM binding site and also improved the protein thermal stability by strengthening the tetramerization of the enzyme.

A Low Concentration of Citreoviridin Prevents Both Intracellular Calcium Deposition in Vascular Smooth Muscle Cell and Osteoclast Activation In Vitro.

Vascular calcification (VC) and osteoporosis are age-related diseases and significant risk factors for the mortality of elderly. VC and osteoporosis may share common risk factors such as renin-angiotensin system (RAS)-related hypertension. In fact, inhibitors of RAS pathway, such as angiotensin type 1 receptor blockers (ARBs), improved both vascular calcification and hip fracture in elderly. However, a sex-dependent discrepancy in the responsiveness to ARB treatment in hip fracture was observed, possibly due to the estrogen deficiency in older women, suggesting that blocking the angiotensin signaling pathway may not be effective to suppress bone resorption, especially if an individual has underlying osteoclast activating conditions such as estrogen deficiency. Therefore, it has its own significance to find alternative modality for inhibiting both vascular calcification and osteoporosis by directly targeting osteoclast activation to circumvent the shortcoming of ARBs in preventing bone resorption in estrogen deficient individuals. In the present study, a natural compound library was screened to find chemical agents that are effective in preventing both calcium deposition in vascular smooth muscle cells (vSMCs) and activation of osteoclast using experimental methods such as Alizarin red staining and Tartrate-resistant acid phosphatase staining. According to our data, citreoviridin (CIT) has both an anti-VC effect and anti-osteoclastic effect in vSMCs and in Raw 264.7 cells, respectively, suggesting its potential as an effective therapeutic agent for both VC and osteoporosis.

Nidus vespae Built by an Invasive Alien Hornet, Vespa velutina nigrithorax, Inhibits Adipose Tissue Expansion in High-Fat Diet-Induced Obese Mice

Nidus vespae, commonly known as the wasp nest, has antioxidative, anti-inflammatory, antimicrobial, and antitumor properties. However, the anti-obesity effects of Nidus vespae extract (NV) have not yet been reported. This study aimed to elucidate the potential anti-obesity effects of NV in vivo and in vitro, using a high-fat diet (HFD)-induced obese mouse model and 3T3-L1 adipocytes, respectively. NV administration to HFD-induced obese mice significantly decreased the mass and plasma lipid content of adipose tissues. Uncoupling protein-1 expression was significantly higher in the inguinal white adipose tissues of NV-treated mice than in those of HFD-fed mice. Furthermore, we found that NV inhibited the differentiation and intracellular lipid accumulation of 3T3-L1 adipocytes by regulating the insulin signaling cascade, including protein kinase B, peroxisome proliferator-activated receptor gamma, CCAAT/enhancer binding protein alpha, and adiponectin. These findings suggest that NV may exhibit therapeutic effects against obesity by suppressing adipose tissue expansion and preadipocyte differentiation, thereby providing critical information for the development of new drugs for disease prevention and treatment. To our knowledge, this study provides the first evidence of the anti-obesity effects of NV.

Cold-pressed oil from Citrus aurantifolia inhibits the proliferation of vascular smooth muscle cells via regulation of PI3K/MAPK signaling pathways.

Vascular occlusive disease is a chronic disease with significant morbidity and mortality. Although a variety of therapies and medications have been developed, the likelihood of disease re-emergence is high and this can be life-threatening. Based on a previous screening experiment related to vascular obstructive diseases using 34 types of essential oils, cold-pressed oil (CpO) from Citrus aurantifolia (lime) has been demonstrated to have the best effect for the inhibition of vascular smooth muscle cells (VSMCs) proliferation. The aim of the present study was to evaluate the effect of lime CpO on the pathological changes of VSMCs. To determine this, the effect of lime CpO on VSMC proliferation, a major cause of vascular disease, was investigated. To determine the safe concentration interval for toxicity of CpO during VSMC culture, a dilution of 1x10-5 was determined using Cell Counting Kit-8 assay, which was confirmed to be non-toxic using a lactate dehydrogenase assay. To examine the effect of lime CpO in cellular signaling pathways, changes in phosphorylation of both the PI3K/AKT/mTOR and extracellular signal-regulated MEK/ERK signaling pathways with serum were investigated. Furthermore, lime CpO with FBS also significantly decreased the expression levels of the cell cycle regulators cyclin D1 and proliferating cell nuclear antigen. Additionally, lime CpO with FBS significantly inhibited the sprouting of VSMCs in an ex vivo culture system. These results suggested that lime CpO inhibited the abnormal proliferation of VSMCs and can be developed as a nature-based therapeutic agent for obstructive vascular disease.

ADSC-Based Cell Therapies for Musculoskeletal Disorders: A Review of Recent Clinical Trials.

Recently published clinical trials involving the use of adipose-derived stem cells (ADSCs) indicated that approximately one-third of the studies were conducted on musculoskeletal disorders (MSD). MSD refers to a wide range of degenerative conditions of joints, bones, and muscles, and these conditions are the most common causes of chronic disability worldwide, being a major burden to the society. Conventional treatment modalities for MSD are not sufficient to correct the underlying structural abnormalities. Hence, ADSC-based cell therapies are being tested as a form of alternative, yet more effective, therapies in the management of MSDs. Therefore, in this review, MSDs subjected to the ADSC-based therapy were further categorized as arthritis, craniomaxillofacial defects, tendon/ligament related disorders, and spine disorders, and their brief characterization as well as the corresponding conventional therapeutic approaches with possible mechanisms with which ADSCs produce regenerative effects in disease-specific microenvironments were discussed to provide an overview of under which circumstances and on what bases the ADSC-based cell therapy was implemented. Providing an overview of the current status of ADSC-based cell therapy on MSDs can help to develop better and optimized strategies of ADSC-based therapeutics for MSDs as well as help to find novel clinical applications of ADSCs in the near future.

Suppressing Pyroptosis Augments Post-Transplant Survival of Stem Cells and Cardiac Function Following Ischemic Injury.

The acute demise of stem cells following transplantation significantly compromises the efficacy of stem cell-based cell therapeutics for infarcted hearts. As the stem cells transplanted into the damaged heart are readily exposed to the hostile environment, it can be assumed that the acute death of the transplanted stem cells is also inflicted by the same environmental cues that caused massive death of the host cardiac cells. Pyroptosis, a highly inflammatory form of programmed cell death, has been added to the list of important cell death mechanisms in the damaged heart. However, unlike the well-established cell death mechanisms such as necrosis or apoptosis, the exact role and significance of pyroptosis in the acute death of transplanted stem cells have not been explored in depth. In the present study, we found that M1 macrophages mediate the pyroptosis in the ischemia/reperfusion (I/R) injured hearts and identified miRNA-762 as an important regulator of interleukin 1ß production and subsequent pyroptosis. Delivery of exogenous miRNA-762 prior to transplantation significantly increased the post-transplant survival of stem cells and also significantly ameliorated cardiac fibrosis and heart functions following I/R injury. Our data strongly suggest that suppressing pyroptosis can be an effective adjuvant strategy to enhance the efficacy of stem cell-based therapeutics for diseased hearts.

Isoliquiritigenin Enhances the Beige Adipocyte Potential of Adipose-Derived Stem Cells by JNK Inhibition.

Human adipose-derived stem cells (hASCs) can be isolated from fat tissue and have attracted interest for their potential therapeutic applications in metabolic disease. hASCs can be induced to undergo adipogenic differentiation in vitro by exposure to chemical agents or inductive growth factors. We investigated the effects and mechanism of differentiating hASC-derived white adipocytes into functional beige and brown adipocytes with isoliquiritigenin (ILG) treatment. Here, we showed that hASC-derived white adipocytes could promote brown adipogenesis by expressing both uncoupling protein 1 (UCP1) and PR/SET Domain 16 (PRDM16) following low-dose ILG treatments. ILG treatment of white adipocytes enhanced the expression of brown fat-specific markers, while the expression levels of c-Jun N-terminal kinase (JNK) signaling pathway proteins were downregulated. Furthermore, we showed that the inhibition of JNK phosphorylation contributed to white adipocyte differentiation into beige adipocytes, which was validated by the use of SP600125. We identified distinct regulatory effects of ILG dose responses and suggested that low-dose ILG induced the beige adipocyte potential of hASCs via JNK inhibition.

Adipose tissue derived stromal vascular fraction as an adjuvant therapy in stroke rehabilitation: Case reports.

INTRODUCTION: Stroke often causes residual hemiparesis, and upper extremity motor impairment is usually more disabling than lower extremity in those who are suffering from post-stroke hemiparesis. Cell therapy is one of the promising therapies to reduce post-stroke disability. PATIENT CONCERNS: Three male participants were included in the study to investigate the feasibility and tolerability of autologous adipose tissue derived stromal vascular fraction. DIAGNOSIS: All participants had hemiparesis after 1st-ever stroke longer than 6 months previously. INTERVENTIONS: Under general anesthesia, liposuction of abdominal subcutaneous fat was performed. Stromal vascular fraction freshly isolated from the adipose tissue extract was injected into the muscles of paretic upper extremity. All participants received inpatient stroke rehabilitation consisted of physical and occupational therapy more than 3 hours a day for 2 months or more. OUTCOMES: The whole procedure did not produce any significant adverse event in all participants. Adipose tissue extracts yielded sufficient stromal cells. One participant showed clinically important change in upper extremity Fugl-Meyer assessment after the injection and it lasted up to 6 months. Functional magnetic resonance imaging showed concomitant increase in ipsilesional cortical activity. The other 2 participants did not show remarkable changes. LESSONS: Intramuscular injection of autologous adipose tissue derived stromal vascular fraction seems to be a safe and tolerable procedure in subjects with chronic stroke, and its utility in rehabilitation needs further investigation.

Differentiation of adipose-derived stem cells into functional chondrocytes by a small molecule that induces Sox9.

Osteoarthritis (OA) is a common joint disease that results from the disintegration of joint cartilage and the underlying bone. Because cartilage and chondrocytes lack the ability to self-regenerate, efforts have been made to utilize stem cells to treat OA. Although various methods have been used to differentiate stem cells into functional chondrocytes, the currently available methods cannot induce stem cells to undergo differentiation into chondrocyte-like cells without inducing characteristics of hypertrophic chondrocytes, which finally lead to cartilage disintegration and calcification. Therefore, an optimized method to differentiate stem cells into chondrocytes that do not display undesired phenotypes is needed. This study focused on differentiating adipose-derived stem cells (ASCs) into functional chondrocytes using a small molecule that regulated the expression of Sox9 as a key factor in cartilage development and then explored its ability to treat OA. We selected ellipticine (ELPC), which induces chondrocyte differentiation of ASCs, using a GFP-Sox9 promoter vector screening system. An in vivo study was performed to confirm the recovery rate of cartilage regeneration with ASC differentiation into chondrocytes by ELPC in a collagenase-induced animal model of OA. Taken together, these data indicate that ellipticine induces ASCs to differentiate into mature chondrocytes without hypertrophic chondrocytes in vitro and in vivo, thus overcoming a problem encountered in previous studies. These results indicate that ELPC is a novel chondrocyte differentiation-inducing drug that shows potential as a cell therapy for OA.

A Combinational Therapy of Articular Cartilage Defects: Rapid and Effective Regeneration by Using Low-Intensity Focused Ultrasound After Adipose Tissue-Derived Stem Cell Transplantation.

BACKGROUND: Although low-intensity pulsed ultrasound has been reported to be potential cartilage regeneration, there still unresolved treatment due to cartilage fibrosis and degeneration by a lack of rapid and high-efficiency treatment. The purpose of this study was to investigate the effect of a combination therapy of focused acoustic force and stem cells at site for fast and efficient healing on cartilage regeneration. METHODS: Using a rat articular cartilage defects model, one million adipose tissue-derived stem cells (ASCs) were injected into the defect site, and low-intensity focused ultrasound (LOFUS) in the range of 100-600 mV was used for 20 min/day for 2 weeks. All experimental groups were sacrificed after 4 weeks in total. The gross appearance score and hematoxylin and eosin (H&E), Alcian blue, and Safranin O staining were used for measuring the chondrogenic potential. The cartilage characteristics were observed, and type II collagen, Sox 9, aggrecan, and type X collagen were stained with immunofluorescence. The results of the comprehensive analysis were calculated using the Mankin scoring method. RESULTS: The gross appearance scores of regenerated cartilage and chondrocyte-like cells in H&E images were higher in LOFUS-treated groups compared to those in negative control or ASC-treated groups. Safranin O and Alcian blue staining demonstrated that the 100 and 300 mV LOFUS groups showed greater synthesis of glycosaminoglycan and proteoglycan. The ASC + LOFUS 300 mV group showed positive regulation of type II collagen, Sox 9 and aggrecan and negative regulation of type X collagen, which indicated the occurrence of cartilage regeneration based on the Mankin score result. CONCLUSION: The combination therapy, which involved treatment with ASC and 300 mV LOFUS, quickly and effectively reduced articular cartilage defects.

Hypoxia Rapidly Induces the Expression of Cardiomyogenic Factors in Human Adipose-Derived Adherent Stromal Cells.

BACKGROUND: The efficacy of interstitial vascular fraction (SVF) transplantation in the treatment of heart disease has been proven in a variety of in vivo studies. In a previous study, we found that bone marrow-derived mesenchymal stem cells (BM-MSCs) altered their expression of several cardiomyogenic factors under hypoxic conditions. METHODS: We hypothesized that hypoxia may also induce obtained adipose-derived adherent stromal cells (ADASs) from SVFs and adipose-derived stem cells (ASCs) to differentiate into cardiomyocytes and/or cells with comparable phenotypes. We examined the differentiation markers of cell lineages in ADASs and ASCs according to time by hypoxic stress and found that only ADASs expressed cardiomyogenic markers within 24 h under hypoxic conditions in association with the expression of hypoxia-inducible factor 1-α (HIF-1α). RESULTS: Differentially secreted proteins in a conditioned medium (CM) from ASCs and ADASs under normoxic or hypoxic conditions were detected using an antibody assay and may be associated with a dramatic increase in the expression of cardiomyogenic markers in only ADASs. Furthermore, the cardiomyogenic factors were expressed more rapidly in ADASs than in ASCs under hypoxic conditions in association with the expression of HIF-1α, and angiogenin, fibroblast growth factor-19 (FGF-19) and/or macrophage inhibitory factor (MIF) are related. CONCLUSIONS: These results provide new insights into the applicability of ADASs preconditioned by hypoxic stress in cardiac diseases.

Anti-apoptotic effects of adipose-derived adherent stromal cells in mesenchymal stem cells exposed to oxidative stress.

Adipose-derived stromal vascular fractions (SVFs) are a heterogeneous collection of cells, and their regenerative modality has been applied in various animal experiments and clinical trials. Despite the attractive advantages of SVFs in clinical interventions, the recent status of clinical studies involving the application of SVFs in many diseases has not been fully evaluated. Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into a variety of cell types despite their low numbers in heart tissue. Here, we sought to determine if SVF implantation into impaired heart tissue affected endogenous MSCs in the heart. Therefore, we investigated the expression levels of proteins associated with oxidation, inflammation, and apoptosis in MSCs co-cultured with adipose-derived adherent stromal cells (ADASs) from 6 donors' SVFs under oxidative stress conditions for their roles in many physiological processes in the heart. Interestingly, p53 pathway proteins and mitogen-activated protein kinase (MAPK) signalling pathway components were up-regulated by H2 O2 but exhibited a downward trend in MSCs co-cultured with ADASs. These data suggest that ADASs may inhibit oxidative stress-induced apoptosis in MSCs via the p53 and MAPK pathways. Our findings also suggest that the positive effects of SVF implantation into damaged heart tissue may be attributed to the various responses of MSCs. This finding may provide new insights for the clinical application of adipose-derived SVF transplantation in cardiac diseases. SIGNIFICANCE OF THE STUDY: We investigated the expression levels of proteins associated with oxidation, inflammation, and apoptosis in MSCs co-cultured with isolated ADASs from 6 donors' SVFs under oxidative stress conditions. Our results imply that isolated ADASs from SVFs may inhibit oxidative stress-induced cell cycle arrest and/or apoptosis in MSCs via a p53-dependent pathway. Furthermore, we identified an anti-apoptotic mechanism involving oxidative stress-induced apoptosis by adipose-derived ADASs in MSCs for the first time. Our findings suggest that the positive effects of SVF implantation into damaged heart tissue may be attributed to the various actions of MSCs.

Rapid Induction of Osteogenic Markers in Mesenchymal Stem Cells by Adipose-Derived Stromal Vascular Fraction Cells.

BACKGROUND/AIMS: Stromal vascular fraction (SVF) cells are a mixed cell population, and their regenerative capacity has been validated in various therapeutic models. The purpose of this study was to investigate the regenerative mechanisms utilized by implanted SVF cells. Using an in vitro co-culture system, we sought to determine whether SVF implantation into impaired tissue affects endogenous mesenchymal stem cell (MSC) differentiation; MSCs can differentiate into a variety of cell types, and they have a strong regenerative capacity despite their low numbers in impaired tissue. METHODS: Adipose-derived SVF cells obtained from four donors were co-cultured with bone marrow-derived MSCs, and the differential expression of osteogenic markers and osteogenic differentiation inducers over time was analyzed in mono-cultured MSCs and MSCs co-cultured with SVF cells. RESULTS: The co-cultivation of MSCs with SVF cells significantly and mutually induced the expression of osteogenic-specific markers via paracrine and/or autocrine regulation but did not induce adipocyte, chondrocyte or myoblast marker expression. More surprisingly, subsequent osteogenesis and/or comparable effects were rapidly induced within 48 h. CONCLUSION: To the best of our knowledge, this is the first study in which osteogenesis and/or comparable effects were rapidly induced in bone marrow-derived MSCs and adipose-derived SVF cells through co-cultivation. Our findings suggest that the positive effects of SVF implantation into impaired bone may be attributed to the rapid induction of MSC osteogenesis, and the transplantation of co-cultured and preconditioned SVF cells and/or MSCs may be more effective than the transplantation of untreated cells for the treatment of bone defects.

Gender-dimorphic effects of adipose-derived stromal vascular fractions on HUVECs exposed to oxidative stress.

Stromal vascular fractions (SVFs) are a heterogeneous collection of cells within adipose tissue that are being studied for various clinical indications. In this study, we aimed to determine whether SVF transplantation into impaired tissues has differential effects on inflammatory and angiogenetic properties with regard to gender. As reactive oxygen species have been implicated in cardiovascular disease development, we investigated differences in gene and protein expression related to inflammation and angiogenesis in HUVECs co-cultured with adipose-derived SVFs from male (M group) and female (F group) individuals under oxidative stress conditions. The expression of several inflammatory (interleukin (IL)-33) and angiogenetic (platelet-derived growth factor (PDGF)) factors differed dramatically between male and female donors. Anti-inflammatory and pro-angiogenetic responses were observed in HUVECs co-cultured with SVFs under oxidative stress conditions, and these characteristics may exhibit partially differential effects according to gender. Using network analysis, we showed that co-culturing HUVECs with SVFs ameliorated pyroptosis/apoptosis via an increase in oxidative stress. Activation of caspase-1 and IL-1B was significantly altered in HUVECs co-cultured with SVFs from female donors. These findings regarding gender-dimorphic regulation of adipose-derived SVFs provide valuable information that can be used for evidence-based gender-specific clinical treatment of SVF transplantation for understanding of cardiovascular disease, allowing for the development of additional treatment.

Extracellular Vesicles as Drug Delivery Vehicles for Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic, systemic and progressive autoimmune disease of connective tissues common in middle age. Dysregulation of the tissue homeostasis involving inflammation is the hallmark of disease pathogenesis, inducing autoimmune insults that frequently lead to permanent disability. Although the advent of immunosuppressive and anti-inflammatory drugs and, more recently, pathogenic TNF-TNF-R axis-targeting biologics significantly delayed progressive joint destruction with significant reduction of disability and physical improvement, a large proportion of RA patients failed to respond to the treatment. In this regard, mesenchymal stem/stromal cells (MSC) are particularly attractive to the refractory patients to the pharmacologic intervention for their immunosuppressive/anti-inflammatory capacity as well as tissue reparative and/or regenerative potential. Local or systemic delivery of MSCs led to promising results in preclinical as well as in clinical studies of RA and thus proposing that these cells can be further exploited for their therapeutic application in RA and other degenerative connective tissue diseases. Mechanistically, paracrine factors appear to be the main contributors of MSC-mediated tissue regeneration in a number of preclinical and clinical studies rather than direct tissue cell replacement. More recently, extracellular vesicles (EVs) released from MSCs emerged as key paracrine messengers that can also participate in the healing process through influencing the local microenvironment with anti-inflammatory effects. It is highly likely that the use of these EVs becomes beneficial in the treatment of RA. Yet, identification of key components involved in the regenerative process needs to be assessed for developing efficient MSC-based strategy of RA treatment.

1H-pyrrole-2,5-dione-based small molecule-induced generation of mesenchymal stem cell-derived functional endothelial cells that facilitate rapid endothelialization after vascular injury.

INTRODUCTION: Despite the success of interventional processes such as drug-eluting stents, complete prevention of restenosis is still hindered by impaired or delayed endothelialization or both. Here, we report that 1H-pyrrole-2,5-dione-based small molecule-generated mesenchymal stem cell-derived functional endothelial cells (MDFECs) facilitated rapid transmural coverage of injured blood vessels. METHODS: Small molecules that induced CD31 expression were screened by principal component analysis (PCA). Rat mesenchymal stem cells (MSCs) were treated with selected small molecules for up to 16 days, and the expression levels of CD90 and CD31 were examined by immunocytochemistry. In vitro functional assays of MDFECs, including tube formation assays and nitric oxide production assays, were performed. After MDFECs (intravenous, 3×10(6) cells per animal) were injected into balloon-injured rats, neointima formation was monitored for up to 21 days. The endothelial coverage of denuded blood vessels was evaluated by Evans Blue staining. The functionality of repaired blood vessels was evaluated by measuring vasorelaxation and hemodynamic changes. Additionally, derivatives of the selected small molecules were examined for their ability to induce endothelial markers. RESULTS: PCA indicated that 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione effectively induced MDFECs. MDFECs inhibited the neointima formation of denuded blood vessels by facilitating more rapid endothelialization. Further examination indicated that derivatives with a 1H-pyrrole-2,5-dione moiety are important for initiating the endothelial cell differentiation of MSCs. CONCLUSIONS: Small molecules with 1H-pyrrole-2,5-dione as a core structure have great potential to improve the efficacy of MSC-based cell therapy for vascular diseases, such as atherosclerosis and restenosis.

Flt3 ligand induces monocyte proliferation and enhances the function of monocyte-derived dendritic cells in vitro.

Flt3 ligand (FL), a potent hematopoietic cytokine, plays an important role in development and activation of dendritic cells (DCs) and natural killer cells (NK). Although some post-receptor signaling events of FL have been characterized, the role of FL on Flt3 expressing human peripheral blood monocyte is unclear. In the current study, we examined the role of FL on cell survival and growth of peripheral blood monocytes and function of monocyte-derived DCs. FL promoted monocyte proliferation in a dose-dependent manner and prevented spontaneous apoptosis. FL induced ERK phosphorylation and a specific ERK inhibitor completely abrogated FL-mediated cellular growth, while p38 MAPK, JNK, and AKT were relatively unaffected. Addition of FL to GM-CSF and IL-4 during DCs generation from monocytes increased the yield of DCs through induction of cell proliferation. DCs generated in the presence of FL expressed more costimulatory molecules on their surfaces and stimulated allogeneic T cell proliferation in MLR to a higher magnitude. Furthermore, FL partially antagonized IL-10-mediated inhibition on DCs function. Further characterization of FL actions may provide new and important information for immunotherapeutic approaches utilizing DCs.

Modulation of Fas-Fas Ligand Interaction Rehabilitates Hypoxia-Induced Apoptosis of Mesenchymal Stem Cells in Ischemic Myocardium Niche.

Mesenchymal stem cells (MSCs) have the potential to repair and regenerate ischemic heart tissue; however, the poor viability of transplanted MSCs in the ischemic region is a major obstacle to their therapeutic use. This cell death is caused by Fas and Fas ligand (FasL) interactions under harsh conditions. To investigate improving the survival and therapeutic effects of MSCs, we focused our research on Fas-FasL-mediated cell death. In this study, we found that the poor viability of transplanted MSCs was caused by Fas-FasL interactions between host ischemic myocardial cells and implanted MSCs. In addition, we found that increased Fas expression and the corresponding decrease of cell survival were in close relation to hypoxic MSCs treated with FasL and H2O2. When MSCs were treated with a recombinant Fas/Fc chimera (Fas/Fc) inhibiting Fas-FasL interactions, the expressions of proapoptotic proteins including caspase-8, caspase-3, Bax, and cytochrome-c were attenuated, and the survival of MSCs was recovered. In ischemia-reperfusion injury models, the interaction between FasL in ischemic heart and Fas in implanted MSCs caused a loss of transplanted MSCs, whereas the inhibition of this interaction by Fas/Fc treatment improved cell survival and restored heart function. Thus, our study suggests that Fas-FasL interactions are responsible for activating cell death signaling in implanted stem cells and could be a potential target for improving therapeutic efficacy of stem cells in treating ischemic heart diseases.