Exosomes derived from mesenchymal stem cells primed with disease-condition-serum improved therapeutic efficacy in a mouse rheumatoid arthritis model via enhanced TGF-ß1 production.

BACKGROUNDS: Rheumatoid arthritis (RA) is a chronic and systemic autoimmune disease characterized by synovial inflammation-mediated progressive destruction of the cartilage and bone, resulting in reduced quality of life. We primed human telomerase reverse transcriptase-overexpressing immortalized human adipose tissue-derived mesenchymal stem cells (iMSCs) with serum derived from a non-human primate RA model and studied the immunomodulatory ability of exosomes obtained from primed iMSCs. METHODS: After immunophenotyping, nanoparticle tracking analysis, and in vitro functional tests, Dulbecco's phosphate-buffered saline (dPBS, Group C), exosomes derived from the supernatant of iMSCs (Exo-FBS, Group E), exosomes derived from the supernatant of iMSCs primed with RA serum (Exo-RA, Group F), and methotrexate (Group M) were administered in collagen-induced arthritis (CIA) model mice. dPBS was administered to the normal (N) group for comparison (n = 10/group). RESULTS: Exo-RA had a significantly higher number of exosomes compared to Exo-FBS when measured with nanoparticle tracking analysis or exosome marker CD81, and Transforming growth factor-ß1 amounts were significantly higher in Exo-RA than in Exo-FBS. When Exo-FBS or Exo-RA was administered to the collagen-induced arthritis model, serum interleukin (IL)-4 and the proportion of Th2 (CD4+CD25+GATA3+) and M2 (CD11c - CD206+ of CD45+CD64+) cells were significantly increased compared to the control group. Furthermore, Exo-RA could alleviate cartilage damage by significantly lowering the concentrations of proinflammatory cytokines such as tumor necrosis factor-α, keratinocyte chemoattractant, and IL-12p70. CONCLUSION: Exosomes derived from disease-condition-serum-primed iMSCs ameliorated cartilage damage in a RA model by enhancing TGF-ß1 production, inducing Th2 and M2 polarization and lowering proinflammatory cytokines, TNF-α, KC, and IL-12p70 in the host. Patient-derived serum can be used as an iMSC priming strategy in iMSC-derived exosome treatment of RA.

Protective Effects of Statin and Angiotensin Receptor Blocker in a Rat Model of Doxorubicin- and Trastuzumab-Induced Cardiomyopathy.

BACKGROUND: Chemotherapy has led to improved survival in patients with breast cancer; however, it is associated with an increased risk of cardiac dysfunction and heart failure. We investigated the protective effects of rosuvastatin and candesartan, alone and in combination, in a doxorubicin- and trastuzumab-induced rat model of cardiomyopathy. METHODS: Forty-two rats were allocated into six groups (G1-G6): G1, control; G2, doxorubicin only; G3, doxorubicin + trastuzumab; G4, doxorubicin + trastuzumab + rosuvastatin; G5, doxorubicin + trastuzumab + candesartan; and G6, doxorubicin + trastuzumab + rosuvastatin + candesartan. Doxorubicin and trastuzumab were sequentially administered for 28 days. Left ventricular end-systolic dimension and longitudinal strain (LS) were assessed via echocardiography. Left ventricular (LV) performance was evaluated using a microcatheter in the LV apex on day 28. Blood for biomarker analysis was collected from the inferior vena cava before sacrifice. RESULTS: Doxorubicin in combination with trastuzumab increased the LV end-systolic dimension but worsened LS compared with the control group (all P < .05). The level of C-reactive protein was lower in the rosuvastatin treatment group (P = .007) than in the controls but not in the candesartan treatment group. Both rosuvastatin and candesartan attenuated the increase in glutathione. Candesartan treatment improved +dP/dt (P = .011), whereas rosuvastatin did not. In the combination treatment group, the worsening of LS was significantly attenuated compared with that in either the rosuvastatin or candesartan group (all P < .05). CONCLUSIONS: In a rat model of doxorubicin- and trastuzumab-induced cardiomyopathy, rosuvastatin alleviated systemic inflammation, while candesartan improved LV performance. Combination therapy with rosuvastatin and candesartan demonstrated additional preventive effects on myocardial strain. The protective mechanisms of rosuvastatin and candesartan appear to be different but complementary in chemotherapy-induced cardiomyopathy.

Inhibition of Smooth Muscle Cell Proliferation and Migration by a Talin Modulator Attenuates Neointimal Formation after Femoral Arterial Injury.

BACKGROUND AND OBJECTIVES: Vascular smooth muscle cell (SMC) proliferation and migration play a critical role in neointimal formation. Focal adhesion is involved in cell proliferation and migration, and talin is known to be a key regulator of these processes. We synthesized a new talin modulator that binds to the talin protein, and investigated its effects on SMCs and neointimal formation after vascular injury. METHODS: Human aortic SMCs (HAoSMCs) were treated with a newly synthesized talin modulator. Apolipoprotein E knockout (ApoE KO) mice were subjected to left femoral arterial injury and orally administered with the talin modulator daily. Laser Doppler imager was used to compare the blood flow, and injured femoral arteries and blood serum were analyzed after 28 days. RESULTS: The talin modulator significantly inhibited cell proliferation in a concentration-dependent manner and suppressed the migration of HAoSMCs. Treatment with a talin modulator resulted in a significant reduction in the phosphorylation of focal adhesion molecules and downstream signaling molecules related to cell proliferation and migration. The effects of the talin modulator in HAoSMCs were found to be reversible, as evidenced by the reactivation of signaling pathways upon its removal. After 28 days of administration of the talin modulator, an improvement in the blood flow and reduction in neointimal formation in the injured femoral arteries were observed. CONCLUSIONS: We demonstrated the inhibitory effects of a talin modulator on SMC proliferation and migration, and that were associated with downregulation of signaling pathways, resulting in the attenuation of neointimal formation in ApoE KO mice.

Thymosin ß4-Enhancing Therapeutic Efficacy of Human Adipose-Derived Stem Cells in Mouse Ischemic Hindlimb Model.

Thymosin ß4 (Tß4) is a G-actin sequestering protein that contributes to diverse cellular activities, such as migration and angiogenesis. In this study, the beneficial effects of combined cell therapy with Tß4 and human adipose-derived stem cells (hASCs) in a mouse ischemic hindlimb model were investigated. We observed that exogenous treatment with Tß4 enhanced endogenous TMSB4X mRNA expression and promoted morphological changes (increased cell length) in hASCs. Interestingly, Tß4 induced the active state of hASCs by up-regulating intracellular signaling pathways including the PI3K/AKT/mTOR and MAPK/ERK pathways. Treatment with Tß4 significantly increased cell migration and sprouting from microbeads. Moreover, additional treatment with Tß4 promoted the endothelial differentiation potential of hASCs by up-regulating various angiogenic genes. To evaluate the in vivo effects of the Tß4-hASCs combination on vessel recruitment, dorsal window chambers were transplanted, and the co-treated mice were found to have a significantly increased number of microvessel branches. Transplantation of hASCs in combination with Tß4 was found to improve blood flow and attenuate limb or foot loss post-ischemia compared to transplantation with hASCs alone. Taken together, the therapeutic application of hASCs combined with Tß4 could be effective in enhancing endothelial differentiation and vascularization for treating hindlimb ischemia.

Cardioprotective effects of genetically engineered cardiac stem cells by spheroid formation on ischemic cardiomyocytes.

BACKGROUND: Sca-1+ cardiac stem cells and their limited proliferative potential were major limiting factors for use in various studies. METHODS: Therefore, the effects of sphere genetically engineered cardiac stem cells (S-GECS) inserted with telomerase reverse transcriptase (TERT) were investigated to examine cardiomyocyte survival under hypoxic conditions. GECS was obtained from hTERT-immortalized Sca-1+ cardiac stem cell (CSC) lines, and S-GECS were generated using poly-HEMA. RESULTS: The optimal conditions for S-GECS was determined to be 1052 GECS cells/mm2 and a 48 h culture period to produce spheroids. Compared to adherent-GECS (A-GECS) and S-GECS showed significantly higher mRNA expression of SDF-1α and CXCR4. S-GECS conditioned medium (CM) significantly reduced the proportion of early and late apoptotic cardiomyoblasts during CoCl2-induced hypoxic injury; however, gene silencing via CXCR4 siRNA deteriorated the protective effects of S-GECS against hypoxic injury. As downstream pathways of SDF-1α/CXCR4, the Erk and Akt signaling pathways were stimulated in the presence of S-GECS CM. S-GECS transplantation into a rat acute myocardial infarction model improved cardiac function and reduced the fibrotic area. These cardioprotective effects were confirmed to be related with the SDF-1α/CXCR4 pathway. CONCLUSIONS: Our findings suggest that paracrine factors secreted from transplanted cells may protect host cardiomyoblasts in the infarcted myocardium, contributing to beneficial left ventricle (LV) remodeling after acute myocardial infarction (AMI).

Fimasartan reduces neointimal formation and inflammation after carotid arterial injury in apolipoprotein E knockout mice.

BACKGROUND: The beneficial effects of angiotensin II type 1 receptor blockers (ARBs) on atherosclerosis have been demonstrated in numerous studies. We investigated the effects of fimasartan on reducing neointimal formation and systemic inflammation after carotid artery (CA) injury in Apolipoprotein E knockout (ApoE KO) mice. METHODS: ApoE KO mice were randomly allocated to Group I (without CA injury), Group II (without CA injury + Fimasartan), Group III (CA injury), and Group IV (CA injury + Fimasartan). Fimasartan was orally administered everyday starting 3 days before iatrogenic left CA injury. RESULTS: At 28 days, neointimal hyperplasia and the inflammatory cytokines including TNFα, IL-6, ICAM, and MMP-9 in the peripheral blood were significantly reduced in Groups II and IV compared to Groups I and III, respectively. All fimasartan-administered groups revealed significant increases of CD4+CD25+Foxp3+ regulatory T (Treg) cells with increased plasma levels of IL-10 and TGFß. In addition, increased CD8+ T cells by fimasartan were correlated with reduced smooth muscle cell (SMC) proliferation in the neointima in Groups II and IV. Furthermore, the populations of Treg and CD8+ T cells in total splenocytes were increased in Groups II and IV compared to Groups I and III, respectively. The enlargement of spleens due to CA injury in the Group III was attenuated by fimasartan, as shown in the Group IV. These data indicate that fimasartan significantly reduced SMC proliferation in neointima and increased Treg cells in ApoE KO CA injury mice. CONCLUSIONS: This study suggests fimasartan could be an efficient strategy for reduction of atherosclerotic progression, with a decrease in immune response and systemic inflammation.

Manipulation of the response of human endothelial colony-forming cells by focal adhesion assembly using gradient nanopattern plates.

Nanotopography plays a pivotal role in the regulation of cellular responses. Nonetheless, little is known about how the gradient size of nanostructural stimuli alters the responses of endothelial progenitor cells without chemical factors. Herein, the fabrication of gradient nanopattern plates intended to mimic microenvironment nanotopography is described. The gradient nanopattern plates consist of nanopillars of increasing diameter ranges [120-200 nm (GP 120/200), 200-280 nm (GP 200/280), and 280-360 nm (GP 280/360)] that were used to screen the responses of human endothelial colony-forming cells (hECFCs). Nanopillars with a smaller nanopillar diameter caused the cell area and perimeter of hECFCs to decrease and their filopodial outgrowth to increase. The structure of vinculin (a focal adhesion marker in hECFCs) was also modulated by nanostructural stimuli of the gradient nanopattern plates. Moreover, Rho-associated protein kinase (ROCK) gene expression was significantly higher in hECFCs cultured on GP 120/200 than in those on flat plates (no nanopillars), and ROCK suppression impaired the nanostructural-stimuli-induced vinculin assembly. These results suggest that the gradient nanopattern plates generate size-specific nanostructural stimuli suitable for manipulation of the response of hECFCs, in a process dependent on ROCK signaling. This is the first evidence of size-specific nanostructure-sensing behavior of hECFCs. SIGNIFICANCE: Nano feature surfaces are of growing interest as materials for a controlled response of various cells. In this study, we successfully fabricated gradient nanopattern plates to manipulate the response of blood-derived hECFCs without any chemical stimulation. Interestingly, we find that the sensitive nanopillar size for manipulation of hECFCs is range between 120 nm and 200 nm, which decreased the area and increased the filopodial outgrowth of hECFCs. Furthermore, we only modulate the nanopillar size to increase ROCK expression can be an attractive method for modulating the cytoskeletal integrity and focal adhesion of hECFCs.

Talin Modulation by a Synthetic N-Acylurea Derivative Reduces Angiogenesis in Human Endothelial Cells.

Talin is a focal adhesion protein that activates integrins and recruits other focal adhesion proteins. Talin regulates the interactions between integrins and the extracellular matrix, which are critical for endothelial cells during angiogenesis. In this study, we successfully synthesized a novel talin modulator, N-((2-(1H-indol-3-yl)ethyl)carbamoyl)-2-(benzo[d][1,3]dioxol-5-yloxy)acetamide, referred to as KCH-1521. KCH-1521 was determined to bind talin and modulate downstream signaling molecules of talin. After 24 h of treatment, KCH-1521 changed the cell morphology of human umbilical vein endothelial cells (HUVECs) and reduced focal adhesion protein expression including vinculin and paxillin. Talin downstream signaling is regulated via focal adhesion kinase (FAK), kinase B (AKT), and extracellular signal-regulated kinase (ERK) pathways, however, treatment with KCH-1521 decreased phosphorylation of FAK, AKT, and ERK, leading to reduction of cell proliferation, survival, and angiogenesis. Interestingly, the expression of various angiogenic genes was significantly decreased after treatment with KCH-1521. Also, in vitro tube forming assay revealed that KCH-1521 reduced angiogenic networks in a time-dependent manner. To investigate the reversibility of its effects, KCH-1521 was removed after treatment. HUVECs recovered their morphology through rearrangement of the cytoskeleton and the expression of angiogenic genes was also recovered. By further optimization and in vivo studies of KCH-1521, a novel drug of talin modulation could be used to achieve therapeutic anti-angiogenesis for vascular diseases and cancers.

Sphere formation of adipose stem cell engineered by poly-2-hydroxyethyl methacrylate induces in vitro angiogenesis through fibroblast growth factor 2.

A number of researchers have been reporting a wide range of in vitro and in vivo studies of cell engraftment to enhance angiogenesis using stem cells. Despite these efforts, studies involving three-dimensional (3D) culture method that mimics in vivo environment have not reached its peak yet. In this study, we investigated the change and effects on cellular angiogenic growth factors through sphere formation of adipose stem cell (ASC) which is engineered by poly-2-hydroxyethyl methacrylate (Poly-HEMA). First of all, we successfully induced sphere formation of ASC (sph-ASC) on Poly-HEMA coated plates. sph-ASC represented significantly higher expression levels of anti-apoptotic and hypoxic factors compared to monolayer adherent ASC (adh-ASC). Interestingly, sph-ASC showed higher mRNA levels of the following genes; CD31, CD144, vWF, IGF-2, MCP-1, PDGF-A, VEGF-A, VEGF-C, and FGF-2. In addition, mRNA expressions of angiogenic growth factor receptors such as Flk1, FGFR1, FGFR2, and Tie2 were elevated in sph-ASC. In protein level, Cytokine/Chemokines antibody array revealed a significant increase of FGF-2 in sph-ASC (3.17-fold) compared to adh-ASC. To investigate the effects of FGF-2 on sph-ASC, Matrigel angiogenic invasion assay showed significant reduced level of FGF-2 in FGF-2 siRNA transfected sph-ASC (2.27-fold) compared to negative control siRNA transfected sph-ASC. These findings suggest that Poly-HEMA coated plates induce sphere formation of ASC which has significantly higher expression of FGF-2, and plays a critical role as a major regulating growth factor of in vitro angiogenesis.