ABSTRACT
Fibroblast growth factor receptor 2 (FGFR2) represents an appealing therapeutic target for multiple cancers, yet no selective FGFR2 inhibitors have been approved for clinical use to date. Here, we report the discovery of a series of new selective, irreversible FGFR2 inhibitors. The representative compound LHQ490 potently inhibited FGFR2 kinase activity with an IC50 of 5.2 nM, and was >61-, >34-, and >293-fold selective against FGFR1, FGFR3, and FGFR4, respectively. LHQ490 also exhibited high selectivity in a panel of 416 kinases. Cell-based studies revealed that LHQ490 efficiently suppressed the proliferation of BaF3-FGFR2 cells with an IC50 value of 1.4 nM, and displayed >70- and >714-fold selectivity against BaF3-FGFR1 and the parental BaF3 cells, respectively. More importantly, LHQ490 potently suppressed the FGFR2 signaling pathways, selectively inhibited FGFR2-driven cancer cell proliferation, and induced apoptosis of FGFR2-driven cancer cells. Taken together, this study provides a potent and highly selective FGFR2 inhibitor for further development of FGFR2-targeted therapeutic agents.
Subject(s)
Cell Proliferation , Dose-Response Relationship, Drug , Drug Discovery , Protein Kinase Inhibitors , Receptor, Fibroblast Growth Factor, Type 2 , Receptor, Fibroblast Growth Factor, Type 2/antagonists & inhibitors , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Humans , Cell Proliferation/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/chemical synthesis , Structure-Activity Relationship , Molecular Structure , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Apoptosis/drug effects , Drug Screening Assays, Antitumor , Cell Line, TumorABSTRACT
In an atomic fountain, atoms in motion can be spatially separated into discrete Zeeman sub-states by magnetically induced Stern-Gerlach effect. With resonant light pulses acting as a shutter, specific states are selected for subsequent experiments. Such separation-selection process in atomic optics is the analogue of a spatial filter in physical optics which selects and purifies the modes of light. This technique is demonstrated by injecting a pulsed current in a circular coil around a vertical atomic fountain, separating the pre-cooled Rubidium atoms by a distance of centimeters in between, and filtering each single sub-state with block pulses. The filtered atoms after the process is highly purified in the desired sub-state. The apparatus of the atomic spatial filter is adaptable in atomic optics and can be integrated into the high-vacuum chamber of an atomic fountain.
ABSTRACT
BACKGROUND: The neurodevelopmental model of obsessive-compulsive disorder (OCD) suggests that the neurodevelopmental changes in the ventral striatal circuit of the prefrontal lobe are associated with the initial symptoms of OCD. Facial morphology is one of the most consistent anatomical phenotypes of neurodevelopmental disorders, which can reflect brain structure and function. Facial deformity, an easily measured index of brain malformation, can reflect abnormal brain structure and function. Therefore, this study aims to explore the relationship between clinical features and neurodevelopment of adolescents with OCD through facial morphology. METHODS: The enrolled study sample comprised 40 adolescents diagnosed with OCD using the Obsessive Compulsive Inventory-Child Version (OCI-CV) and 38 healthy controls (HCs). Facial photos, 21 facial diameters, and 9 facial angles were collected using image software. RESULTS: In males, lower lip red height was significantly lower in OCD patients than in HCs (P < 0.025); no significant differences were observed in other facial indicators (all P > 0.025). In females, the nasolabial angle was smaller in OCD patients than in HCs (P < 0.025); no significant differences were observed in other facial indicators (all P > 0.025). The difference in lower lip red height between the OCD group and HC group was positively correlated with neutralizing symptoms (r = 0.401, P < 0.05). CONCLUSIONS: Male OCD patients had a thinner lower lip and female OCD patients had smaller nasolabial angles. The facial features of adolescents with OCD were positively correlated with lower lip redness and neutralizing symptoms.
Subject(s)
Obsessive-Compulsive Disorder , Adolescent , Brain , Female , Humans , Male , Obsessive-Compulsive Disorder/diagnosisABSTRACT
Inhibition of transforming growth factor ß (TGF-ß) type 1 receptor (ALK5) provides a feasible approach for the treatment of fibrotic diseases and malignant tumors. In this study, we designed and synthesized a new series of 4-(pyridin-4-oxy)-3-(3,3-difluorocyclobutyl)-pyrazole derivatives, and evaluated biologically as TGF-ß type 1 receptor inhibitors. The most potent compound 15r inhibited the ALK5 enzyme and NIH3T3 cell viability with IC50 values of 44 and 42.5 nM, respectively. Compound 15r also displayed better oral plasma exposure and excellent bioavailability than LY-3200882, and in vivo inhibited 65.7% of the tumor growth in a CT26 xenograft mouse model.
Subject(s)
Antineoplastic Agents/chemical synthesis , Fibrosis/drug therapy , Neoplasms/drug therapy , Protein Kinase Inhibitors/chemical synthesis , Pyrazoles/chemical synthesis , Receptor, Transforming Growth Factor-beta Type I/antagonists & inhibitors , Amino Acid Sequence , Animals , Antineoplastic Agents/pharmacology , Biological Availability , Cell Survival/drug effects , Drug Screening Assays, Antitumor , Female , Heterografts , Humans , Mice , Mice, Inbred BALB C , Models, Molecular , NIH 3T3 Cells , Neoplasms, Experimental/drug therapy , Protein Kinase Inhibitors/pharmacokinetics , Pyrazoles/pharmacokinetics , Structure-Activity RelationshipABSTRACT
Background: Adipose-derived stem cells (ASCs) assisted lipotransfer have been considered to facilitate the survival of fat grafts. However, emerging evidence of insufficient vascularization is another obstacle for fat graft survival in cell-assisted lipotransfer. Objectives: This study evaluated if endothelial phenotype ASCs with fat lipoaspirate improves survival and neovascularization in fat transplantation. Methods: ASCs were isolated from human periumbilical fat tissue and cultured in endothelial growth medium for 2 weeks. Fat lipoaspirate was mixed with fresh adipose stroma vascular fraction (SVF), endothelial differentiated ASCs (EC/ASCs), and fat lipoaspirate alone. Three fat mixtures were subcutaneously injected into the adult male Sprague-Dawley rat's dorsum at 3 locations. At 8 weeks after transplantation, the grafted fat lipoaspirates were harvested, and the extracted fat was evaluated using photographic, survival weights measurements and histological examination. Neo-vascularization was quantified by immunofluorescence and real-time RT-PCR. Results: Grafts from the EC/ASC assisted group had a higher survival rate, morphologic integrity, and most uniform lipid droplets. They also revealed less inflammation and fibrosis with increased number of vessels by histological and immunofluorescence analysis. Quantitative RT-PCR analysis indicated that the expression levels of EC-specific markers of CD31 and vWF were higher in the EC/ASC group compared with in the control and fat with SVF transplants. Conclusions: These results indicated that co-implantation of fat lipoaspirate with ASCs differentiated toward an endothelial phenotype improves both survival and neovascularization of the transplanted fat lipoaspirate, which might provide benefits and represents a promising strategy for clinical application in autologous fat transplantation.
Subject(s)
Graft Survival/physiology , Neovascularization, Physiologic/physiology , Stem Cell Transplantation/methods , Subcutaneous Fat, Abdominal/transplantation , Adult , Animals , Cell Culture Techniques/methods , Cell Differentiation/physiology , Cell Survival/physiology , Cells, Cultured , Culture Media , Endothelial Cells/physiology , Female , Humans , Male , Middle Aged , Models, Animal , Rats , Rats, Sprague-Dawley , Subcutaneous Fat, Abdominal/cytology , Transplantation, Heterologous/methodsABSTRACT
Soil depth plays an important role in landslide disaster prevention and is a key factor in slopeland development and management. Existing soil depth maps are outdated and incomplete in Taiwan. There is a need to improve the accuracy of the map. The Kriging method, one of the most frequently adopted estimation approaches for soil depth, has room for accuracy improvements. An appropriate soil depth estimation method is proposed, in which soil depth is estimated using Bayesian Maximum Entropy method (BME) considering space distribution of measured soil depth and impact of physiographic factors. BME divides analysis data into groups of deterministic and probabilistic data. The deterministic part are soil depth measurements in a given area and the probabilistic part contains soil depth estimated by a machine learning-based soil depth estimation model based on physiographic factors including slope, aspect, profile curvature, plan curvature, and topographic wetness index. Accuracy of estimates calculated by soil depth grading, very shallow (<20 cm), shallow (20-50 cm), deep (50-90 cm), and very deep (>90 cm), suggests that BME is superior to the Kriging method with estimation accuracy up to 82.94%. The soil depth distribution map of Hsinchu, Taiwan made by BME with a soil depth error of ±5.62 cm provides a promising outcome which is useful in future applications, especially for locations without soil depth data.
ABSTRACT
The approval of the erythropoietin (EPO) mimetic peptide drug peginesatide in 2012 was a breakthrough for the treatment of secondary anemia. However, due to severe allergic reactions, peginesatide was recalled a year later. In this study, 12 novel peptides were designed and synthesized by substituting specific amino acids of the monomeric peptide in peginesatide, with the aim of obtaining new EPO mimetic peptides with higher activities and lower side effects than the parent compound. Their cell proliferation activities were evaluated, and the structure-activity relationships were analyzed. Five compounds had equal cell proliferation activity to the control peptide. Among them, one compound showed a higher in vivo activity than the control peptide, with no obvious side effects.
Subject(s)
Drug Design , Erythroblasts/drug effects , Peptides/pharmacology , Animals , Body Weight/drug effects , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Humans , Injections, Subcutaneous , Mice , Molecular Structure , Peptides/administration & dosage , Peptides/chemical synthesis , Structure-Activity RelationshipABSTRACT
INTRODUCTION: Neoadjuvant chemotherapy prior to lumpectomy or mastectomy for breast cancer challenges wound healing. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, has been shown to work synergistically with paclitaxel in vitro and in preclinical studies. In addition, our laboratory has demonstrated that SAHA treatment decreases paclitaxel-associated stem cell toxicity, modulates inflammatory response, and promotes wound healing in injured fibroblast cells. Our goal was to determine if combined SAHA and paclitaxel treatment would improve wound healing in an in vivo full-thickness murine model, without altering antitumor effect. METHODS: Thirty-two nude athymic mice received intraperitoneal injections of paclitaxel (20 mg/kg), SAHA (25 mg/kg), paclitaxel + SAHA (20 mg/kg + 25 mg/kg), or no treatment for 2 weeks prior to surgery. Under general anesthesia, 8-mm full-thickness dorsal wounds were created in all animals, and a silicone splint was attached to minimize wound contraction. The wounds were measured twice a week with a surgical caliper until healing was complete. To evaluate the in vivo effect of drug treatment, 16 athymic nude mice with MDA-MB-231 xenografts received the treatments described previously, following which tumor volumes were compared between groups. RESULTS: Average wound healing time was prolonged in mice treated with paclitaxel (20 ± 1.9 days), and combination SAHA + paclitaxel therapy improved average wound healing time (17.0 ± 1.8 days). In the xenograft model, the antitumor effect of SAHA and paclitaxel (average tumor volume 43.9 ± 34.1 mm) was greater than paclitaxel alone (105.8 ± 73.8 mm). CONCLUSIONS: The addition of SAHA to taxane chemotherapy improves the therapeutic effect on triple-negative breast cancer while decreasing the detrimental effect of paclitaxel on wound healing. This may have substantial implications on improving outcomes in breast reconstruction following chemotherapy.
Subject(s)
Back Injuries/drug therapy , Histone Deacetylase Inhibitors/pharmacology , Paclitaxel/pharmacology , Vorinostat/pharmacology , Wound Healing/drug effects , Animals , Disease Models, Animal , Mice , Mice, NudeABSTRACT
OBJECTIVE: The purpose of our study was to investigate the effect of adipose-derived stem cells (ASCs), endothelial-differentiated ASCs (EC/ASCs), and various conditioned media (CM) on wound healing in a diabetic swine model. We hypothesized that ASC-based therapies would accelerate wound healing. METHODS: Diabetes was induced in four Yorkshire swine through intravenous injection of streptozotocin. ASCs were harvested from flank fat and cultured in either M199 or EGM-2 medium. A duplicate series of seven full-thickness dorsal wounds were surgically created on each swine. The wounds in the cellular treatment group underwent injection of low-dose or high-dose ASCs or EC/ASCs on day 0, with a repeat injection of one half of the initial dose on day 15. Wounds assigned to the topical CM therapy were covered with 2 mL of either serum-free M199 primed by ASCs or human umbilical vein endothelial cells every 3 days. Wounds were assessed at day 0, 10, 15, 20, and 28. The swine were sacrificed on day 28. ImageJ software was used to evaluate the percentage of wound healing. The wounded skin underwent histologic, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay examinations to evaluate markers of angiogenesis and inflammation. RESULTS: We found an increase in the percentage of wound closure rates in cell-based treatments and topical therapies at various points compared with the untreated control wounds (P < .05). The results from the histologic, messenger RNA, and protein analyses suggested the treated wounds displayed increased angiogenesis and a diminished inflammatory response. CONCLUSIONS: Cellular therapy with ASCs, EC/ASCs, and topical CM accelerated diabetic wound healing in the swine model. Enhanced angiogenesis and immunomodulation might be key contributors to this process.
Subject(s)
Adipose Tissue/cytology , Cell Differentiation , Culture Media, Conditioned/pharmacology , Diabetes Mellitus, Experimental/complications , Endothelial Progenitor Cells/transplantation , Multipotent Stem Cells/transplantation , Skin/blood supply , Wound Healing , Wounds, Penetrating/therapy , Administration, Topical , Animals , Cell Differentiation/drug effects , Cells, Cultured , Culture Media, Conditioned/metabolism , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Endothelial Progenitor Cells/metabolism , Gene Expression Regulation , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Male , Multipotent Stem Cells/metabolism , Neovascularization, Physiologic , Phenotype , Skin/drug effects , Skin/injuries , Skin/metabolism , Sus scrofa , Time Factors , Wound Healing/drug effects , Wounds, Penetrating/etiology , Wounds, Penetrating/metabolism , Wounds, Penetrating/pathologyABSTRACT
BACKGROUND: Chronic wounds are a common surgical problem exacerbated by diabetes and ischemia. Although adipose-derived stem cells (ASCs) have shown promise as a wound healing therapy, their function and proliferation are hindered in diabetes. This study examines the ability of the human umbilical vein endothelial cell (HUVEC) secretome to reverse the deleterious effects of high glucose concentrations on ASCs through priming, thereby enhancing their ability to participate in angiogenesis and wound healing. METHODS: Institutional review board-approved human ASCs were cultured in M199 medium with or without glucose (30 mmol/L). HUVEC were grown in 30 mmol/L glucose-containing M199 medium; the resulting conditioned medium (HUVEC-CM) was collected every 3 days and used to prime ASCs. An aliquot of HUVEC-CM was heated (85°C for 30 minutes) to produce thermal denaturation of protein. Viability, proliferation, and endothelial differentiation were measured by MTT assays, growth curves, and quantitative polymerase chain reaction, respectively. A Matrigel assay was used to assess the ability of primed ASCs to participate in capillary-like tube formation. An Institutional Animal Care and Use Committee-approved in vivo murine model of diabetic and ischemic hindlimbs was used to evaluate the angiogenic potential of primed stem cells. Human ASCs were cultured with either control M199 or HUVEC-CM. Mice were randomized to a control group, an unprimed ASC group, or a HUVEC-primed ASC group. Cellular therapies were injected into the ischemic muscle. Thirty days later, slides were made. Microvessels were counted by three blinded observers. RESULTS: MTT assays revealed that HUVEC-priming induced a 1.5 times increase in cell viability over diabetic controls. This promoting effect was lost with heated HUVEC-CM (P < .001), indicating that the active molecules are of protein origin. After 9 days, ASCs cultured in 30 mmol/L glucose solution showed a 14% reduction in growth from nondiabetic controls (P = .013) and exhibited atrophic morphology. Conversely, diabetic HUVEC-primed stem cells demonstrated a nearly four-fold increase in proliferation (P < .05) and took on a fusiform, endothelial-like phenotype. Polymerase chain reaction demonstrated enhanced expression of CD31 messenger RNA by 4.7-fold after 14 days in the HUVEC-primed group, and endothelial nitric oxide synthase messenger RNA messenger RNA was increased 20.1-fold from controls. Unlike unprimed controls, HUVEC-primed ASCs readily formed capillary-like tube networks on Matrigel. Diabetic mice that were injected with HUVEC-primed ASCs demonstrated greater vessel density than both controls (2.1-fold) and unprimed stem cell treatments (P < .001). CONCLUSIONS: HUVECs secrete protein factors that significantly increase proliferation and endothelial differentiation of ASCs under diabetic conditions. Injection of ischemic hindlimbs in diabetic mice with HUVEC-primed ASCs leads to enhanced angiogenesis.
Subject(s)
Adipose Tissue/cytology , Diabetic Angiopathies/surgery , Endothelial Progenitor Cells/metabolism , Endothelial Progenitor Cells/transplantation , Human Umbilical Vein Endothelial Cells/metabolism , Ischemia/surgery , Muscle, Skeletal/blood supply , Neovascularization, Physiologic , Wound Healing , Angiogenic Proteins/metabolism , Animals , Blood Glucose/metabolism , Cell Differentiation , Cell Movement , Cell Proliferation , Cells, Cultured , Culture Media, Conditioned/metabolism , Cytokines/metabolism , Diabetic Angiopathies/metabolism , Diabetic Angiopathies/pathology , Diabetic Angiopathies/physiopathology , Female , Hindlimb , Humans , Ischemia/metabolism , Ischemia/pathology , Ischemia/physiopathology , Male , Mice, Inbred C57BL , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Paracrine Communication , Phenotype , Signal Transduction , Time FactorsABSTRACT
PURPOSE: This study aimed to determine the prevalence and the socio-demographic and clinical correlates of sleep disturbance in first-episode individuals with schizophrenia in rural China and the factors that impact sleep among individuals with schizophrenia. DESIGN AND METHODS: A total of 104 first-episode individuals with schizophrenia were randomly selected in rural areas in Ningxia, China, in 2015 as the study sample. FINDINGS: In first-episode individuals with schizophrenia, the prevalence of sleep disturbance was 78.8% (82/104). Sleep disturbance was significantly associated with economic status, living situation, educational level, depressive symptoms, and anxiety symptoms. PRACTICE IMPLICATIONS: Sleep disturbance is common in first-episode individuals with schizophrenia in rural China and more attention should be paid in clinical practice to improve the sleep quality for individuals with schizophrenia.
Subject(s)
Anxiety/epidemiology , Depression/epidemiology , Rural Population/statistics & numerical data , Schizophrenia/epidemiology , Sleep Wake Disorders/epidemiology , Socioeconomic Factors , Adult , China/epidemiology , Comorbidity , Female , Humans , Male , Middle Aged , PrevalenceABSTRACT
BACKGROUND: Traumatic brain injury (TBI) is a common cause of morbidity and mortality in the civilian population. The purpose of this study was to examine the effect(s) of adipose-derived stem cell (ASC) treatment on cellular and functional recovery in TBI via both in vitro and in vivo methods. METHODS: Cultured neuroblastoma cells, SH-SY5Y, were scratched to mimic TBI in an in vitro model. The effect of ASC-conditioned medium (CM) on cell death, mitochondrial function, and expression of inflammatory cytokines (tumor necrosis factor α [TNF-α], interleukin 1ß [IL-1ß], and IL-6), as well as apoptosis marker FAS, was measured. In our in vivo model, Sprague-Dawley rats underwent TBI via a frontal, closed-head injury model. Animals randomly received either intravenous human-derived ASCs or intravenous saline within 3 hours of injury and were compared with a sham group. Functional recovery was evaluated via accelerating Rotarod method. On post-TBI Day 3, brain tissue was harvested and assessed for cellular damage via enzyme-linked immunosorbent assay for TNF-α, as well as immunohistochemical staining for ß-amyloid precursor protein (ß-APP). RESULTS: Our in vitro data show that ASC treatment imparted reduced cell death (ratio to control: 1.21 ± 0.066 vs. 1.01 ± 0.056, p = 0.017), increased cell viability (ratio to control: 0.86 ± 0.009 vs. 1.09 ± 0.01, p = 0.0001), increased mitochondrial function (percentage of control: 78 ± 6% vs. 68 ± 3%), and significantly decreased levels of inflammatory cytokine IL-1ß. In our in vivo study, compared with TBI alone, ASC-treated animals showed no difference in functional recovery, lower levels of expressed TNF-α (ratio to total protein, 0.47 ± 0.01 vs. 0.67 ± 0.04; p < 0.01), and lower levels of ß-amyloid precursor protein (fluorescence ratio, 0.43 ± 0.05 vs. 0.69 ± 0.03; p < 0.01). CONCLUSIONS: Adipose-derived stem cell treatment results in improved cell survival, decreased inflammatory marker release, and decreased evidence of neural injury. No difference in functional recovery was seen. These data suggest the potential for ASC treatment to aid in cellular protection and recovery in neural cells following TBI.
Subject(s)
Adipocytes/cytology , Brain Injuries, Traumatic/therapy , Nerve Regeneration/physiology , Neurons/pathology , Stem Cell Transplantation/methods , Stem Cells/cytology , Animals , Brain Injuries, Traumatic/metabolism , Brain Injuries, Traumatic/pathology , Cells, Cultured , Cytokines/metabolism , Disease Models, Animal , Humans , Male , Rats , Rats, Sprague-Dawley , Treatment OutcomeABSTRACT
INTRODUCTION: Paclitaxel improves the oncologic response of breast cancer resections; however, it may negatively affect the wound-healing potential of human adipose-derived stem cells (hASCs) for fat grafting and reconstructive surgery. Histone deacetylase inhibitors (HDACis) modify the epigenetic regulation of gene expression and stabilize microtubules similarly to paclitaxel, thus, creating a synergistic mechanism of cell cycle arrest. We aim to combine these drugs to enhance cytotoxicity towards breast cancer cells, while preserving the wound-healing function of hASCs for downstream reconstructive applications. METHODS: Triple negative breast cancer cells (MBA-MB-231) and hASCs (institutional review board-approved clinical isolates) were treated with a standard therapeutic dose of paclitaxel (1.0 µM) or with low-dose paclitaxel (0.1 µM) combined with the HDACi suberoylanilide hydroxamic acid or trichostatin A. Cell viability, gene expression, apoptosis, and wound-healing/migration were measured via methylthiazol tetrazolium assay, quantitative real-time polymerase chain reaction, annexin V assay, and fibroblast scratch assay, respectively. RESULTS: Combined HDACi and low-dose paclitaxel therapy maintained cytotoxicity towards breast cancer cells and preserved adipose-derived stem cell viability. Histone deacetylase inhibitor demonstrated selective anti-inflammatory effects on adipose-derived stem cell gene expression and decreased expression of the proapoptotic gene FAS. Furthermore, HDACi therapy did not increase relative apoptosis within hASCs. A scratch assay demonstrated enhanced wound healing among injured fibroblasts indirectly co-cultured with HDACi-treated hASCs. CONCLUSIONS: Combining HDACi with low-dose paclitaxel improved cytotoxicity towards breast cancer cells and preserved hASC viability. Furthermore, enhanced wound healing was observed by improved migration in a fibroblast scratch assay. These results suggest that the addition of HDACi to taxane chemotherapy regimens may improve oncologic results and wound-healing outcomes after reconstructive surgery.
Subject(s)
Adipose Tissue/cytology , Breast Neoplasms/drug therapy , Histone Deacetylase Inhibitors/pharmacology , Paclitaxel/pharmacology , Stem Cells/drug effects , Wound Healing/drug effects , Apoptosis/drug effects , Breast Neoplasms/surgery , Cell Movement/drug effects , Cell Survival/drug effects , Gene Expression/drug effects , Histone Deacetylase Inhibitors/administration & dosage , Humans , Mammaplasty , Paclitaxel/administration & dosage , Real-Time Polymerase Chain Reaction , Tumor Cells, CulturedABSTRACT
OBJECTIVE: Adipose-derived stem cells (ASCs) are a potential adult mesenchymal stem cell source for restoring endothelial function in patients with critical limb ischemia. Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF) play a major role in angiogenesis and wound healing. This study evaluated the effects of FGF and VEGF on the proliferation, migration, and potential endothelial differentiation of human ASCs with regards to their use as endothelial cell substitutes. METHODS: ASCs were isolated from clinical lipoaspirates and cultured in M199 medium with fetal bovine serum (10%), FGF2 (10 ng/mL), VEGF (50 ng/mL), or combinations of FGF2 and VEGF. Cell proliferation rates, viability, and migration were measured by growth curves, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and scratch assays. For cell attachment determinations, ASCs were seeded onto a scaffold of small intestinal submucosa for 5 days. Endothelial differentiation capabilities of ASCs were confirmed by expression of endothelial cell-specific markers using quantitative polymerase chain reaction, immunofluorescence staining, and cord formation on Matrigel (BD Biosciences, San Jose, Calif). PD173074, a selective inhibitor of FGF receptor, was used to confirm the importance of FGF signaling. RESULTS: ASCs treated with FGF or combinations of FGF and VEGF showed increased proliferation rates and consistent differentiation toward an endothelial cell lineage increase in platelet endothelial cell adhesion molecule (CD31), von Willebrand factor, endothelial nitric oxide synthase, and vascular endothelial cadherin message, and in protein and cord formation on Matrigel. FGF and VEGF stimulated ASC migration and increased the attachment and retention after seeding onto a matrix graft of small intestinal submucosa. Blockade of FGF signaling with PD173074 abrogated ASC endothelial cell differentiation potential. CONCLUSIONS: These results indicate that FGF and VEGF are ASC promoters for proliferation, migration, attachment, and endothelial differentiation. FGF and VEGF have a costimulatory effect on ASC endotheliogenesis. These results further suggest that ASCs with enhanced FGF signaling may potentially be used for tissue engineering and cell-based therapies in patients with critical limb ischemia.
Subject(s)
Adipose Tissue/cytology , Angiogenesis Inducing Agents/pharmacology , Cell Differentiation/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Endothelial Progenitor Cells/drug effects , Fibroblast Growth Factor 2/pharmacology , Mesenchymal Stem Cells/drug effects , Neovascularization, Physiologic/drug effects , Vascular Endothelial Growth Factor A/pharmacology , Biomarkers/metabolism , Cell Adhesion/drug effects , Cell Survival/drug effects , Cells, Cultured , Endothelial Progenitor Cells/metabolism , Extracellular Matrix/metabolism , Humans , Intestine, Small/metabolism , Mesenchymal Stem Cells/metabolism , Phenotype , Time Factors , Tissue ScaffoldsABSTRACT
BACKGROUND AIMS: Adipose-derived stem cells (ASCs) are considered to play a positive role in wound healing as evidenced by their increasing use in breast reconstructive procedures. After chemotherapy for breast cancer, poor soft tissue wound healing is a major problem. In the present study, the functional capabilities and recovery of ASCs after exposure to chemotherapeutic agent paclitaxel (PTX) using in vitro and ex vivo models were demonstrated. METHODS: Human ASCs were isolated from periumbilical fat tissue and treated with PTX at various concentrations. Adult Sprague-Dawley rats were given intravenous injections with PTX. Two and four weeks after the initial PTX treatment, ASCs were isolated from rat adipose tissue. Proliferation, cell viability, apoptosis and cell migration rates were measured by growth curves, MTT assays, flow cytometry and scratch assays. ASCs were cultured in derivative-specific differentiation media with or without PTX for 3 weeks. Adipogenic, osteogenic and endothelial differentiation levels were measured by quantitative reverse transcriptase polymerase chain reaction and histological staining. RESULTS: PTX induced apoptosis, decreased the proliferation and cell migration rates of ASCs and inhibited ASCs multipotent differentiation in both in vitro human ASC populations and ex vivo rat ASC populations with PTX treatment. Furthermore, after cessation of PTX, ASCs exhibited recovery potential of differentiation capacity in both in vitro and animal studies. CONCLUSIONS: Our results provide insight into poor soft tissue wound healing and promote further understanding of the potential capability of ASCs to serve as a cell source for fat grafting and reconstruction in cancer patients undergoing chemotherapy treatment.
Subject(s)
Adipose Tissue/cytology , Adult Stem Cells/drug effects , Adult Stem Cells/physiology , Paclitaxel/pharmacology , Abdominal Fat/cytology , Abdominal Fat/drug effects , Adipose Tissue/drug effects , Adipose Tissue/pathology , Adult , Animals , Apoptosis/drug effects , Breast Neoplasms/pathology , Breast Neoplasms/rehabilitation , Cell Differentiation/drug effects , Cell Movement/drug effects , Cell Survival/drug effects , Cells, Cultured , Female , Humans , Male , Osteogenesis/drug effects , Osteogenesis/physiology , Rats , Rats, Sprague-Dawley , Recovery of Function/drug effects , Wound Healing/drug effectsABSTRACT
In breast reconstructive procedures, adipose-derived stem cells (ASCs) that are present in clinical fat grafting isolates are considered to play the main role in improving wound healing. In patients following chemotherapy for breast cancer, poor soft tissue wound healing is a major problem. However, it is unclear if tamoxifen (TAM) as the most widely used hormonal therapeutic agent for breast cancer treatment, affects the ASCs and ultimately wound healing. This study evaluated whether TAM exposure to in vitro human ASCs modulate cellular functions. Human ASCs were isolated and treated with TAM at various concentrations. The effects of TAM on cell cycle, cell viability and proliferation rates of ASCs were examined by growth curves, MTT assay and BrdU incorporation, respectively. Annexin V and JC-1 Mitochondrial Membrane Potential assays were used to analyze ASC apoptosis rates. ASCs were cultured in derivative-specific differentiation media with or without TAM (5 uM) for 3 weeks. Adipogenic and osteogenic differentiation levels were measured by quantitative RT-PCR and histological staining. TAM has cytotoxic effects on human ASCs through apoptosis and inhibition of proliferation in dose- and time-dependent manners. TAM treatment significantly down-regulates the capacity of ASCs for adipogenic and osteogenic differentiation (p<0.05 vs. control), and inhibit the ability of the ASCs to subsequently formed cords in Matrigel. This study is the first findings to our knowledge that demonstrated that TAM inhibited ASC proliferation and multi-lineage ASC differentiation rates. These results may provide insight into the role of TAM with associated poor soft tissue wound healing and decreased fat graft survival in cancer patients receiving TAM.
Subject(s)
Adipose Tissue/transplantation , Stem Cells/cytology , Tamoxifen/therapeutic use , Wound Healing/drug effects , Wounds and Injuries/therapy , Adipose Tissue/cytology , Adult , Aged , Apoptosis , Cell Differentiation , Cell Proliferation/drug effects , Cell Survival , Cells, Cultured , Estrogen Antagonists/therapeutic use , Female , Humans , Middle Aged , Stem Cell Transplantation , Stem Cells/drug effects , Wounds and Injuries/metabolism , Wounds and Injuries/pathologyABSTRACT
BACKGROUND: Protein Kinase C (PKC) dysfunction is implicated in a variety of smooth muscle disorders including detrusor overactivity associated with frequency and urgency of micturition. In this study, we aimed to evaluate the modulatory effects of endogenous PKC-dependent pathways on bladder storage and emptying function. METHODS: We utilized in vivo cystometry and in vitro organ bath studies using isolated bladder muscle strips (BMS) from rats to measure contractility, intravesical pressure, and voided volume. Both in vitro and in vivo results were statistically analyzed using one-way repeated measures ANOVA between the groups followed by Bonferroni's post-test, as appropriate (Systat Software Inc., San Jose, CA). RESULTS: Effects of PKC activators, phorbol-12,13-dibutyrate (PDBu), and phorbol-12,13-myristate (PMA), were concentration-dependent, with high concentrations increasing frequency of micturition, and sensitivity of intramural nerves to electrical field stimulation (EFS), in vitro, while lower concentrations had no effect on BMS sensitivity to EFS. The PKC inhibitors, bisindolylmaleimide1 (Bim-1), (28 nM), and Ro318220 (50 µM) triggered an increase in the number of non-voiding contractions (NVC), and a decrease in the voided volume associated with reduced ability to maintain contractile force upon EFS, but did not affect peak force in vitro. Both low (50 nM) and high PDBu 1 micromolar (1 uM) decreased the sensitivity of BMS to carbachol. Application of a low concentration of PDBu inhibited spontaneous contractions, in vitro, and Bim-1-induced NVC, and restored normal voiding frequency during urodynamic recordings in vivo. CONCLUSIONS: In summary, the effects of low PKC stimulation include inhibition of smooth muscle contractile responses, whereas high levels of PKC stimulation increased nerve-mediated contractions in vitro, and micturition contractions in vivo. These results indicate that endogenous PKC signaling displays a concentration-dependent contraction profile in the urinary bladder via both smooth muscle and nerve-mediated pathways.
Subject(s)
Muscle Contraction/drug effects , Muscle Contraction/physiology , Protein Kinase C/physiology , Urinary Bladder/drug effects , Urinary Bladder/physiology , Urination/drug effects , Urination/physiology , Animals , Dose-Response Relationship, Drug , Male , Phorbol 12,13-Dibutyrate/pharmacology , Phorbol Esters/pharmacology , Rats, Sprague-DawleyABSTRACT
BACKGROUND: Cancer patients with chemotherapy-induced immunosuppression have poor surgical site wound healing. Prior literature supports the use of human adipose-derived stem cell (hASC) lipoinjection to improve wound healing. It has been established that multipotent hASCs facilitate neovascularization, accelerate epithelialization, and quicken wound closure in animal models. Although hASC wound therapy may benefit surgical cancer patients, the chemotherapeutic effects on hASCs are unknown. We hypothesized that paclitaxel, a chemotherapeutic agent, impairs hASC growth, multipotency, and induces apoptosis. METHODS: hASCs were isolated and harvested from consented, chemotherapy and radiation naive patients. Growth curves, MTT (3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide), and EdU (5-ethynyl-2-deoxyguridine) assays measured cytotoxicity and proliferation. Oil Red O stain, Alizarin Red stain, matrigel tube formation assay, and quantitative polymerase chain reaction analyzed hASC differentiation. Annexin V assay measured apoptosis. Immunostaining and Western blot determined tumor necrosis factor α (TNF-α) expression. RESULTS: hASCs were selectively more sensitive to paclitaxel (0.01-30 µM) than fibroblasts (P < 0.05). After 12 d, paclitaxel caused hASC growth arrest, whereas control hASCs proliferated (P = 0.006). Paclitaxel caused an 80.6% reduction in new DNA synthesis (P < 0.001). Paclitaxel severely inhibited endothelial differentiation and capillary-like tube formation. Differentiation markers, lipoprotein lipase (adipogenic), alkaline phosphatase (osteogenic), CD31, and van Willebrand factor (endothelial), were significantly decreased (all P < 0.05) confirming paclitaxel impaired differentiation. Paclitaxel was also found to induce apoptosis and TNF-α was upregulated in paclitaxel-treated hASCs (P < 0.001). CONCLUSIONS: Paclitaxel is more cytotoxic to hASCs than fibroblasts. Paclitaxel inhibits hASC proliferation, differentiation, and induces apoptosis, possibly through the TNF-α pathway. Paclitaxel's severe inhibition of endothelial differentiation indicates neovascularization disruption, possibly causing poor wound healing in cancer patients receiving chemotherapy.
Subject(s)
Adult Stem Cells/drug effects , Antineoplastic Agents, Phytogenic/adverse effects , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Paclitaxel/adverse effects , Adipose Tissue/cytology , Adult Stem Cells/metabolism , Apoptosis/drug effects , Cell Line , Endothelial Cells/cytology , Fibroblasts/drug effects , Humans , Osteogenesis/drug effects , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Smith-Lemli-Opitz syndrome (SLOS) is a congenital, autosomal recessive metabolic and developmental disorder caused by mutations in the enzyme which catalyzes the reduction of 7-dehydrocholesterol (7DHC) to cholesterol. Herein we show that dermal fibroblasts obtained from SLOS children display increased basal levels of LC3B-II, the hallmark protein signifying increased autophagy. The elevated LC3B-II is accompanied by increased beclin-1 and cellular autophagosome content. We also show that the LC3B-II concentration in SLOS cells is directly proportional to the cellular concentration of 7DHC, suggesting that the increased autophagy is caused by 7DHC accumulation secondary to defective DHCR7. Further, the increased basal LC3B-II levels were decreased significantly by pretreating the cells with antioxidants implicating a role for oxidative stress in elevating autophagy in SLOS cells. Considering the possible source of oxidative stress, we examined mitochondrial function in the SLOS cells using JC-1 assay and found significant mitochondrial dysfunction compared to mitochondria in control cells. In addition, the levels of PINK1 which targets dysfunctional mitochondria for removal by the autophagic pathway are elevated in SLOS cells, consistent with mitochondrial dysfunction as a stimulant of mitophagy in SLOS. This suggests the increase in autophagic activity may be protective, i.e., to remove dysfunctional mitochondria. Taken together, these studies are consistent with a role for mitochondrial dysfunction leading to increased autophagy in SLOS pathophysiology.
ABSTRACT
BACKGROUND: Adipose tissue provides a readily available source of autologous stem cells. Adipose-derived stem cells (ASCs) have been proposed as a source for endothelial cell substitutes for lining the luminal surface of tissue engineered bypass grafts. Endothelial nitric oxide synthase (eNOS) is a key protein in endothelial cell function. Currently, endothelial differentiation from ASCs is limited by poor eNOS expression. The goal of this study was to investigate the role of three molecules, sphingosine-1-phosphate (S1P), bradykinin, and prostaglandin-E1 (PGE1) in ASC endothelial differentiation. Endothelial differentiation markers (CD31, vWF and eNOS) were used to evaluate the level of ASCs differentiation capability. RESULTS: ASCs demonstrated differentiation capability toward to adipose, osteocyte and endothelial like cell phenotypes. Bradykinin, S1P and PGE were used to promote differentiation of ASCs to an endothelial phenotype. Real-time PCR showed that all three molecules induced significantly greater expression of endothelial differentiation markers CD31, vWF and eNOS than untreated cells. Among the three molecules, S1P showed the highest up-regulation on endothelial differentiation markers. Immunostaining confirmed presence of more eNOS in cells treated with S1P than the other groups. Cell growth measurements by MTT assay, cell counting and EdU DNA incorporation suggest that S1P promotes cell growth during ASCs endothelial differentiation. The S1P1 receptor was expressed in ASC-differentiated endothelial cells and S1P induced up-regulation of PI3K. CONCLUSIONS: S1P up-regulates endothelial cell markers including eNOS in ASCs differentiated to endothelial like cells. This up-regulation appears to be mediated by the up-regulation of PI3K via S1P1 receptor. ASCs treated with S1P offer promising use as endothelial cell substitutes for tissue engineered vascular grafts and vascular networks.