Synergistic effect of adipose-derived stem cell therapy and bone marrow progenitor recruitment in ischemic heart

Human multipotent adipose-derived stem cells (hMADSCs) have recently been isolated featuring extensive expansion capacity ex vivo. We tested the hypothesis that hMADSC transplantation might contribute to cardiac functional recovery by its direct or indirect effect on myocardial infarction (MI). Nude rats were either transplanted with hMADSCs or PBS (control) in ischemic myocardium immediately following MI. Echocardiographical assessment of cardiac function after MI with hMADSCs showed significant improvement of each parameter compared to that with PBS. Histological analysis also showed significantly reduced infarct size and increased capillary density in peri-infarct myocardium by hMADSC treatment. However, remarkable transdifferentiation of hMADSCs into cardiac or vascular lineage cells was not observed. Despite the less transdifferentiation capacity, hMADSCs produced robust multiple pro-angiogenic growth factors and chemokines, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and stromal cell-derived factor-1α (SDF-1α). Specifically, hMADSC-derived SDF-1α had a crucial role for cooperative angiogenesis, with the paracrine effect of hMADSCs and Tie2-positive bone marrow (BM) progenitor recruitment in ischemic myocardium. hMADSCs exhibit a therapeutic effect on cardiac preservation following MI, with the production of VEGF, bFGF, and SDF-1α showing paracrine effects and endogenous BM stem/progenitor recruitment to ischemic myocardium rather than its direct contribution to tissue regeneration.     

Conformational alterations in unidirectional ion transport of a light-driven chloride pump revealed using X-ray free electron lasers

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10-µs and 1-ms time points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied by a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3.

Microbial ion-pumping rhodopsins are integral membrane proteins that actively transport ions across membranes upon light stimulation (1). Bacteriorhodopsin (bR) and halorhodopsin (HR) are well-known microbial ion-pumping rhodopsins found in halophilic archaea (2, 3). bR is a light-driven outward proton pump and HR is a light-driven inward anion pump, specific for chloride ion. Microbial ion-pumping rhodopsins possess common structural features consisting of seven α-helices with an all-trans retinal covalently bound to a lysine residue as the chromophore, despite the transport of different ions (4). The retinal undergoes photoisomerization from the all-trans to 13-cis configuration, which initiates the photocycle accompanied by several intermediates to export ions (4, 5). Its light-controllable function is suitable for optogenetics applications for manipulating cells, such as neurons, by changing the ion concentration inside or outside the membrane (6, 7). In fact, microbial rhodopsins, including channelrhodopsins and HRs, are employed as optogenetic tools (8–10).Nonlabens marinus rhodopsin-3 (NM-R3) is a light-driven chloride pump recently discovered in a marine flavobacterium (11). It is a distinct chloride pump from HRs and shows low amino acid sequence homology with HRs (11). To date, HR-type chloride pumps have been found in haloarchaea, marine bacteria, and cyanobacteria, including Halobacterium salinarum, Natronomonas pharaonis, and Mastigocladopsins repens, with sequence identities of 20%, 21%, and 20% to NM-R3, respectively (3, 12–15). Interestingly, NM-R3 has higher sequence identity (36%) to Krokinobacter rhodopsin 2 (KR2), a sodium pump found in Krokinobacter eikastus (16). NM-R3 possesses a unique NTQ motif (Asn98, Thr102, Gln109) in the third helix (helix C), which corresponds to key residues (DTD motif, Asp85, Thr89, Asp96) for proton transport in bR (11, 17, 18) (SI Appendix, Table S1). Asp85 acts as the primary proton acceptor of bR from the protonated Schiff-base (PSB), with assistance from Thr89 and Asp96, which is the proton donor (5, 17, 18). HRs from haloarchaea have a highly conserved TSA (Thr, Ser, Ala) motif, while the Ala residue is replaced by Asp in HR from cyanobacteria (19). In the X-ray crystal structure of NM-R3 (SI Appendix, Fig. S1A), a chloride ion located between the PSB and Asn98 (SI Appendix, Fig. S1B) is stabilized by the positive charge of the PSB (20). The position of this chloride ion is similar to those in the H. salinarum HR and N. pharaonis HR (NpHR) structures except for Thr and Ser, which correspond to Asn98 and Thr102 in NM-R3, respectively (20–22). Several amino acid residues around the retinal, including Arg95, Trp99, Trp201, and Asp231, are highly conserved among ion-pumping rhodopsins. Previous spectroscopic studies suggested that NM-R3 displays a similar sequence of intermediates, with K-, L-, N-, and O-like species, as in other HRs (23) (Fig. 1A). Recently, intermediate structures of NM-R3 obtained by low-temperature trapping X-ray crystallography and serial femtosecond crystallography (SFX) have been reported (24, 25). However, the detailed ion-pump mechanism still remains unclear, due to the lack of dynamic structures of anion transport at atomic resolution.Open in a separate windowFig. 1.TR-visible absorption spectroscopy for microcrystals. (A) Photocycle model of NM-R3 in the 1 M NaCl buffer solution (23). (B) TR difference spectra ΔA upon the 532-nm excitation. The difference was calculated by subtracting the spectrum of NM-R3. (C) Global fitting analysis with two exponentials. The A₁ and A₂ amplitude spectra correspond to the differences of [ΔA_O – ΔA_{10 µs}] and [ΔA_{200 ms} − ΔA_O], respectively. Here, ΔA_O represents the difference spectrum of the O intermediate minus NM-R3. (D) The isomeric forms of the retinal chromophore in bacterial-type rhodopsins.Time-resolved serial femtosecond crystallography (TR-SFX) is a powerful tool for visualizing reactions and motions in proteins at the atomic level (26–28). In SFX, myriads of microcrystals are continuously injected by a sample injector into an irradiation point of X-ray free electron lasers (XFELs) at room temperature, thus providing diffraction patterns before the onset of radiation damage by the intense X-ray pulse. Combined with a visible-light pump laser for reaction initiation, TR-SFX has been applied to light-driven ion pumps to observe the structural dynamics during the ion transfer. While TR-SFX has revealed femto-to-millisecond structural dynamics in light-driven cation pumps, including bR and KR2 (29–31), TR-SFX studies of anion pumps have been limited to early-stage structures adopted at picoseconds after light illumination (32). In addition, although NM-R3 pumps a chloride ion (Cl⁻) as a physiological substrate, it can also transport bromide (Br⁻), iodide (I⁻), and other anions from the extracellular side to the cytoplasmic side (23). I⁻ or Br⁻ serves as a marker for tracking the positions of ions, due to the greater number of electrons, whereas Cl⁻ is less distinguishable in X-ray crystallography. Therefore, TR-SFX experiments using I⁻ or Br⁻ are expected to directly visualize the process of ion transport.Here, we report the conformational alterations in NM-R3 during Br⁻ or I⁻ pumping, obtained by both TR-SFX and time-resolved spectroscopy of crystals. The resulting sequence of movements in NM-R3 demonstrates how the chloride pump transports anions with a large ionic radius and prevents the backflow of anions from the cytoplasmic side.     

A novel microsatellite polymorphism in the human OB gene: a highly polymorphic marker for linkage analysis

Summary The mouse ob gene and its human homologue OB have recently been cloned. The mutations in the ob gene are known to be associated with extreme obesity. The relationship between the human OB gene and disease, however, is largely unknown due to the lack of suitable markers within or adjacent to the OB gene. To obtain informative markers, we searched for simple tandem repeat polymorphisms in the genomic sequence of the human OB gene and identified a novel tetranucleotide repeat in the 3′ flanking region. Fifteen alleles were detected in this marker with a heterozygosity of 0.85 and polymorphism information content of 0.83, indicating a highly informative nature of this marker. Two-point linkage mapping in two Centre Etude Polymorphisme Humaine (CEPH) reference families suggested that this marker is located in the interval between D7S514 and D7S530, the same interval where the OB gene is located (recombination fractions with D7S514 and D7S530 were 0.026 and 0.034, respectively). Although allele frequency distributions of this marker did not differ between 84 control subjects and 69 NIDDM patients, there was a tendency to higher body weight in control subjects with class I/class I genotype than in those without this genotype (68.8 ± 11.1 vs 60.8 ± 10.3 kg, p = 0.05). The highly polymorphic nature of this marker and its location in the OB gene makes this marker useful for linkage studies of the OB gene with a number of phenotypes, such as obesity, non-insulin-dependent diabetes mellitus, hypertension and the insulin resistance syndrome. [Diabetologia (1996) 36: 1398–1401] Received: 9 July 1996 and in revised form: 7 August 1996     

Downregulation of leptin by free fatty acids in rat adipocytes: effects of triacsin C, palmitate, and 2-bromopalmitate

Free fatty acid (FFA) has been reported to decrease leptin mRNA levels in 3T3-L1 adipocytes. When using this cell line, it is difficult to determine the protein levels because a very small amount of leptin is secreted into the medium. The effect of FFA on leptin secretion from adipocytes has not yet been determined. In addition, in vivo studies have failed to demonstrate a FFA-induced decrease in plasma leptin levels. To clarify the effect of FFA on leptin production, we investigated the leptin protein level in the medium and the mRNA level in primary cultured rat adipocytes treated with triacsin C, which is a potent inhibitor of acyl-coenzyme A (CoA) synthetase, palmitate, and 2-bromopalmitate. Triacsin C (0 to 5 x 10(-5) mol/L) decreased leptin concentrations in the culture medium in a dose-dependent manner. Leptin mRNA levels were decreased to 10% of the control in the presence of triacsin C. The concentration of triacsin C needed to suppress leptin production was similar to the Ki value (approximately 10(-5) mol/L) for inhibition of acyl-CoA synthetase. Both palmitate and 2-bromopalmitate decreased leptin concentra-tions but did not affect the triacsin C-induced decrease in leptin additively. In conclusion, both protein and mRNA levels of leptin were decreased by triacsin C and FFA in primary cultured rat adipocytes. Our findings suggest that FFA is involved in the regulation of leptin production in adipocytes.     

Prevalence of and risk factors for low bone mineral density in Japanese female patients with systemic lupus erythematosus

To examine the prevalence of and risk factors for low bone mineral density (BMD) (osteoporosis or osteopenia) in Japanese female patients with systemic lupus erythematosus (SLE). We performed BMD measurements by dual X-ray absorptiometry at the lumbar spine and the hip and collected basic and lifestyle-related, clinical and treatment characteristics among 58 SLE patients. Odds ratios (ORs) and their 95% confidence intervals (CIs) were assessed for associations between low BMD and selected factors among SLE patients. The mean BMD?±?SD was 0.90?±?0.17?g/cm² at the lumbar spine and 0.76?±?0.17?g/cm² at the hip. The prevalence of osteopenia (2.5 SD?<?T score?<?1 SD) was 50.0% and that of osteoporosis (T score?<?2.5 SD) was 13.8% in our SLE patients. After adjustment for age and disease duration, we found the number of deliveries (OR?=?5.58, 95% CI?=?1.31?C26.06; P?=?0.02) to be a risk factor for overall low BMD (T score?<?1 SD) and a maximal dosage of >50?mg/day of oral corticosteroids (OR?=?0.25, 95% CI?=?0.07?C0.91; P?=?0.035) as a preventive factor for low BMD at the lumbar spine. Reduced BMD, especially in spinal trabecular bone, was pronounced in Japanese female patients with SLE, particular in those with a history of delivery. A history of high-dose oral corticosteroids was associated with the preservation of BMD at the lumbar spine, however, further study is needed considering the limited sample size.     

Alpha‐actinin‐4 (ACTN4) gene amplification is a predictive biomarker for adjuvant chemotherapy with tegafur/uracil in stage I lung adenocarcinomas

Although adjuvant tegafur/uracil (UFT) is recommended for patients with completely resected stage I non‐small‐cell lung cancer (NSCLC) in Japan, only one‐third of cases has received adjuvant chemotherapy (ADJ) according to real‐world data. Therefore, robust predictive biomarkers for selecting ADJ or observation (OBS) without ADJ are needed. Patients who underwent complete resection of stage I lung adenocarcinoma with or without adjuvant UFT were enrolled. The status of ACTN4 gene amplification was analyzed by FISH. Statistical analyses to determine whether the status of ACTN4 gene amplification affected recurrence‐free survival (RFS) were carried out. Formalin‐fixed, paraffin‐embedded samples from 1136 lung adenocarcinomas were submitted for analysis of ACTN4 gene amplification. Ninety‐nine (8.9%) of 1114 cases were positive for ACTN4 gene amplification. In the subgroup analysis of patients aged 65 years or older, the ADJ group had better RFS than the OBS group in the ACTN4‐positive cohort (hazard ratio [HR], 0.084, 95% confidence interval [CI], 0.009‐0.806; P = .032). The difference in RFS between the ADJ group and the OBS group was not significant in ACTN4‐negative cases (all ages: HR, 1.214; 95% CI, 0.848‐1.738; P = .289). Analyses of ACTN4 gene amplification contributed to the decision regarding postoperative ADJ for stage I lung adenocarcinomas, preventing recurrence, improving the quality of medical care, preventing the unnecessary side‐effects of ADJ, and saving medical costs.     

Metabolic cardiovascular disease risk factors and their clustering in subclinical hypothyroidism

Objective A possible association between subclinical hypothyroidism and cardiovascular disease (CVD) has been reported. Monitoring of atomic‐bomb survivors for late effects of radiation exposure at the Radiation Effects Research Foundation has provided the opportunity to examine associations between subclinical hypothyroidism and metabolic CVD risk factors. The objective of the study was to evaluate associations between subclinical hypothyroidism and metabolic CVD risk factors, and a cluster of these factors. Design and participants This was a cross‐sectional study of 3549 subjects (mean age 70 years; 1221 men and 2328 women) between 2000 and 2003 comprising 306 subjects with subclinical hypothyroidism and 3243 control euthyroid subjects in Japan. Measurements We investigated associations between subclinical hypothyroidism and metabolic CVD risk factors such as hypertension, diabetes mellitus, dyslipidaemia and hyperuricaemia, and a cluster of these factors. Results Subclinical hypothyroidism was not significantly associated with either hypertension, diabetes mellitus or hyperuricaemia defined by taking into account the use of medications in both men and women, but in men it was associated with dyslipidaemia (P = 0·02). We observed a significantly increased odds ratio (OR) for the presence of three or more metabolic CVD risk factors in men with subclinical hypothyroidism after adjusting for age, body mass index (BMI), and smoking status [OR: 1·83, 95% confidence interval (CI): 1·13–2·94, P = 0·01]. The significant associations remained after an additional adjustment for atomic‐bomb radiation dose. Conclusions There appears to be a significant increase in a cluster of metabolic CVD risk factors among people with subclinical hypothyroidism.     

Apoptosis, cell proliferation and modulation of cyclin-dependent kinase inhibitor p21cip1 in vascular remodelling during vein arterialization in the rat

Neo-intima development and atherosclerosis limit long-term vein graft use for revascularization of ischaemic tissues. Using a rat model, which is technically less challenging than smaller rodents, we provide evidence that the temporal morphological, cellular, and key molecular events during vein arterialization resemble the human vein graft adaptation. Right jugular vein was surgically connected to carotid artery and observed up to 90 days. Morphometry demonstrated gradual thickening of the medial layer and important formation of neo-intima with deposition of smooth muscle cells (SMC) in the subendothelial layer from day 7 onwards. Transmission electron microscopy showed that SMCs switch from the contractile to synthetic phenotype on day 3 and new elastic lamellae formation occurs from day 7 onwards. Apoptosis markedly increased on day 1, while α-actin immunostaining for SMC almost disappeared by day 3. On day 7, cell proliferation reached the highest level and cellular density gradually increased until day 90. The relative magnitude of cellular changes was higher in the intima vs . the media layer (100 vs . 2 times respectively). Cyclin-dependent kinase inhibitors (CDKIs) p27^Kip1 and p16^INKA remained unchanged, whereas p21^Cip1 was gradually downregulated, reaching the lowest levels by day 7 until day 90. Taken together, these data indicate for the first time that p21^Cip1 is the main CDKI protein modulated during the arterialization process the rat model of vein arterialization that may be useful to identify and validate new targets and interventions to improve the long-term patency of vein grafts.