Tbx18-dependent differentiation of brown adipose tissue-derived stem cells toward cardiac pacemaker cells.

A cell-sourced biological pacemaker is a promising therapeutic approach for sick sinus syndrome (SSS) or severe atrial ventricular block (AVB). Adipose tissue-derived stem cells (ATSCs), which are optimal candidate cells for possible use in regenerative therapy for acute or chronic myocardial injury, have the potential to differentiate into spontaneous beating cardiomyocytes. However, the pacemaker characteristics of the beating cells need to be confirmed, and little is known about the underlying differential mechanism. In this study, we found that brown adipose tissue-derived stem cells (BATSCs) in mice could differentiate into spontaneous beating cells in 15% FBS Dulbecco's modified Eagle's medium (DMEM) without additional treatment. Subsequently, we provide additional evidence, including data regarding ultrastructure, protein expression, electrophysiology, and pharmacology, to support the differentiation of BATSCs into a cardiac pacemaker phenotype during the course of early cultivation. Furthermore, we found that silencing Tbx18, a key transcription factor in the development of pacemaker cells, terminated the differentiation of BATSCs into a pacemaker phenotype, suggesting that Tbx18 is required to direct BATSCs toward a cardiac pacemaker fate. The expression of Tbx3 and shox2, the other two important transcription factors in the development of pacemaker cells, was decreased by silencing Tbx18, which suggests that Tbx18 mediates the differentiation of BATSCs into a pacemaker phenotype via these two downstream transcription factors.

[Blood vessels and nerves surrounding the seminal vesicles: A clinical anatomic study].

OBJECTIVE: To investigate the precise locations of the blood vessels and nerves surrounding the seminal vesicles (SV) in men and provide some anatomical evidence for SV-related minimally invasive surgery. METHODS: We observed the courses and distribution of the blood vessels and nerves surrounding SVs and obtained the data for positioning the SV neuroplexes in 20 male pelvises. RESULTS: One branch of the neuroplexes was distributed to the SVs bilaterally with the neurovascular bundles, (2.85 ± 0.18) cm from the median sulcus of the prostate (MSP), while another branch ran through the Denonvillier fascia behind the SV, (0.81 ± 0.06) cm from the MSP. The arterial SVs (ASV) originated from the inferior vesical artery and fell into 4 types, 55% going directly to the SVs as one branch, 15% running between the SV and the ampulla of the deferent duct as another branch, 25% downward as 2 branches to the SV and between the SV and the ampulla of the deferent duct respectively, and 5% as the other ASVs. The shortest distance from the ASV through the prostatic neuroplexus to the posterior SV was (1.08 ± 0.09) cm. CONCLUSION: In SV resection, neuroplexus injury can be reduced with a bilateral distance of < 2.85 cm and a posterior distance of < 0.81 cm from the MSP, and so can bleeding by vascular ligation between the SV and the ampulla of the deferent duct.

Interferon regulatory factor-1 together with reactive oxygen species promotes the acceleration of cell cycle progression by up-regulating the cyclin E and CDK2 genes during high glucose-induced proliferation of vascular smooth muscle cells.

BACKGROUND: The high glucose-induced proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development of diabetic vascular diseases. In a previous study, we confirmed that Interferon regulatory factor-1 (Irf-1) is a positive regulator of the high glucose-induced proliferation of VSMCs. However, the mechanisms remain to be determined. METHODS: The levels of cyclin/CDK expression in two cell models involving Irf-1 knockdown and overexpression were quantified to explore the relationship between Irf-1 and its downstream effectors under normal or high glucose conditions. Subsequently, cells were treated with high glucose/NAC, normal glucose/H2O2, high glucose/U0126 or normal glucose/H2O2/U0126 during an incubation period. Then proliferation, cyclin/CDK expression and cell cycle distribution assays were performed to determine whether ROS/Erk1/2 signaling pathway was involved in the Irf-1-induced regulation of VSMC growth under high glucose conditions. RESULTS: We found that Irf-1 overexpression led to down-regulation of cyclin D1/CDK4 and inhibited cell cycle progression in VSMCs under normal glucose conditions. In high glucose conditions, Irf-1 overexpression led to an up-regulation of cyclin E/CDK2 and an acceleration of cell cycle progression, whereas silencing of Irf-1 suppressed the expression of both proteins and inhibited the cell cycle during the high glucose-induced proliferation of VSMCs. Treatment of VSMCs with antioxidants prevented the Irf-1 overexpression-induced proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression in high glucose conditions. In contrast, under normal glucose conditions, H2O2 stimulation and Irf-1 overexpression induced cell proliferation, up-regulated cyclin E/CDK2 expression and promoted cell cycle acceleration. In addition, overexpression of Irf-1 promoted the activation of Erk1/2 and when VSMCs overexpressing Irf-1 were treated with U0126, the specific Erk1/2 inhibitor abolished the proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression under high glucose or normal glucose/H2O2 conditions. CONCLUSIONS: These results demonstrate that the downstream effectors of Irf-1 are cyclin E/CDK2 during the high glucose-induced proliferation of VSMCs, whereas they are cyclin D1/CDK4 in normal glucose conditions. The Irf-1 overexpression-induced proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression are associated with ROS/Erk1/2 signaling pathway under high glucose conditions.

Endothelin-induced differentiation of Nkx2.5⁺ cardiac progenitor cells into pacemaking cells.

The mechanisms governing the development of cardiac pacemaking and conduction system are not well understood. In order to provide evidence for the derivation of pacemaking cells and the signal that induce and maintain the cells in the developing heart, Nkx2.5(+) cardiac progenitor cells (CPCs) were isolated from embryonic heart tubes of rats. Endothelin-1 was subsequently added to the CPCs to induce differentiation of them towards cardiac pacemaking cells. After the treatment, Nkx2.5(+) CPCs displayed spontaneous beating and spontaneously electrical activity as what we have previously described. Furthermore, RT-PCR and immunofluorescence staining demonstrated that Tbx3 expression was increased and Nkx2.5 expression was decreased in the induced cells 4 days after ET-1 treatment. And the significantly increased expression of Hcn4 and connexin-45 were detected in the induced cells 10 days after the treatment. In addition, Nkx2.5(+) CPCs were transfected with pGCsi-Tbx3 4 days after ET-1 treatment in an attempt to determine the transcription regulatory factor governing the differentiation of the cells into cardiac pacemaking cells. The results showed that silencing of Tbx3 decreased the pacemaking activity and led to down-regulation of pacemaker genes in the induced cells. These results confirmed that Nkx2.5(+) CPCs differentiated into cardiac pacemaking cells after being treated with ET-1 and suggested that an ET-1-Tbx3 molecular pathway govern/mediate this process. In conclusion, our study support the notion that pacemaking cells originate from Nkx2.5(+) CPCs present in embryonic heart tubes and endothelin-1 might be involved in diversification of cardiomyogenic progenitors toward the cells.

Thermosensitive injectable decellularized nucleus pulposus hydrogel as an ideal biomaterial for nucleus pulposus regeneration.

Extracellular matrix loss is one of the early manifestations of intervertebral disc degeneration. Stem cell-based tissue engineering creates an appropriate microenvironment for long term cell survival, promising for NP regeneration. We created a decellularized nucleus pulposus hydrogel (DNPH) from fresh bovine nucleus pulposus. Decellularization removed NP cells effectively, while highly preserving their structures and major biochemical components, such as glycosaminoglycan and collagen II. DNPH could be gelled as a uniform grid structure in situ at 37°C for 30 min. Adding adipose marrow-derived mesenchymal stem cells into the hydrogel for three-dimensional culture resulted in good bioactivity and biocompatibility in vitro. Meanwhile, NP-related gene expression significantly increased without the addition of exogenous biological factors. In summary, the thermosensitive and injectable hydrogel, which has low toxicity and inducible differentiation, could serve as a bio-scaffold, bio-carrier, and three-dimensional culture system. Therefore, DNPH has an outstanding potential for intervertebral disc regeneration.

Isolation, culture and characterization of cardiac progenitor cells derived from human embryonic heart tubes.

A variety of studies have reported on the isolation and expansion of cardiac stem cells from adult hearts. However, there is little information concerning cardiac stem/progenitor cells derived from embryonic hearts/heart tubes. To provide more evidence for embryonic heart-derived stem/progenitor cells, Nkx2.5+ human cardiac progenitorcells (hCPCs) were isolated and cloned from human heart tubes. The cells stained positive for Nkx2.5 and Oct-4, and negative for alpha-smooth muscle actin (alpha-SMA), cytokeratin, factor-VIII, alpha-sarcomeric actin and c-Kit. GATA-4 expression of Nkx2.5+ hCPCs was higher than that of embryonic limb bud mesenchymal cells of the control group (p < 0.05). These cells were passaged continuously for >3 months (23 passages) and proliferated actively in vitro. After being treated with 5-azacytidine, Nkx2.5+ hCPCs underwent cardiomyogenic differentiation. Ultrastructural observation confirmed that the longitudinal section of these cardiomyogenic differentiation cells clearly revealed typical sarcomeres and intercalated discs. alpha-MHC, alpha-sarcomeric actin and GATA-4 levels were increased in Nkx2.5+ hCPCs treated with 5-azacytidine compared to untreated cells. Nkx2.5+ hCPCs exhibited positive staining and had a higher expression for alpha-SMA when cocultured with canine vascular endothelial cells. After Nkx2.5+ hCPCs were treated with endothelin-1, all cells displayed spontaneous electrical activity and spontaneous beating. Connexin-40 and -45 were stained positive in the treated cells. In conclusion, Nkx2.5+ hCPCs derived from heart tubes have been isolated and cloned in vitro. These cells are capable of long-term self-renewal and possess a potential to differentiate into cardiac muscle-like cells, cardiac pacemaking cells and smooth muscle-like cells. They could have a significant impact on cardiac regeneration medicine and developmental biology.

High-cholesterol diet augments endothelial dysfunction via elevated oxidative stress and reduced tetrahydrobiopterin in Ins2(Akita) mice, an autosomal dominant mutant type 1 diabetic model.

1. Oxidative stress contributes to endothelial dysfunction and atherogenesis in diabetes. The present study tested the hypothesis that a high-cholesterol diet accelerates endothelial dysfunction in Ins2(Akita) mice, a Type 1 diabetic model with a spontaneous autosomal preproinsulin gene (Ins2 gene) mutation, through further increase of superoxide production. 2. The Ins2(Akita) diabetic mice were fed a high-cholesterol diet (1.25% cholesterol) for 4 months. Some Ins2(Akita) mice were also treated for 4 months with the selective NADPH oxidase inhibitor apocynin (4 mg/kg per day in drinking water). Oxidative stress markers, tetrahydrobiopterin (BH4) levels, GTP cyclohydrolase I activity and endothelial function were determined in serum or arteries afterwards. 3. Serum lipid peroxidation and arterial superoxide levels were increased, whereas arterial BH(4) levels and GTP cyclohydrolase I activity were decreased, in Ins2(Akita) mice on a high-cholesterol diet, resulting in impaired endothelium-dependent nitric oxide-mediated relaxation in response to acetylcholine. 4. In vivo treatment with apocynin not only blunted serum lipid peroxidation and arterial superoxide levels, but also increased BH4 levels and GTP cyclohydrolase I activity, resulting in improved endothelium-dependent relaxation. 5. These results suggest that NADPH oxidase may play a potential role in oxidative stress-induced arterial BH4 and GTP cyclohydrolase I deficiency, resulting in endothelial dysfunction in Ins2(Akita) Type 1 diabetic mice fed a high-cholesterol diet.

Increased superoxide contributes to enhancement of vascular contraction in Ins2(Akita) diabetic mice, an autosomal dominant mutant model.

Superoxide has been reported to be involved in vascular dysfunction in diabetes. The Ins2(Akita) mouse is an autosomal dominant mutant diabetic model that can serve as an excellent substitute for the Type 1 diabetic mouse model induced by chemical diabetogens. The purpose of the present study was to investigate the role of superoxide on vascular dysfunction using this new diabetic model. Compared with age-matched normal C57BL/6 mice, in Ins2(Akita) diabetic mice arterial superoxide, lipid peroxidation production (1.2 +/- 0.1 vs 17.4 +/- 1.9 mmol/mg tissue, respectively; P < 0.01) and plasma lipid peroxidation production (0.08 +/- 0.02 vs 0.40 +/- 0.03 mmol/L, respectively; P < 0.01) were increased. Meanwhile, expression of vascular adhesion molecule-1, E-selectin and monocyte chemoattractant protein-1 in the aorta and/or plasma was elevated. The contraction of carotid arteries to U46619 in Ins2(Akita) diabetic mice was significantly enhanced compared with control mice (P < 0.05). Tempol (a scavenger of superoxide), apocynin (an inhibitor of NADPH oxidase) and allopurinol (an inhibitor of xanthine oxidase) all not only decreased superoxide in carotid arteries, but also suppressed arterial contractions to U46619 in Ins2(Akita) diabetic mice. Indomethacin, an inhibitor of cyclo-oxygenase, and chelerythrine, an inhibitor of protein kinase C, also suppressed the enhanced vascular contraction. These results suggest that increased arterial superoxide generated from diverse sources may potentiate the contractions of carotid arteries in Ins2(Akita) diabetic mice.

Comparison of mouse brown and white adipose­derived stem cell differentiation into pacemaker­like cells induced by TBX18 transduction.

The present study aimed to compare brown adipose-derived stem cell (BASC) and white adipose-derived stem cell (WASC) differentiation into pacemaker­like cells following T­box (TBX)18 transduction. Mouse BASCs and WASCs were induced to differentiate into pacemaker­like cells by adenovirus­TBX18 transduction in vitro. The transduction rate was determined by fluorescence microscopy and cell ultrastructural changes were observed by transmission electron microscopy at 48 h post­transduction. The mRNA and protein expression of pacemaker cell­associated markers, including TBX18, TBX3, sarcomeric α­actinin (Sr) and hyperpolarization­activated cyclic nucleotide­gated channel 4 (HCN4), were detected by reverse transcription­quantitative polymerase chain reaction, immunofluorescence staining and western blot analysis. The results demonstrated that no significant difference was observed in the transduction rate between BASCs and WASCs. The ultrastructure of BASCs was observed to be more complex than that of WASCs, indicating that BASCs may possess a better structural foundation to differentiate into pacemaker­like cells. TBX18, TBX3, Sr and HCN4 mRNA and protein expression in differentiated stem cells was significantly increased compared with the respective control groups. Furthermore, the expression levels were significantly higher in TBX18­BASCs compared with TBX18­WASCs. In conclusion, TBX18 gene transduction may facilitate the differentiation of BASCs and WASCs into pacemaker­like myocardial cells, and BASCs may have a higher capacity than WASCs for this differentiation. TBX18 gene may therefore act as an efficient candidate in cell transplantation therapy for diseases and for future research into the cardiovascular system.

Carbon monoxide as a promising molecule to promote nerve regeneration after traumatic brain injury.

Carbon monoxide (CO) is known as a toxic gas. Although there have been many studies on both toxic and protective effects of CO, most of these studies lack novelty, except for Eng H Lo team's study on the therapeutic effect of CO on brain injuries. In this commentary, we summarize the potential application value of CO in the treatment of some clinical diseases, especially its protective effect and nerve regeneration in brain injuries, hoping that our interest in CO could promote related clinical application studies.

Functional analysis in vivo of engineered valved venous conduit with decellularized matrix and two bone marrow-derived progenitors in sheep.

Tissue engineering has been considered a promising approach for creating grafts to replace autologous venous valves. Here, ovine bone marrow-derived endothelial progenitor cells (EPCs) and multipotent adult progenitor cells (MAPCs) were harvested and then loaded into decellularized venous matrix to create tissue-engineered (TE) valved vein. Subsequently, the ovine femoral veins containing the valve were removed and replaced by TE grafts or acellular matrix only. The morphology and function were analysed for up to 1 year by ultrasonography, angiography, H&E staining and scanning electron microscopy (SEM). The differentiation of seeded cells was traced immunofluorochemically. The results showed that decellularized venous matrix could initially and feebly attract endogenous cells, but failed afterwards and were insufficient to restore valve function. On the contrary, the seeded cells differentiated into endothelial cells (ECs) in vivo and formed a monolayer endothelium, and smooth muscle cells within the scaffold therefore produced TE grafts comparable to the native vein valve. This TE graft remained patent and sufficient after implantation into the venous circuit of the ovine lower extremity for at least 6 months. Unfortunately, cells seeded on the luminal surface and both sides of the leaflets lost their biological functions at 12 months, resulting in thrombosis formation and leading to complete occlusion of the TE grafts and impotent venous valves. These findings suggest that this TE valved venous conduit can function physiologically in vivo in the medium term. Before translating this TE venous valve into clinical practice, the durability should be improved and thrombogenicity should be suppressed. Copyright © 2016 John Wiley & Sons, Ltd.

Biocompatibility evaluation of tissue-engineered valved venous conduit by reseeding autologous bone marrow-derived endothelial progenitor cells and multipotent adult progenitor cells into heterogeneous decellularized venous matrix.

Clinical treatment of chronic deep venous insufficiency remains difficult despite the availability of various therapies. Previous experimental efforts have demonstrated that the tissue-engineered valvedvenous conduit (TEVV) is a promising option to replace the damaged venous valve. The aim of the present study was to evaluate the TEVV by reseeding bone marrow-derived endothelial progenitor cells and multipotent adult progenitor cells into acellular matrix according to International Standard ISO10993, and to clarify their interactions with blood, the local effect after implantation both in vitro and vivo, and immunogenicity. The results showed that the 2-cm long TEVV did not cause haemolysis in vitro and remained patent without thrombosis formation in vivo. However, the luminal surface of TEVV was partially covered by multilayer cells. Compared with the native ovine femoral vein segment, the TEVV beneath the mouse skin produced significant mononuclear cell infiltration, with serum interleukin-6 and tumour necrosis factor-α similar to normal. The TEVV maintained its structural integrity, while the native ovine femoral vein segments fell apart at postoperative week nine. The TEVV implantation did not change serum immunoglobulin G. In addition, the seeds and extracts of the scaffold did not affect the proliferation of mouse lymphocytes. These findings suggest that the histocompatibility, haemocompatibility and immunogenicity of this TEVV are acceptable owing to complete removal of the cellular components of autologous seeds and residues of chemical regents, thus providing an experimental basis for further clinical translation. Copyright © 2014 John Wiley & Sons, Ltd.

Gene transfer of human guanosine 5'-triphosphate cyclohydrolase I restores vascular tetrahydrobiopterin level and endothelial function in low renin hypertension.

BACKGROUND: We recently reported that arterial superoxide (O2-) is augmented by increased endothelin-1 (ET-1) in deoxycorticosterone acetate (DOCA)-salt hypertension, a model of low renin hypertension. Tetrahydrobiopterin (BH4), a potent reducing molecule with antioxidant properties and an essential cofactor for endothelial nitric oxide synthase, protects against O2--induced vascular dysfunction. However, the interaction between O2- and BH4 on endothelial function and the underlying mechanisms are unknown. METHODS AND RESULTS: The present study tested the hypothesis that BH4 deficiency due to ET-1-induced O2- leads to impaired endothelium-dependent relaxation and that gene transfer of human guanosine 5'-triphosphate (GTP) cyclohydrolase I (GTPCH I), the first and rate-limiting enzyme for BH4 biosynthesis, reverses such deficiency and endothelial dysfunction in carotid arteries of DOCA-salt rats. There were significantly increased arterial O2- levels and decreased GTPCH I activity and BH4 levels in DOCA-salt compared with sham rats. Treatment of arteries of DOCA-salt rats with the selective ETA receptor antagonist ABT-627, NADPH oxidase inhibitor apocynin, or superoxide dismutase (SOD) mimetic tempol abolished O2- and restored BH4 levels. Basal arterial NO release and endothelium-dependent relaxations were impaired in DOCA-salt rats, conditions that were improved by apocynin or tempol treatment. Gene transfer of GTPCH I restored arterial GTPCH I activity and BH4 levels, resulting in reduced O2- and improved endothelium-dependent relaxation and basal NO release in DOCA-salt rats. CONCLUSIONS: These results indicate that a BH4 deficiency resulting from ET-1-induced O2- via an ETA/NADPH oxidase pathway leads to endothelial dysfunction, and gene transfer of GTPCH I reverses the BH4 deficiency and endothelial dysfunction by reducing O2- in low renin mineralocorticoid hypertension.

Preserved postischemic heart function in sucrose-fed type 2 diabetic OLETF rats.

Cardiovascular disease is one of the most important causes of morbidity and mortality in diabetes mellitus, but there has been controversy over functional impairment of diabetic hearts and their tolerance to ischemia. We studied ischemic heart function in type 2 diabetic rats with different degrees of hyperglycemia and its relationship with cardiac norepinephrine release. Otsuka Long-Evans Tokushima Fatty rats (OLETF) and age-matched Long-Evans Tokushima Otsuka normal rats (LETO) were used. One group of OLETF rats was given 30% sucrose in drinking water (OLETF-S). Hearts were isolated and perfused in a working heart preparation and subjected to 30 min ischemia followed by 40 min reperfusion at age of 12 months. Hemodynamics and coronary norepinephrine overflow were examined. Fasting plasma glucose in OLETF increased markedly at 12 months and sucrose administration exacerbated hyperglycemia in diabetic rats (LETO 6.6 +/- 0.5, OLETF 8.3 +/- 0.7, OLETF-S 15.0 +/- 1.7 mmol/L, P < 0.01). Basic cardiac output in OLETF was decreased as compared with LETO and OLETF-S (LETO 29.4 +/- 2.5, OLETF 24.0 +/- 2.4, OLETF-S 27.0 +/- 0.9 ml/min/g, P < 0.05) and remained very low after ischemia, while in OLETF-S it was well preserved (OLETF 4.2 +/- 2.1, OLETF-S 13.7 +/- 2.6 ml/min/g, P < 0.01). Correspondently, cardiac norepinephrine released during ischemia and reperfusion was lower in OLETF-S (OLETF 2.3 +/- 1.0, OLETF-S 0.7 +/- 0.1 pmol/ml, P < 0.01). Thus, OLETF hearts were more vulnerable to ischemia but sucrose feeding rendered their hearts resistant to ischemia. Less norepinephrine release may play a role in preventing postischemic functional deterioration in sucrose-fed diabetic hearts.

Effects of long-term treatment with ketanserin on blood pressure variability and end-organ damage in spontaneously hypertensive rats.

It has been proposed that instability of blood pressure may produce organ damage. Ketanserin is an anti-hypertensive drug with an ability to reduce blood pressure variability (BPV) in acute experiments in spontaneously hypertensive rats (SHRs). The present work was designed to observe the effects of long-term treatment with ketanserin on BPV and end-organ damage in SHRs. Ketanserin was mixed in rat chow at an estimated dose of 10 mg/kg/d. After 5 months of drug administration, BP was continuously recorded in conscious, freely moving rats for 24 h. The heart, kidneys, and abdominal aorta were then isolated and examined by using histologic methods and computer image analysis. In another work, the effects of hydralazine (40 mg/kg/d, for 5 months) on BP, BPV, and organ damage were observed in SHRs. Ketanserin significantly decreased BP and BPV, ameliorated impaired arterial baroreflex function, and significantly prevented the target organs of SHRs from being damaged. This preventive effect was characterized by decrease in left ventricular hypertrophy, diminution of glomerulus damage, and amelioration in vascular lesion. Hydralazine decreased BP but did not lower BPV. No organ protection was found in hydralazine-treated rats. In conclusion, long-term treatment with ketanserin reduced hypertensive organ damage. Lowering BP, decreasing BPV, and ameliorating arterial baroreflex function may contribute together to this effect.