Development of multifunctional pyrrole-imidazole polyamides that increase hepatocyte growth factor and suppress transforming growth factor-ß1.

PURPOSE: The DNA recognition peptide compounds pyrrole-imidazole (PI) polyamides bind to the minor groove and can block the binding of transcription factors to target sequences. To develop more PI polyamides as potential treatments for fibrotic diseases, including chronic renal failure, we developed multifunctional PI polyamides that increase hepatocyte growth factor (HGF) and decrease transforming growth factor (TGF)-ß1. METHODS: We designed seven PI polyamides (HGF-1 to HGF-7) that bind to the chicken ovalbumin upstream promoter transcription factor-1 (COUP-TF1) binding site of the HGF promoter sequence. We selected PI polyamides that increase HGF and suppress TGF-ß1 in human dermal fibroblasts (HDFs). FINDINGS: Gel shift assays showed that HGF-2 and HGF-4 bound the appropriate dsDNAs. HGF-2 and HGF-4 significantly inhibited the TGF-ß1 mRNA expression in HDFs stimulated by phorbol 12-myristate 13-acetate. HGF-2 and HGF-4 significantly inhibited the TGF-ß1 protein expression in HDFs with siRNA targeting HGF, indicating that HGF-2 and HGF-4 directly inhibited the expression of TGF-ß1. CONCLUSION: The designed and synthetic HGF PI polyamides targeting the HGF promoter, which increased the expression of HGF and suppressed the expression of TGF-ß, will be a potential practical medicine for fibrotic diseases, including progressive renal diseases.

Syntheses of 25-Adamantyl-25-alkyl-2-methylidene-1α,25-dihydroxyvitamin D3 Derivatives with Structure-Function Studies of Antagonistic and Agonistic Active Vitamin D Analogs.

The active form of vitamin D3, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], is a major regulator of calcium homeostasis through activation of the vitamin D receptor (VDR). We have previously synthesized vitamin D derivatives with large adamantane (AD) rings at position 24, 25, or 26 of the side chain to study VDR agonist and/or antagonist properties. One of them-ADTK1, with an AD ring and 23,24-triple bond-shows a high VDR affinity and cell-selective VDR activity. In this study, we synthesized novel vitamin D derivatives (ADKM1-6) with an alkyl group substituted at position 25 of ADTK1 to develop more cell-selective VDR ligands. ADKM2, ADKM4, and ADKM6 had VDR transcriptional activity comparable to 1,25(OH)2D3 and ADTK1, although their VDR affinities were weaker. Interestingly, ADKM2 has selective VDR activity in kidney- and skin-derived cells-a unique phenotype that differs from ADTK1. Furthermore, ADKM2, ADKM4, and ADKM6 induced osteoblast differentiation in human dedifferentiated fat cells more effectively than ADTK1. The development of vitamin D derivatives with bulky modifications such as AD at position 24, 25, or 26 of the side chain is useful for increased stability and tissue selectivity in VDR-targeting therapy.

Intravenous Semaphorin 3A Administration Maintains Cardiac Contractility and Improves Electrical Remodeling in a Mouse Model of Isoproterenol-Induced Heart Failure.

The effects of recombinant semaphorin 3A (Sema3A) on myocardial contractility and electrical remodeling in mice with isoproterenol (ISP) -induced heart failure were investigated.C57BL/6J mice intraperitoneally received ISP (480 mg/kg/day, ISP group; n = 24) or saline (control group; n = 31) for 14 days. Twenty-one ISP-treated mice received 0.5 mg/kg Sema3A intravenously on days 7 and 11 (ISP+Sema3A group). The sympathetic nervous system was activated upon ISP treatment, but was reduced upon Sema3A administration. Greater myocardial tissue fibrosis was observed in the ISP group than in the control group. However, fibrosis was not significantly different between the ISP+Sema3A and control groups. Fractional shortening of the left ventricle was lower in the ISP group than in the control group and was restored in the ISP+Sema3A group (control, 53 ± 8%; ISP, 37 ± 7%; ISP+Sema3A, 48 ± 3%; P < 0.05). Monophasic action potential duration at 20% repolarization (MAPD20) was prolonged in the ISP group (compared to control group), but this was reversed upon Sema3A administration (control, 29 ± 3 ms; ISP, 35 ± 6 ms; ISP+Sema3A, 29 ± 3 ms; P < 0.05). qPCR revealed Kv4.3, KChIP2, and SERCA2 downregulation in the ISP group and upregulation in the ISP+Sema3A group; however, Western blotting revealed similar changes only for Kv4.3 (P < 0.05).Intravenous Sema3A may maintain myocardial contractility by suppressing the sympathetic innervation of the myocardium and reducing myocardial tissue damage, in addition to restoring MAPD via Kv4.3 upregulation.

Dedifferentiated fat cells administration ameliorates abnormal expressions of fatty acids metabolism-related protein expressions and intestinal tissue damage in experimental necrotizing enterocolitis.

Neonatal necrotizing enterocolitis (NEC) is a serious disease of premature infants that necessitates intensive care and frequently results in life-threatening complications and high mortality. Dedifferentiated fat cells (DFATs) are mesenchymal stem cell-like cells derived from mature adipocytes. DFATs were intraperitoneally administrated to a rat NEC model, and the treatment effect and its mechanism were evaluated. The NEC model was created using rat pups hand fed with artificial milk, exposed to asphyxia and cold stress, and given oral lipopolysaccharides after cesarean section. The pups were sacrificed 96 h after birth for macroscopic histological examination and proteomics analysis. DFATs administration significantly improved the survival rate from 25.0 (vehicle group) to 60.6% (DFAT group) and revealed a significant reduction in macroscopical, histological, and apoptosis evaluation compared with the vehicle group. Additionally, the expression of C-C motif ligand 2 was significantly decreased, and that of interleukin-6 decreased in the DFAT group. DFAT administration ameliorated 93 proteins mainly related to proteins of fatty acid metabolism of the 436 proteins up-/down-regulated by NEC. DFATs improved mortality and restored damaged intestinal tissues in NEC, possibly by improving the abnormal expression of fatty acid-related proteins and reducing inflammation.

Bone marrow-derived dedifferentiated fat cells exhibit similar phenotype as bone marrow mesenchymal stem cells with high osteogenic differentiation and bone regeneration ability.

BACKGROUND: Mesenchymal stem cells (MSCs) are known to have different differentiation potential depending on the tissue of origin. Dedifferentiated fat cells (DFATs) are MSC-like multipotent cells that can be prepared from mature adipocytes by ceiling culture method. It is still unknown whether DFATs derived from adipocytes in different tissue showed different phenotype and functional properties. In the present study, we prepared bone marrow (BM)-derived DFATs (BM-DFATs), BM-MSCs, subcutaneous (SC) adipose tissue-derived DFATs (SC-DFATs), and adipose tissue-derived stem cells (ASCs) from donor-matched tissue samples. Then, we compared their phenotypes and multilineage differentiation potential in vitro. We also evaluated in vivo bone regeneration ability of these cells using a mouse femoral fracture model. METHODS: BM-DFATs, SC-DFATs, BM-MSCs, and ASCs were prepared from tissue samples of knee osteoarthritis patients who received total knee arthroplasty. Cell surface antigens, gene expression profile, and in vitro differentiation capacity of these cells were determined. In vivo bone regenerative ability of these cells was evaluated by micro-computed tomography imaging at 28 days after local injection of the cells with peptide hydrogel (PHG) in the femoral fracture model in severe combined immunodeficiency mice. RESULTS: BM-DFATs were successfully generated at similar efficiency as SC-DFATs. Cell surface antigen and gene expression profiles of BM-DFATs were similar to those of BM-MSCs, whereas these profiles of SC-DFATs were similar to those of ASCs. In vitro differentiation analysis revealed that BM-DFATs and BM-MSCs had higher differentiation tendency toward osteoblasts and lower differentiation tendency toward adipocytes compared to SC-DFATs and ASCs. Transplantation of BM-DFATs and BM-MSCs with PHG enhanced bone mineral density at the injection sites compared to PHG alone in the mouse femoral fracture model. CONCLUSIONS: We showed that phenotypic characteristics of BM-DFATs were similar to those of BM-MSCs. BM-DFATs exhibited higher osteogenic differentiation potential and bone regenerative ability compared to SC-DFATs and ASCs. These results suggest that BM-DFATs may be suitable sources of cell-based therapies for patients with nonunion bone fracture.

Development of gene silencer pyrrole-imidazole polyamides targeting GSK3ß for treatment of polycystic kidney diseases.

The cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB)-glycogen synthase kinase 3ß (GSK3ß) signaling pathway was reported to be involved in the progression of autosomal dominant polycystic kidney diseases (ADPKD). We designed and synthesized pyrrole-imidazole (PI) polyamides as novel gene-silencers to prevent binding of CREB on the GSK3ß gene promoter and examined the effects of the PI polyamides on proliferation and cyst formation of mouse collecting duct M1 cells. The GSK3ß PI polyamides significantly inhibited expression of GSK3ß mRNA in M1 cells with forskolin. To obtain cells as collecting ducts from ADPKD, the PKD1 gene was knocked down by shRNA. Lower concentrations of forskolin significantly stimulated proliferation of PKD1 knock-down M1 cells, whereas GSK3ß PI polyamide significantly inhibited proliferation of PKD1 knock-down M1 cells with forskolin. Stimulation with forskolin for 5 days induced enlargement of cysts from PKD1 knock-down M1 cells. GSK3ß PI polyamides significantly suppressed the enlargement of cysts with forskolin stimulation in PKD1 knock-down M1 cells. Thus, the present study showed that transcriptional suppression of the GSK3ß gene by PI polyamides targeting the binding of CREB inhibited the proliferation and cyst formation of PKD1 knock-down M1 cells. The GSK3ß PI polyamides may potentially be novel medicines for ADPKD.

Effects of dedifferentiated fat cells on neurogenic differentiation and cell proliferation in neuroblastoma cells.

PURPOSE: Mesenchymal stem cells (MSCs) can induce differentiation of neuroblastoma (NB) cells. Properties of dedifferentiated fat cells (DFATs) are similar to those of MSCs. Here, we investigated whether DFATs can induce NB cell differentiation and suppress cell proliferation. METHODS: DFATs were obtained from mature adipocytes isolated from adipose tissue from a ceiling culture. NB cells were cultured in a medium with or without DFATs and, subsequently, cultured in a DFAT-conditioned medium (CM) with or without phosphatidylinositol 3-kinase (PI3K) inhibitor. The neurite lengths were measured, and mRNA expression levels of the neurofilament (NF) and tubulin beta III (TUBß3) were assessed using quantitative real-time RT-PCR. Cell viability was assessed using the WST-1 assay. RESULTS: NB cells cultured with DFATs caused elongation of the neurites and upregulated the expression of NF and Tubß3. NB cells cultured in DFAT-CM demonstrated increased cell viability. NB cells cultured with DFAT-CM and PI3K inhibitors suppressed cell viability. NB cells cultured with DFAT-CM and PI3K inhibitor demonstrated increased neurite length and expression, and upregulation of Tubß3. CONCLUSION: The combined use of DFAT-CM and PI3K inhibitors suppresses the proliferation of NB cells and induces their differentiation. Thus, DFAT may offer new insights into therapeutic approaches in neuroblastoma.

Implantation of dedifferentiated fat cells ameliorated antineutrophil cytoplasmic antibody glomerulonephritis by immunosuppression and increases in tumor necrosis factor-stimulated gene-6.

INTRODUCTION: The implantation of dedifferentiated fat (DFAT) cells has been shown to exert immunosuppressive effects. To develop DFAT cell therapy for antineutrophil cytoplasmic antibody (ANCA) glomerulonephritis, the effects of the implantation of DFAT cells on ANCA glomerulonephritis were investigated in mice. METHODS: PKH26-labeled DFAT cells (105) were infused through the posterior orbital venous plexus to investigate delivery of DFAT cells in ICR mice. DFAT cells (105) were also implanted in SCG mice as a model for ANCA glomerulonephritis. Expression of tumor necrosis factor-stimulated gene-6 (TSG-6) mRNA and protein in kidney was evaluated, and the expression of microRNAs associated with TSG-6 in plasma, lung and kidney was analyzed. Expressions of CD44, prostaglandin (PG) E2, interleukin (IL)-10, IL-1ß, tumor necrosis factor (TNF)-α mRNAs, C-C motif chemokine ligand 17 (CCL-17) and monocyte chemoattractant protein (MCP)-1 proteins were measured in kidney from SCG mice implanted with DFAT cells. RESULTS: After their intravenous infusion, almost all DFAT cells were trapped in the lung and not delivered into the kidney. Implantation of DFAT cells in SCG mice suppressed glomerular crescent formation, decreased urinary protein excretions and increased expression of TSG-6 mRNA, protein and immunostaining in kidney from these mice. Increased expression of microRNA 23b-3p in plasma, kidney and lung; decreased expression of CD44 mRNA; and increased expression of PGE2 and IL-10 mRNAs were also observed in kidney from these mice. Implantation of DFAT cells also decreased the expression of TNF-α and MCP-1 proteins and increased that of CCL-17 protein in kidney from the SCG mice. Survival rates were higher in SCG mice implanted with DFAT cells than in SCG mice without implantation. CONCLUSION: Mechanisms underlying the effects of improvement of ANCA glomerulonephritis are associated with immunosuppressive effects by TSG-6 and the transition of M1-M2 macrophages, suggesting that implantation of DFAT cells may become a cell therapy for ANCA glomerulonephritis.

Transplantation of Mature Adipocyte-Derived Dedifferentiated Fat Cells Facilitates Periodontal Tissue Regeneration of Class II Furcation Defects in Miniature Pigs.

Adipose tissue is composed mostly of adipocytes that are in contact with capillaries. By using a ceiling culture method based on buoyancy, lipid-free fibroblast-like cells, also known as dedifferentiated fat (DFAT) cells, can be separated from mature adipocytes with a large single lipid droplet. DFAT cells can re-establish their active proliferation ability and transdifferentiate into various cell types under appropriate culture conditions. Herein, we sought to compare the regenerative potential of collagen matrix alone (control) with autologous DFAT cell-loaded collagen matrix transplantation in adult miniature pigs (microminipigs; MMPs). We established and transplanted DFAT cells into inflammation-inducing periodontal class II furcation defects. At 12 weeks after cell transplantation, a marked attachment gain was observed based on the clinical parameters of probing depth (PD) and clinical attachment level (CAL). Additionally, micro computed tomography (CT) revealed hard tissue formation in furcation defects of the second premolar. The cemento-enamel junction and alveolar bone crest distance was significantly shorter following transplantation. Moreover, newly formed cellular cementum, well-oriented periodontal ligament-like fibers, and alveolar bone formation were observed via histological analysis. No teratomas were found in the internal organs of recipient MMPs. Taken together, these findings suggest that DFAT cells can safely enhance periodontal tissue regeneration.

Urethral injection of dedifferentiated fat cells ameliorates sphincter damage and voiding dysfunction in a rat model of persistence stress urinary incontinence.

PURPOSE: Dedifferentiated fat (DFAT) cells are mature adipocyte-derived multipotent cells that can be applicable to cell-based therapy for stress urinary incontinence (SUI). This study developed a persistence SUI model that allows long-term evaluation using a combination of vaginal distention (VD) and bilateral ovariectomy (OVX) in rats. Then, the therapeutic effects of DFAT cell transplantation in the persistence SUI model was examined. METHODS: In total, 48 Sprague-Dawley rats were divided into four groups and underwent VD (VD group), bilateral OVX (OVX group), VD and bilateral OVX (VD + OVX group), or sham operation (Control group). At 2, 4, and 6 weeks after injury, leak point pressure (LPP) and histological changes of the urethral sphincter were evaluated. Next, 14 rats undergoing VD and bilateral OVX were divided into two groups and administered urethral injection of DFAT cells (DFAT group) or fibroblasts (Fibroblast group). At 6 weeks after the injection, LPP and histology of the urethral sphincter were evaluated. RESULTS: The VD + OVX group retained a decrease in LPP with sphincter muscle atrophy at least until 6 weeks after injury. The LPP and urethral sphincter muscle atrophy in the DFAT group recovered better than those in the fibroblast group. CONCLUSIONS: The persistence SUI model was created by a combination of VD and bilateral OVX in rats. Urethral injection of DFAT cells inhibited sphincter muscle atrophy and improved LPP in the persistence SUI model. These findings suggest that the DFAT cells may be an attractive cell source for cell-based therapy to treat SUI.

Phenotypic and functional properties of dedifferentiated fat cells derived from infrapatellar fat pad.

INTRODUCTION: Mature adipocyte-derived dedifferentiated fat cells (DFATs) are mesenchymal stem cell (MSC)-like cells with high proliferative ability and multilineage differentiation potential. In this study, we first examined whether DFATs can be prepared from infrapatellar fat pad (IFP) and then compared phenotypic and functional properties of IFP-derived DFATs (IFP-DFATs) with those of subcutaneous adipose tissue (SC)-derived DFATs (SC-DFATs). METHODS: Mature adipocytes isolated from IFP and SC in osteoarthritis patients (n = 7) were cultured by ceiling culture method to generate DFATs. Obtained IFP-DFATs and SC-DFATs were subjected to flow cytometric and microarray analysis to compare their immunophenotypes and gene expression profiles. Cell proliferation assay and adipogenic, osteogenic, and chondrogenic differentiation assays were performed to evaluate their functional properties. RESULTS: DFATs could be prepared from IFP and SC with similar efficiency. IFP-DFATs and SC-DFATs exhibited similar immunophenotypes (CD73+, CD90+, CD105+, CD31-, CD45-, HLA-DR-) and tri-lineage (adipogenic, osteogenic, and chondrogenic) differentiation potential, consistent with the minimal criteria for defining MSCs. Microarray analysis revealed that the gene expression profiles in IFP-DFATs were very similar to those in SC-DFATs, although there were certain number of genes that showed different levels of expression. The proliferative activity in IFP-DFATs was significantly (p < 0.05) higher than that in the SC-DFATs. IFP-DFATs showed higher chondrogenic differentiation potential than SC-DFATs in regard to production of soluble galactosaminogalactan and gene expression of type II collagen. CONCLUSIONS: IFP-DFATs showed higher cellular proliferative potential and higher chondrogenic differentiation capacity than SC-DFATs. IFP-DFAT cells may be an attractive cell source for chondrogenic regeneration.

Effect of Rivaroxaban and Clopidogrel Combination Therapy on In-Stent Responses After Everolimus-Eluting Stent Implantation in a Porcine Coronary Model.

AIM: According to recent clinical trials, a combination of direct oral anticoagulants with antiplatelet drugs is often recommended for atrial fibrillation patients who receive drug-eluting stents (DESs). Although the optimal combination comprises direct factor Xa inhibitors and a P2Y12 receptor antagonist (or aspirin), their influence on vascular responses to DESs remains unclear. METHODS: Pigs were given either aspirin and clopidogrel (dual antiplatelet therapy [DAPT] group), aspirin and rivaroxaban (AR group), or clopidogrel and rivaroxaban (CR group), followed by everolimus-eluting stent (Promus Element) implantation into the coronary artery. Stented coronary arteries were evaluated via intravascular optical coherence tomography (OCT) and histological analysis at 1 and 3 months. RESULTS: OCT revealed lower neointimal thickness in the DAPT group and comparable thickness among all groups at 1 and 3 months, respectively. Histological analyses revealed comparable neointimal area among all groups and the smallest neointimal area in the CR group at 1 and 3 months, respectively. In the DAPT and AR groups, the neointima continued to grow from 1 to 3 months. A shortened time course for neointima growth was observed in the CR group, with rapid growth within a month (maintained for 3 months). A higher incidence of in-stent thrombi was observed in the AR group at 1 month; no thrombi were found in either group at 3 months. More smooth muscle cells with contractile features were found in the CR group at both 1 and 3 months. CONCLUSIONS: Our results proved the noninferiority of the combination of rivaroxaban with an antiplatelet drug, particularly the dual therapy using rivaroxaban and clopidogrel, compared to DAPT after DES implantation.

Antinuclear antibodies produced in HLA-DR transgenic humanized mice developed chronic graft-versus-host disease.

BACKGROUND: Chronic graft versus host disease (GVHD) has been reported in humanized mice after the implantation of human hematopoietic stem cells (hu-HSC). As such, humanized mice have been applied to a mouse model of chronic GVHD; however, B-cell activation and autoantibody production did not occur, and the clinical features of chronic GVHD were not sufficiently reproduced. The purpose of this study was to establish an improved humanized mouse model with chronic GVHD using HLA-DR transgenic NOD/Shi-scid, IL-2RγKO (NOG) mice. METHODS: CD34-positive cells were isolated from blood extracted from HLA-DRB1∗0405-positive umbilical cords using magnetic cell isolation. Then these were transplanted into NOG-Iab KO, HLA-DR 0405 Tg mice aged 8-16 weeks. GVHD symptoms were observed 26 weeks after transplantation. Histological findings of the skin, lung, liver, and spleen were compared with those of non-humanized mice. Antinuclear antibodies (ANA) were measured by indirect immunofluorescence using sera isolated 26 weeks after transplantation. RESULTS: Although GVHD symptoms were not observed in humanized (hu-HSC) NOG-Iab KO, HLA-DR 0405 Tg mice during the observation period, histological findings of human T-cell infiltration were observed in the skin, liver, and lung, suggesting that GVDH was present; human tingible body macrophages or clusters of BCL-6-positive human B-cells were observed in the spleen. Furthermore, human IgG ANA with peripheral or homogeneous staining patterns were also detected in the sera. CONCLUSION: Hu-HSC NOG-Iab KO, HLA-DR 0405 Tg mice differed from conventional models in terms of B-cell activation and ANA production. This study is the first to report on B-cell activation and autoantibody production in humanized mice with chronic GVHD, suggesting that hu-HSC NOG-Iab KO, HLA-DR 0405 Tg mice could be applied to a new humanized mouse model of chronic GVHD.

Quantitative Analysis of Factors Regulating Angiogenesis for Stem Cell Therapy.

(1) Background: The control of angiogenesis is essential in disease treatment. We investigated angiogenesis-promoting or -suppressing factors and their molecular mechanisms. (2) Methods: Angiogenesis from HUVECs was quantitatively analyzed using the Angiogenesis Analysis Kit (Kurabo, Osaka, Japan). Human rAng-1-producing 107-35 CHO cells or mouse DFAT-D1 cells were co-cultured with HUVEC. Antioxidant polyphenols were added to the culture. Gene expression was analyzed by RT-PCR. (3) Results: The addition of rAng-1-producing cells, their culture supernatant, or commercially available rAng-1 showed a promoting effect on angiogenesis. The co-culture of DFAT-D1 cells promoted angiogenesis. Polyphenols showed a dose-dependent inhibitory effect on angiogenesis. Luteolin and quercetin showed remarkable anti-angiogenic effects. The expression of vWF, Flk1, and PECAM-1 was increased by adding rAng-1-producing cell culture supernatant. Polyphenols suppressed these genes. Apigenin and luteolin markedly suppressed α-SMA and Flk1. Resveratrol and quercetin enhanced the expression of PPARγ, and luteolin suppressed the expression of COX-1. The expression of endothelial nitric oxide synthase (eNOS), an oxidative stress-related gene, was slightly increased by luteolin. These results suggest that polyphenols induce ROS reduction. (4) Conclusions: We showed the promoting effect of Ang-1 or DFAT and the suppressing effect of polyphenols on angiogenesis and studied their molecular mechanisms. These results help control angiogenesis in regenerative therapy.

Transcriptional Suppression of Diabetic Nephropathy with Novel Gene Silencer Pyrrole-Imidazole Polyamides Preventing USF1 Binding to the TGF-ß1 Promoter.

Upstream stimulatory factor 1 (USF1) is a transcription factor that is increased in high-glucose conditions and activates the transforming growth factor (TGF)-ß1 promoter. We examined the effects of synthetic pyrrole-imidazole (PI) polyamides in preventing USF1 binding on the TGF-ß1 promoter in Wistar rats in which diabetic nephropathy was established by intravenous administration of streptozotocin (STZ). High glucose induced nuclear localization of USF1 in cultured mesangial cells (MCs). In MCs with high glucose, USF1 PI polyamide significantly inhibited increases in promoter activity of TGF-ß1 and expression of TGF-ß1 mRNA and protein, whereas it significantly decreased the expression of osteopontin and increased that of h-caldesmon mRNA. We also examined the effects of USF1 PI polyamide on diabetic nephropathy. Intraperitoneal injection of USF1 PI polyamide significantly suppressed urinary albumin excretion and decreased serum urea nitrogen in the STZ-diabetic rats. USF1 PI polyamide significantly decreased the glomerular injury score and tubular injury score in the STZ-diabetic rats. It also suppressed the immunostaining of TGF-ß1 in the glomerulus and proximal tubules and significantly decreased the expression of TGF-ß1 protein from kidney in these rats. These findings indicate that synthetic USF1 PI polyamide could potentially be a practical medicine for diabetic nephropathy.

Umbilical artery tissue contains p75 neurotrophin receptor-positive pericyte-like cells that possess neurosphere formation capacity and neurogenic differentiation potential.

INTRODUCTION: The p75 neurotrophin receptor (p75NTR) is known as an efficient marker for the prospective isolation of mesenchymal stem cells (MSCs) and neural crest-derived stem cells (NCSCs). To date, there is quite limited information concerning p75NTR-expressing cells in umbilical cord (UC), although UC is known as a rich source of MSCs. We show for the first time the localization, phenotype, and functional properties of p75NTR+ cells in UC. METHODS: Human UC tissue sections were subjected to immunohistochemistry for MSC markers including p75NTR. Enzymatically isolated umbilical artery (UA) cells containing p75NTR+ cells were assessed for immunophenotype, clonogenic capacity, and differentiation potential. To identify the presence of neural crest-derived cells in the UA, P0-Cre/Floxed-EGFP reporter mouse embryos were used, and immunohistochemical analysis of UC tissue was performed. RESULTS: Immunohistochemical analysis revealed that p75NTR+ cells were specifically localized to the subendothelial area of the UA and umbilical vein. The p75NTR+ cells co-expressed PDGFRß, CD90, CD146, and NG2, phenotypic markers of MSCs and pericytes. Isolated UA cells possessed the potential to form neurospheres that further differentiated into neuronal and glial cell lineages. Genetic lineage tracing analysis showed that EGFP+ neural crest-derived cells were detected in the subendothelial area of UA with p75NTR immunoreactivity. CONCLUSIONS: These results show that UA tissue harbors p75NTR+ pericyte-like cells in the subendothelial area that have the capacity to form neurospheres and the potential for neurogenic differentiation. The lineage tracing data suggests the p75NTR+ cells are putatively derived from the neural crest.

Soluble factors released by dedifferentiated fat cells reduce the functional activity of iPS cell-derived cardiomyocytes.

Interactions between tissues such as epicardial adipose (EAT), and myocardial tissues is important in the pathogenesis of heart failure. Changes in adipose tissues in obesity or diabetes impair preadipocyte differentiation. Furthermore, proinflammatory cytokine secretion is higher in preadipocytes than in mature adipocytes in diabetes and obesity. However, how undifferentiated cells committed to the adipose lineage directly influence cardiomyocytes is not yet understood. We used human-derived dedifferentiated fat (DFAT) cells as models of undifferentiated cells committed to an adipose lineage. Here, we evaluated the effects of soluble factor interactions in indirect cocultures of DFAT cells and induced pluripotent stem cell-derived cardiomyocytes. Our RNA sequencing findings showed that these interactions were predominantly inflammatory responses. Furthermore, proinflammatory cytokines secreted by DFAT cells reduced myocardial functions such as contraction frequency and catecholamine sensitivity, and simultaneously increased apoptosis, decreased antioxidative stress tolerance, and reduced oxygen consumption rates in cardiomyocytes. These adverse effects might be attributable to monocyte chemoattractant protein-1, chemokine (C-X-C motif) ligands 1 (CXCL1), and 12, granulocyte colony-stimulating factor, interleukins 6 and 8, macrophage migration inhibitory factor (MIF), and plasminogen activator inhibitor 1-A among the proinflammatory mediators secreted by DFAT cells. Our results could be useful for understanding the pathogenesis of EAT-related heart failure in terms of the involvement of undifferentiated cells committed to the adipose lineage. Furthermore, we suggest the importance of focusing on surrounding adipose tissues as a strategy with which to maximize the survival and function of transplanted stem cell-derived cardiomyocytes.

Effect of the dipeptidyl peptidase-4 inhibitor linagliptin on atherosclerotic lesions in Watanabe heritable hyperlipidemic rabbits: iMap-IVUS and pathological analysis.

Dipeptidyl peptidase-4 (DPP-4) inhibitors have potential as a treatment for atherosclerosis. However, it is unclear whether DPP-4 inhibitors stabilize atherosclerotic plaque or alter the composition of complex plaque. Sixteen Watanabe heritable hyperlipidemic rabbits aged 10-12 weeks with atherosclerotic plaque in the brachiocephalic artery detected by iMap™ intravascular ultrasound (IVUS) were divided into a DPP-4 inhibitor group and a control group. Linagliptin was administered to the DPP-4 inhibitor group via nasogastric tube at a dose of 10 mg/kg/day for 16 weeks, and control rabbits received the same volume of 0.5% hydroxyethylcellulose. After evaluation by IVUS at 16 weeks, the brachiocephalic arteries were harvested for pathological examination. IVUS revealed that linagliptin significantly reduced the plaque volume and vessel volume (control group vs. DPP-4 inhibitor group: ∆plaque volume, 1.02 ± 0.96 mm3 vs. - 3.59 ± 0.92 mm3, P = 0.004; ∆vessel volume, - 1.22 ± 2.36 mm3 vs. - 8.66 ± 2.33 mm3, P = 0.04; %change in plaque volume, 6.90 ± 5.62% vs. - 15.06 ± 3.29%, P = 0.005). With regard to plaque composition, linagliptin significantly reduced the volume of fibrotic, lipidic, and necrotic plaque (control group vs. DPP-4 inhibitor group: ∆fibrotic volume, 0.56 ± 1.27 mm3 vs. - 5.57 ± 1.46 mm3, P = 0.04; ∆lipidic volume, 0.24 ± 0.24 mm3 vs. - 0.42 ± 0.16 mm3 P = 0.04; ∆necrotic volume, 0.76 ± 0.54 mm3 vs. - 0.84 ± 0.25 mm3, P = 0.02). Pathological examination did not show any significant differences in the %smooth muscle cell area or %fibrotic area, but infiltration of macrophages into plaque was reduced by linagliptin treatment (%macrophage area: 12.03% ± 1.51% vs. 7.21 ± 1.65%, P < 0.05). These findings indicate that linagliptin inhibited plaque growth and stabilized plaque in Watanabe heritable hyperlipidemic rabbits.

Functional recurrent laryngeal nerve regeneration using a silicon tube containing a collagen gel in a rat model.

In this study, we examined the effect of differing gap lengths on regeneration of transected recurrent laryngeal nerves using silicon tubes containing type I collagen gel and the ability of this regeneration to result in restoration of vocal fold movements in rats. We simulated nerve gaps in Sprague-Dawley rats by transecting the left recurrent laryngeal nerves and bridged the nerve stumps using silicon tubes containing type 1 collagen gel. Three experimental groups, in which the gap lengths between the stumps were 1, 3, or 5 mm, were compared with a control group in which the nerve was transected but was not bridged. After surgery, we observed vocal fold movements over time with a laryngoscope. At week 15, we assessed the extent of nerve regeneration in the tube, histologically and electrophysiologically. We also assessed the degree of atrophy of the thyroarytenoid muscle (T/U ratio). Restoration of vocal fold movements was observed in 9 rats in the 1-mm group, in 6 rats in the 3-mm group, and in 3 rats in the 5-mm group. However, in most rats, restoration was temporary, with only one rat demonstrating continued vocal fold movements at week 15. In electromyograph, evoked potentials were observed in rats in the 1-mm and 3-mm groups. Regenerated tissue in the tube was thickest in the 1-mm group, followed by the 3-mm and 5-mm groups. The regenerated tissue showed the presence of myelinated and unmyelinated nerve fibers. In assessment of thyroarytenoid muscle atrophy, the T/U ratio was highest in the 1-mm group, followed by the 3-mm and 5-mm groups. We successfully regenerated the nerves and produced a rat model of recurrent laryngeal nerve regeneration that demonstrated temporary recovery of vocal fold movements. This rat model could be useful for assessing novel treatments developing in the future.