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.

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.

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.

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.

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.

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.

Role of complement 3 in renin generation during the differentiation of mesenchymal stem cells to smooth muscle cells.

We showed that increased expression of complement 3 (C3) induces dedifferentiation of mesenchymal cells and epithelial mesenchymal transition, which activate the local renin-angiotensin system (RAS) that contributes to cardiovascular and renal remodeling in spontaneously hypertensive rats (SHRs). In the present study, to investigate contributions of C3 to the development of the pathogenesis of hypertension, we evaluated the formation of renin-producing cells and roles of C3 in renin generation during differentiation of primary bone marrow-mesenchymal stem cells (MSCs) from C57BL/6 mice, Wistar-Kyoto (WKY) rats, and SHRs to smooth muscle cells (SMCs) with transforming growth factor-ß1. The expression of renin transiently increased with increases in transcription factor liver X receptor α (LXRα), and expression of C3 and Krüppel-like factor 5 (KLF5) increased during differentiation of MSCs from C57BL/6 mice, WKY rats, and SHRs to SMCs. Exogenous C3a stimulated renin and LXRα expression accompanied by nuclear translocation of LXRα. C3a receptor antagonist SB290157 suppressed renin and LXRα expression, with inhibition of nuclear translocation of LXRα during the differentiation of mouse MSCs to SMCs. The expression of C3 and KLF5 was significantly higher in the differentiated cells from SHRs compared with the cells from WKY rats during differentiation. Renin-producing cells were formed during differentiation of MSCs to SMCs, and renin generation was observed in undifferentiated SMCs, in which transient expression of renin in the differentiated cells with lower differentiation stage was stronger from SHRs than that from WKY rats. Expression and nuclear localization of LXRα in the differentiated cells from SHRs were stronger than that from WKY rats. C3 was important in forming and maintaining this undifferentiated state of SMCs from MSCs to generate renin with increases in transcription factor LXRα and KLF5. Increases in C3 expression maintain the undifferentiated state of SMCs from MSCs to generate renin that activates RAS and contributes to the pathogenesis of hypertension in SHRs.

Therapeutic potential of mature adipocyte-derived dedifferentiated fat cells for inflammatory bowel disease.

PURPOSE: Our previous studies demonstrated that mature adipocyte-derived dedifferentiated fat (DFAT) cells possess similar multipotency as mesenchymal stem cells. Here, we examined the immunoregulatory potential of DFAT cells in vitro and the therapeutic effect of DFAT cell transplantation in a mouse inflammatory bowel disease (IBD) model. METHODS: The effect of DFAT cell co-culture on T cell proliferation and expression of immunosuppression-related genes in DFAT cells were evaluated. To create IBD, CD4+CD45RBhigh T cells were intraperitoneally injected into SCID mice. One week later, DFAT cells (1 × 105, DFAT group) or saline (Control group) were intraperitoneally injected. Subsequently bodyweight was measured every week and IBD clinical and histological scores were evaluated at 5 weeks after T cell administration. RESULTS: The T cell proliferation was inhibited by co-cultured DFAT cells in a cell density-dependent manner. Gene expression of TRAIL, IDO1, and NOS2 in DFAT cells was upregulated by TNFα stimulation. DFAT group improved IBD-associated weight loss, IBD clinical and histological scores compared to Control group. CONCLUSION: DFAT cells possess immunoregulatory potential and the cell transplantation promoted recovery from colon damage and improved clinical symptoms in the IBD model. DFAT cells could play an important role in the treatment of IBD.

Identification of novel polymorphisms and two distinct haplotype structures in dog leukocyte antigen class I genes: DLA-88, DLA-12 and DLA-64.

The current information on the polymorphism variation and haplotype structure of the domestic dog leukocyte antigen (DLA) genes is limited in comparison to other experimental animals. In this paper, to better elucidate the degree and types of polymorphisms and genetic differences for DLA-88, DLA-12 and DLA-64, we genotyped four families of 38 beagles and another 404 unrelated dogs representing 49 breeds by RT-PCR based Sanger sequencing. We also sequenced and analyzed the genomic organization of the DLA-88 and DLA-12 gene segments to better define these two-gene DLA haplotypes more precisely. We identified 45 alleles for DLA-88, 15 for DLA-12 and six for DLA-64, of which 20, 14 and six, respectively, were newly described alleles. Therefore, this study shows that the DLA-12 and DLA-64 loci are far more polymorphic than previously reported. Phylogenetic analysis strongly supported that the DLA-88, DLA-12 and DLA-64 alleles were independently generated after the original divergence of the DLA-79 alleles. Two distinct DLA-88 and DLA-12 haplotype structures, tentatively named DLA-88-DLA-12 and DLA-88-DLA-88L, were identified, and the novel haplotype DLA-88-DLA-88L contributed to 32.7% of the unrelated dogs. Quantitative real-time PCR analysis showed that the gene expression levels of DLA-88L and DLA-88 were similar, and that the gene expression level of DLA-12 was significantly lower. In addition, haplotype frequency estimations using frequently occurring alleles revealed 45 different DLA-class I haplotypes (88-88L/12-64) overall, and 22 different DLA-class I haplotypes in homozygous dogs for 18 breeds and mongrels.

Transplantation of dedifferentiated fat cell-derived micromass pellets contributed to cartilage repair in the rat osteochondral defect model.

BACKGROUND: Mature adipocyte-derived dedifferentiated fat (DFAT) cells possesses the ability to proliferate effectively and the potential to differentiate into multiple linages of mesenchymal tissue; similar to adipose-derived stem cells (ASCs). The purpose of this study is to examine the effects of DFAT cell transplantation on cartilage repair in a rat model of osteochondral defects. METHODS: Full-thickness osteochondral defects were created in the knees of Sprague-Dawley rats bilaterally. Cartilage-like micromass pellets were prepared from green fluorescent protein (GFP)-labeled rat DFAT cells and subsequently transplanted into the affected right knee of these rats. Defects in the left knee were used as a control. Macroscopic and microscopic changes of treated and control defects were evaluated up to 12 weeks post-treatment with DFAT cells. To observe the transplanted cells, sectioned femurs were immunostained for GFP and type II collagen. RESULTS: DFAT cells formed micromass pellets expressing characteristics of immature cartilage in vitro. In the DFAT cell-transplanted limbs, the defects were completely filled with white micromass pellets as early as 2 weeks post-treatment. These limbs became smooth at 4 weeks. Conversely, the defects in the control limbs were still not repaired by 4 weeks. Macroscopic ICRS scores at 2 and 4 weeks were significantly higher in the DFAT cells-transplanted limbs compared to those of the control limbs. The modified O'Driscol histological scores for the DFAT cell-transplanted limbs were significantly higher than those of the control limbs at corresponding time points. GFP-positive DAFT cells were detected in the transplanted area at 2 weeks but hardly visible at 12 weeks post-operation. CONCLUSIONS: Transplantation of DFAT cell-derived micromass pellets contribute to cartilage repair in a rat osteochondral defect model. DFAT cell transplantation may be a viable therapeutic strategy for the repair of osteochondral injuries.

Transplantation of dedifferentiation fat cells promotes intervertebral disc regeneration in a rat intervertebral disc degeneration model.

Our group has reported that mature adipocyte-derived dedifferentiated fat (DFAT) cells show multilineage differentiation potential similar to that observed in mesenchymal stem cells. In the present study, we examined whether DFAT cell transplantation could contribute to intervertebral disc regeneration using a rat intervertebral disc degeneration (IDD) model. The IDD was created in Sprague-Dawley rats by puncturing at level of caudal intervertebral disc under fluoroscopy. One week after injury, rat DFAT cells (5 × 104, DFAT group, n = 13) or phosphate-buffered saline (PBS, control group, n = 13) were injected into the intervertebral disc. Percent disc height index (%DHI) was measured every week and histology of injured disc was evaluated at 8 weeks after transplantation. Radiographic analysis revealed that the %DHI in the DFAT group significantly higher than that in the control group at 2-3 weeks after transplantation. Histological analysis revealed that ectopic formation of nucleus pulposus (NP)-like tissue at the outer layer of annulus fibrosus was frequently observed in the DFAT group but not in the control group. Transplantation experiments using green fluorescent protein (GFP)-labeled DFAT cells revealed that the ectopic NP-like tissue was positive for GFP, suggesting direct differentiation of DFAT cells into NP-like cells. In conclusion, DFAT cell transplantation promoted the regeneration of intervertebral disc and improved intervertebral disc height in the rat IDD model. Because adipose tissue is abundant and easily accessible, DFAT cell transplantation may be an attractive therapeutic strategy against IDD.

Dedifferentiated Fat Cells as a Novel Source for Cell Therapy to Target Neonatal Hypoxic-Ischemic Encephalopathy.

Neonatal hypoxic-ischemic (HI) encephalopathy (HIE) remains a major cause of mortality and persistent neurological disabilities in affected individuals. At present, hypothermia is considered to be the only applicable treatment option, although growing evidence suggests that cell-based therapy might achieve better outcomes. Dedifferentiated fat (DFAT) cells are derived from mature adipocytes via a dedifferentiation strategy called ceiling culture. Their abundance and ready availability might make them an ideal therapeutic tool for the treatment of HIE. In the present study, we aimed to determine whether the outcome of HIE can be improved by DFAT cell treatment. HI injury was achieved by ligating the left common carotid artery in 7-day-old rat pups, followed by 1-h exposure to 8% O2. Subsequently, the severity of damage was assessed by diffusion-weighted magnetic resonance imaging to assign animals to equivalent groups. 24 h after hypoxia, DFAT cells were injected at 105 cells/pup into the right external jugular vein. To evaluate brain damage in the acute phase, a group of animals was sacrificed 48 h after the insult, and paraffin sections of the brain were stained to assess several acute injury markers. In the chronic phase, the behavioral outcome was measured by performing a series of behavioral tests. From the 24th day of age, the sensorimotor function was examined by evaluating the initial forepaw placement on a cylinder wall and the latency to falling from a rotarod treadmill. The cognitive function was tested with the novel object recognition (NOR) test. In vitro conditioned medium (CM) prepared from cultured DFAT cells was added at various concentrations to neuronal cell cultures, which were then exposed to oxygen-glucose deprivation (OGD). The number of cells that stained positive for the apoptosis marker active caspase-3 decreased by 73 and 52% in the hippocampus and temporal cortex areas of the brain, respectively, in the DFAT-treated pups. Similarly, the numbers of ED-1-positive cells (activated microglia) decreased by 66 and 44%, respectively, in the same areas in the DFAT-treated group. The number of cells positive for the oxidative stress marker 4-hydroxyl-2-nonenal decreased by 68 and 50% in the hippocampus and the parietal cortex areas, respectively, in the DFAT-treated group. The HI insult led to a motor deficit according to the rotarod treadmill and cylinder test, where it significantly affected the vehicle group, whereas no difference was confirmed between the DFAT and sham groups. However, the NOR test indicated no significant differences between any of the groups. DFAT treatment did not reduce the infarct volume, which was confirmed immunohistochemically. According to in vitro experiments, the cell death rates in the DFAT-CM-treated cells were significantly lower than those in the controls when DFAT-CM was added 48 h prior to OGD. The treatment effect of adding DFAT-CM 24 h prior to OGD was also significant. Our results indicate that intravenous injection with DFAT cells is effective for ameliorating HI brain injury, possibly via paracrine effects.

Transplantation of mature adipocyte-derived dedifferentiated fat cells promotes locomotor functional recovery by remyelination and glial scar reduction after spinal cord injury in mice.

Mature adipocyte-derived dedifferentiated fat cells (DFAT) have a potential to be useful as new cell-source for cell-based therapy for spinal cord injury (SCI), but the mechanisms remain unclear. The objective of this study was to examine whether DFAT-induced functional recovery is achieved through remyelination and/or glial scar reduction in a mice model of SCI. To accomplish this we subjected adult female mice (n=22) to SCI. On the 8th day post-injury locomotor tests were performed, and the mice were randomly divided into two groups (control and DFAT). The DFAT group received stereotaxic injection of DFAT, while the controls received DMEM medium. Functional tests were conducted at repeated intervals, until the 36th day, and immunohistochemistry or staining was performed on the spinal cord sections. DFAT transplantation significantly improved locomotor function of their hindlimbs, and promoted remyelination and glial scar reduction, when compared to the controls. There were significant and positive correlations between promotion of remyelination or/and reduction of glial scar, and recovery of locomotor function. Furthermore, transplanted DFAT expressed markers for neuron, astrocyte, and oligodendrocyte, along with neurotrophic factors, within the injured spinal cord. In conclusion, DFAT-induced functional recovery in mice after SCI is probably mediated by both cell-autonomous and cell-non-autonomous effects on remyelination of the injured spinal cord.

2α-Substituted Vitamin D Derivatives Effectively Enhance the Osteoblast Differentiation of Dedifferentiated Fat Cells.

The active form of vitamin D3, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], is a principal regulator of calcium homeostasis through activation of the vitamin D receptor (VDR). Previous studies have shown that 2α-(3-hydroxypropyl)-1,25D3 (O1C3) and 2α-(3-hydroxypropoxy)-1,25D3 (O2C3), vitamin D derivatives resistant to inactivation enzymes, can activate VDR, induce leukemic cell differentiation, and increase blood calcium levels in rats more effectively than 1,25(OH)2D3. In this study, to further investigate the usefulness of 2α-substituted vitamin D derivatives, we examined the effects of O2C3, O1C3, and their derivatives on VDR activity in cells and mouse tissues and on osteoblast differentiation of dedifferentiated fat (DFAT) cells, a cell type with potential therapeutic application in regenerative medicine. In cell culture experiments using kidney-derived HEK293 cells, intestinal mucosa-derived CaCO2 cells, and osteoblast-derived MG63 cells, and in mouse experiments, O2C2, O2C3, O1C3, and O1C4 had a weaker effect than or equivalent effect to 1,25(OH)2D3 in VDR transactivation and induction of the VDR target gene CYP24A1, but they enhanced osteoblast differentiation in DFAT cells equally to or more effectively than 1,25(OH)2D3. In long-term treatment with the compound without the medium change (7 days), the derivatives enhanced osteoblast differentiation more effectively than 1,25(OH)2D3. O2C3 and O1C3 were more stable than 1,25(OH)2D3 in DFAT cell culture. These results indicate that 2α-substituted vitamin D derivatives, such as inactivation-resistant O2C3 and O1C3, are more effective than 1,25(OH)2D3 in osteoblast differentiation of DFAT cells, suggesting potential roles in regenerative medicine with DFAT cells and other multipotent cells.

Therapeutic potential of dedifferentiated fat cells in a rat model of osteoarthritis of the knee.

Introduction: Mature adipocyte-derived dedifferentiated fat cells (DFATs) represent a subtype of multipotent cells that exhibit comparable phenotypic and functional characteristics to adipose-derived stem cells (ASCs). In this study, we assessed the chondroprotective properties of intra-articularly administrated DFATs in a rat model of osteoarthritis (OA). We also investigated in vitro the expression of anti-inflammatory and chondroprotective genes in DFATs prepared from the infrapatellar fat pad (IFP) and subcutaneous adipose-tissue (SC) of human origin. Methods: In the cell transplantation experiment, rats were assigned to the DFAT and Control group (n = 10 in each group) and underwent anterior cruciate ligament transection (ACLT) accompanied by medial meniscus resection (MMx) to induce OA. One week later, they received intra-articular injections of 1 × 106 DFATs (DFAT group) or PBS (control group) four times, with a weekly administration frequency. Macroscopic and microscopic evaluations were conducted five weeks post-surgery. In the in vitro experiments. DFATs derived from the IFP (IFP-DFATs) and SC (SC-DFATs) were prepared from donor-matched tissue samples (n = 3). The gene expression of PTGS2, TNFAIP6, PRG4, BMP2, and BMP6 under TNF-α or IFN-γ stimulation in these cells was evaluated using RT-PCR. Furthermore, the effect of co-culturing synovial fibroblasts with DFATs on the gene expression of ADAMTS4 and IL-6 were evaluated. Results: Intra-articular injections of DFATs significantly inhibited cartilage degeneration in the rat OA model induced by ACLT and MMx. RT-PCR analysis revealed that both IFP-DFATs and SC-DFATs upregulated the expression of genes involved in immune regulation, anti-inflammation, and cartilage protection such as PTGS2, TNFAIP6, and BMP2, under stimulation by inflammatory cytokines. Co-culture with DFATs suppressed the expression of ADAMTS4 and IL6 in synovial fibroblasts. Conclusions: The intra-articular injection of DFATs resulted in chondroprotective effects in the rat OA model. Both SC-DFATs and IFP-DFATs induced the expression of anti-inflammatory and chondroprotective genes in vitro. These results indicate that DFATs appear to possess therapeutic potential in inhibiting cartilage degradation and could serve as a promising cellular resource for OA treatment.

Abnormal epigenetic memory of mesenchymal stem and progenitor cells caused by fetal malnutrition induces hypertension and renal injury in adulthood.

Fetal malnutrition has been reported to induce hypertension and renal injury in adulthood. We hypothesized that this hypertension and renal injury would be associated with abnormal epigenetic memory of stem and progenitor cells contributing to organization in offspring due to fetal malnutrition. We measured blood pressure (BP) for 60 weeks in offspring of pregnant rats fed a normal protein diet (Control), low-protein diet (LP), and LP plus taurine (LPT) in the fetal period. We used western blot analysis to evaluate the expression of αSMA and renin in CD44-positive renal mesenchymal stem cells (MSCs) during differentiation by TGF-ß1. We measured kidney label-retaining cells (LRCs) at 11 weeks of age and formation of endothelial progenitor cells (EPCs) at 60 weeks of age from the offspring with fetal malnutrition. Epigenetics of the renal MSCs at 14 weeks were investigated by ATAC-sequence and RNA-sequence analyses. BP was significantly higher in LP than that in Control and LPT after 45-60 weeks of age. Numbers of LRCs and EPC colonies were significantly lower in LP than in Control. Renal MSCs from LP already showed expression of h-caldesmon, αSMA, LXRα, and renin before their differentiation. Epigenetic analyses identified PAR2, Chac1, and Tspan6 genes in the abnormal differentiation of renal MSCs. These findings suggested that epigenetic abnormalities of stem and progenitor cell memory cause hypertension and renal injury that appear in adulthood of offspring with fetal malnutrition.

Complement 3 activates the renal renin-angiotensin system by induction of epithelial-to-mesenchymal transition of the nephrotubulus in mice.

We have demonstrated that mesenchymal cells from spontaneously hypertensive rats genetically express complement 3 (C3). Mature tubular epithelial cells can undergo epithelial-to-mesenchymal transition (EMT) that is linked to the pathogenesis of renal fibrosis and injury. In this study, we investigated the contribution of C3 in EMT and in the renal renin-angiotensin (RA) systems associated with hypertension. C3a induced EMT in mouse TCMK-1 epithelial cells, which displayed increased expression of renin and Krüppel-like factor 5 (KLF5) and nuclear localization of liver X receptor α (LXRα). C3 and renin were strongly stained in the degenerated nephrotubulus and colocalized with LXRα and prorenin receptor in unilateral ureteral obstruction (UUO) kidneys from wild-type mice. In C3-deficient mice, hydronephrus and EMT were suppressed, with no expression of renin and C3. After UUO, systolic blood pressure was increased in wild-type but not C3-deficient mice. In wild-type mice, intrarenal angiotensin II (ANG II) levels were markedly higher in UUO kidneys than normal kidneys and decreased with aliskiren. There were no increases in intrarenal ANG II levels after UUO in C3-deficient mice. Thus C3 induces EMT and dedifferentiation of epithelial cells, which produce renin through induction of LXRα. These data indicate for the first time that C3 may be a primary factor to activate the renal RA systems to induce hypertension.

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.