Differentiation of human adipose tissue-derived mesenchymal stromal cells into steroidogenic cells by adenovirus-mediated overexpression of NR5A1 and implantation into adrenal insufficient mice.

BACKGROUND AIMS: Cell therapy for adrenal insufficiency is a potential method for physiological glucocorticoid and mineralocorticoid replacement. We have previously shown that mouse mesenchymal stromal cells (MSCs) differentiated into steroidogenic cells by the viral vector-mediated overexpression of nuclear receptor subfamily 5 group A member 1 (NR5A1), an essential regulator of steroidogenesis, and their implantation extended the survival of bilateral adrenalectomized (bADX) mice. METHODS: In this study, we examined the capability of NR5A1-induced steroidogenic cells prepared from human adipose tissue-derived MSCs (MSC [AT]) and the therapeutic effect of the implantation of human NR5A1-induced steroidogenic cells into immunodeficient bADX mice. RESULTS: Human NR5A1-induced steroidogenic cells secreted adrenal and gonadal steroids and exhibited responsiveness to adrenocorticotropic hormone and angiotensin II in vitro. In vivo, the survival time of bADX mice implanted with NR5A1-induced steroidogenic cells was significantly prolonged compared with that of bADX mice implanted with control MSC (AT). Serum cortisol levels, which indicate hormone secretion from the graft, were detected in bADX mice implanted with steroidogenic cells. CONCLUSIONS: This is the first report to demonstrate steroid replacement by the implantation of steroid-producing cells derived from human MSC (AT). These results indicate the potential of human MSC (AT) to be a source of steroid hormone-producing cells.

Xenotransplantation of neonatal porcine bone marrow-derived mesenchymal stem cells improves diabetic wound healing by promoting angiogenesis and lymphangiogenesis.

BACKGROUND: Some clinical trials have shown the usefulness of stem cell therapy for diabetic foot ulcers. However, the donor supply is limited, and the process is time consuming and expensive. This study assessed the therapeutic effects of neonatal porcine bone marrow-derived mesenchymal stem cell (npBM-MSC) xenotransplantation using diabetic wound model mice. METHODS: All layers of back skin were removed from streptozotocin-induced diabetic mice. In the npBM-MSCs group, npBM-MSCs were transplanted to the wound, and syngeneic mouse bone marrow-derived mesenchymal stem cells (mBM-MSCs) were transplanted to the wound in the mBM-MSCs group. The control group comprised diabetic mice that did not receive cellular therapy. The therapeutic effects of the transplantation were evaluated according to the rate of wound closure and the promotion of neovascularization in the wound. RESULTS: The wound closure rate was significantly improved in the npBM-MSCs group compared with the control group (p < .001 at postoperative day [POD] 4 and p < .01 at POD 7) and mBM-MSCs groups (p < .05 at POD 4). Prominent promotion of both angiogenesis and lymphangiogenesis was observed in the npBM-MSCs group. Furthermore, the expression of murine Prox1 and both porcine and murine Vegfs and Tgfb1 in the wounds was enhanced until POD 4 by npBM-MSCs transplantation. The amounts of vascular endothelial growth factor (VEGF) A, VEGFC, and transforming growth factor ß1 secreted from npBM-MSCs were higher than those from mBM-MSCs (p < .05). CONCLUSION: Xenotransplantation of npBM-MSCs improved diabetic wound healing by promoting both angiogenesis and lymphangiogenesis.

Xenotransplantation of neonatal porcine bone marrow-derived mesenchymal stem cells improves murine hind limb ischemia through lymphangiogenesis and angiogenesis.

BACKGROUND: The clinical utility of stem cell therapy for peripheral artery disease has not been fully discussed, and one obstacle is limited donor supplies. In this study, we attempted to rescue mouse ischemic hind limb by xenotransplantation of neonatal porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs). METHODS: Neonatal porcine bone marrow-derived mesenchymal stem cells were transplanted to ischemic hind limbs of male C57BL/6J mice (npBM-MSCs group). Mice with syngeneic transplantation of mouse BM-MSCs (mBM-MSCs group) were also prepared for comparison. The angiogenic effects were evaluated by recovery of blood flow on laser Doppler imaging, histologic findings, and genetic and protein levels of angiogenic factors. RESULTS: Regarding laser Doppler assessments, blood flow in the hind limb was rapidly recovered in the npBM-MSCs group, compared with that in the mBM-MSCs group (P = .016). Compared with the mBM-MSCs group, the npBM-MSCs group had early and prominent lymphangiogenesis [P < .05 on both post-operative days (PODs) 3 and 7] but had similar angiogenesis. Regarding genomic assessments, xenotransplantation of npBM-MSCs enhanced the expressions of both porcine and murine Vegfc in the hind limbs by POD 3. Interestingly, the level of murine Vegfc expression was significantly higher in the npBM-MSCs group than in the mBM-MSCs group on PODs 3 and 7 (P < .001 for both). Furthermore, the secreted VEGFC protein level was higher from npBM-MSCs than from mBM-MSCs (P < .001). CONCLUSION: Xenotransplantation of npBM-MSCs contributed to the improvement of hind limb ischemia by both angiogenesis and lymphangiogenesis, especially promotion of the latter. npBM-MSCs may provide an alternative to autologous and allogeneic MSCs for stem cell therapy of critical limb ischemia.

Lymphangiogenesis and angiogenesis rescue murine ischemic hindlimb via transient receptor potential vanilloid 4.

In this study, we assessed the regulation of transient receptor potential vanilloid 4 (TRPV4) promoting lymphangio/angiogenesis to improve the ischemic hindlimb animal model, and revealed that (1) a TRPV4 agonist improved the blood flow of ischemic hindlimbs by inducing both angiogenesis and lymphangiogenesis; (2) excessive TRPV4 expression was detected on lymphatic endothelial cells (LECs) in the ischemic hindlimb; and (3) hypoxic conditions promoted Ca2+ influx into LECs via TRPV4. It is considered that the upregulation of both lymphatic and blood vessels by activating TRPV4 would be a promising therapeutic strategy for peripheral artery disease.

Development and Characteristics of Pancreatic Epsilon Cells.

Pancreatic endocrine cells expressing the ghrelin gene and producing the ghrelin hormone were first identified in 2002. These cells, named ε cells, were recognized as the fifth type of endocrine cells. Differentiation of ε cells is induced by various transcription factors, including Nk2 homeobox 2, paired box proteins Pax-4 and Pax6, and the aristaless-related homeobox. Ghrelin is generally considered to be a "hunger hormone" that stimulates the appetite and is produced mainly by the stomach. Although the population of ε cells is small in adults, they play important roles in regulating other endocrine cells, especially ß cells, by releasing ghrelin. However, the roles of ghrelin in ß cells are complex. Ghrelin contributes to increased blood glucose levels by suppressing insulin release from ß cells and is also involved in the growth and proliferation of ß cells and the prevention of ß cell apoptosis. Despite increasing evidence and clarification of the mechanisms of ε cells over the last 20 years, many questions remain to be answered. In this review, we present the current evidence for the participation of ε cells in differentiation and clarify their characteristics by focusing on the roles of ghrelin.

CD44 Promotes Inflammation and Extracellular Matrix Production During Arteriovenous Fistula Maturation.

OBJECTIVE: Arteriovenous fistulae (AVF) remain the optimal conduit for hemodialysis access but continue to demonstrate poor patency and poor rates of maturation. We hypothesized that CD44, a widely expressed cellular adhesion molecule that serves as a major receptor for extracellular matrix components, promotes wall thickening and extracellular matrix deposition during AVF maturation. APPROACH AND RESULTS: AVF were created via needle puncture in wild-type C57BL/6J and CD44 knockout mice. CD44 mRNA and protein expression was increased in wild-type AVF. CD44 knockout mice showed no increase in AVF wall thickness (8.9 versus 26.8 µm; P=0.0114), collagen density, and hyaluronic acid density, but similar elastin density when compared with control AVF. CD44 knockout mice also showed no increase in vascular cell adhesion molecule-1 expression, intercellular adhesion molecule-1 expression, and monocyte chemoattractant protein-1 expression in the AVF compared with controls; there were also no increased M2 macrophage markers (transglutaminase-2: 81.5-fold, P=0.0015; interleukin-10: 7.6-fold, P=0.0450) in CD44 knockout mice. Delivery of monocyte chemoattractant protein-1 to CD44 knockout mice rescued the phenotype with thicker AVF walls (27.2 versus 14.7 µm; P=0.0306), increased collagen density (2.4-fold; P=0.0432), and increased number of M2 macrophages (2.1-fold; P=0.0335). CONCLUSIONS: CD44 promotes accumulation of M2 macrophages, extracellular matrix deposition, and wall thickening during AVF maturation. These data show the association of M2 macrophages with wall thickening during AVF maturation and suggest that enhancing CD44 activity may be a strategy to increase AVF maturation.

The Spleen as an Optimal Site for Islet Transplantation and a Source of Mesenchymal Stem Cells.

This review demonstrates the unique potential of the spleen as an optimal site for islet transplantation and as a source of mesenchymal stem cells. Islet transplantation is a cellular replacement therapy used to treat severe diabetes mellitus; however, its clinical outcome is currently unsatisfactory. Selection of the most appropriate transplantation site is a major factor affecting the clinical success of this therapy. The spleen has long been studied as a candidate site for islet transplantation. Its advantages include physiological insulin drainage and regulation of immunity, and it has recently also been shown to contribute to the regeneration of transplanted islets. However, the efficacy of transplantation in the spleen is lower than that of intraportal transplantation, which is the current representative method of clinical islet transplantation. Safer and more effective methods of islet transplantation need to be established to allow the spleen to be used for clinical transplantation. The spleen is also of interest as a mesenchymal stem cell reservoir. Splenic mesenchymal stem cells contribute to the repair of damaged tissue, and their infusion may thus be a promising therapy for autoimmune diseases, including type 1 diabetes mellitus and Sjogren’s syndrome.

Islet-derived damage-associated molecular pattern molecule contributes to immune responses following microencapsulated neonatal porcine islet xenotransplantation in mice.

BACKGROUND: Clinical allogeneic islet transplantation has become an attractive procedure for type 1 diabetes mellitus treatment. However, there is a severe shortage of human donors. Microencapsulated neonatal porcine islet (NPI) xenotransplantation may be an alternative transplantation procedure. Currently, the efficacy of microencapsulated NPI xenotransplantation into the peritoneal cavity is limited because of early non-function resulting from inflammation, which is a serious hindrance to promoting this procedure as a standard therapy. Previously, we have demonstrated that high-mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) molecule, was released from transplanted islets and triggered inflammatory reactions leading to early loss of intrahepatic syngeneic islet grafts in mice. In this study, we hypothesized that the inflammatory reaction in the peritoneal cavity following the transplantation of microencapsulated NPIs is more severe than that of empty capsules. Additionally, we predicted that HMGB1 released from transplanted microencapsulated NPIs triggers further inflammatory reactions in mice. Finally, we hypothesized that microencapsulated NPI xenotransplantation efficacy would be improved by treatment-targeting inflammatory reactions in a mouse model. METHODS: A total of 10 000 empty capsules (alginate-poly-L-ornithine-alginate) or 10 000 IEQ microencapsulated NPIs were transplanted into the peritoneal cavity of streptozotocin-induced diabetic C57BL/6 mice. RESULTS: The numbers of mononuclear cells in the peritoneal cavity following empty capsule or microencapsulated NPI transplantation were 4.8 × 10(6)  ± 0.9 × 10(6) and 13.6 × 10(6)  ± 3.0 × 10(6) , respectively (P < 0.05). Fluorescence-activated cell sorting (FACS) analysis revealed that tumor necrosis factor (TNF)-α-, interleukin (IL)-6-, interferon (IFN)-γ-, and/or IL-12-positive macrophages, neutrophils, and dendritic cells had infiltrated the peritoneal cavity after empty capsules or microencapsulated NPIs administration. IL-6 concentrations in the peritoneal lavage fluids on 7 days after empty capsule or microencapsulated NPI transplantation were 18.5 ± 10.0 and 157.4 ± 46.3 pg/ml, respectively (P < 0.001), while TNF-α concentrations were 4.6 ± 1.4 and 19.8 ± 8.4 pg/ml, respectively (P < 0.01). In addition, HMGB1 concentrations were 37.6 ± 6.6 and 117.4 ± 8.1 ng/ml, respectively (P < 0.0001). In vitro experiments revealed that the total amount of released HMGB1 into the culture medium of empty capsule (200 capsules/dish) and microencapsulated NPI (200 IEQ/dish) after hypoxic culture (1% O2 , 5% CO2 , and 94% N2 ) was 0 and 8.6 ± 2.2 ng, respectively (P < 0.001). FACS analysis revealed that TNF-α- and IL-6-positive macrophages were also observed in the peritoneal cavity following intraperitoneal injection of HMGB1 itself. Anti-TNF-α antibody treatment was associated with slightly prolonged graft survival and improved glucose tolerance 30 days after transplantation, but none of the recipients were remained normoglycemic. CONCLUSIONS: In conclusion, early inflammatory reactions might be therapeutic targets for the prolongation of microencapsulated NPIs graft survival. Thus, treatment-targeting inflammation might improve the efficiency of clinical microencapsulated NPI xenotransplantation.

Treatment for salivary gland hypofunction at both initial and advanced stages of Sjögren-like disease: a comparative study of bone marrow therapy versus spleen cell therapy with a 1-year monitoring period.

BACKGROUND AIMS: Non-obese diabetic mice (NOD) exhibit autoimmune Sjögren-like disease (SS-like). We reported previously that a combined-therapy consisting of immuno- and cell-based therapy rescued NOD from SS-like. However, therapies tested to date on NOD mice were aimed at the initial phase of SS-like. It is unknown whether therapies are effective in restoring salivary function when given at an advanced phase of SS-like. METHODS: The efficacy of two therapies (bone marrow versus spleen cells) was compared head-to-head for halting/reversing salivary hypofunction at two critical time points of SS-like (7-week-old NOD with normal saliva output and 20-week-old NOD with minimal saliva). NOD mice were divided into four groups: (i) control, (ii) complete Freund's adjuvant (CFA), (iii) bone marrow transplants with CFA or (iv) spleen cell transplants with CFA. Mice were monitored 8-12 months after therapy. RESULTS: Both cell therapies were effective during the initial phase of SS-like; salivary flow rates were maintained between 80-100% of pre-symptomatic levels. Spleen cell therapy was better than bone marrow when administered in the initial phase of SS-like. When cell therapies were given at an advanced phase of SS-like (20 weeks and older), salivary flow rates improved but were at best 50% of pre-symptomatic levels. Both cell therapies decreased tumor necrosis factor-α, transforming growth factor-ß1 levels and T and B cells while increasing epidermal growth factor and regulatory T cells. Elevated serum epidermal growth factor levels were measured in spleen-treated mice. CONCLUSIONS: A therapeutic effect in advanced phase disease, albeit in mice, holds promise for humans in which Sjögren syndrome is generally not diagnosed until a late stage.

Influence of relatively short-term culture on adult porcine islets for xenotransplantation.

Porcine islet xenotransplantation is a promising therapy for severe diabetes mellitus. Maintenance of the quality and quantity of porcine islets is important for the success of this treatment. Here, we aimed to elucidate the influence of relatively short-term (14 days) culture on adult porcine islets isolated from three micro-minipigs (P111, P112 and P121). Morphological characteristics of islets changed little after 14 days of culture. The viability of cultured islets was also maintained at a high level (> 80%). Furthermore, cultured islets exhibited similar glucose-stimulated insulin secretion and insulin content at Day 14 were preserved comparing with Day 1, while the expressions of Ins, Gcg and Sst were attenuated at Day 14. Xenotransplantation using diabetic nude mice showed no normalization of blood glucose but increased levels of plasma porcine C-peptide after the transplantation of 14 day cultured porcine islets. Histological assessment revealed that relatively short-term cultured porcine islets were successfully engrafted 56 days following transplantation. These data show that relatively short-term culture did not impair the quality of adult porcine islets in regard to function, morphology, and viability. Prevention of impairment of gene correlated with endocrine hormone is warranted for further improvement.

The porcine islet-derived organoid showed the characteristics as pancreatic duct.

Organoid is a tissue-engineered organ-like structure that resemble as an organ. Porcine islet-derived organoid might be used as an alternative donor of porcine islet xenotransplantation, a promising therapy for severe diabetes. In this study, we elucidated the characteristics of porcine islet organoids derived from porcine islets as a cell source for transplantation. Isolated porcine islets were 3D-cultured using growth factor-reduced matrigel in organoid culture medium consist of advanced DMEM/F12 with Wnt-3A, R-spondin, EGF, Noggin, IGF-1, bFGF, nicotinamide, B27, and some small molecules. Morphological and functional characteristics of islet organoids were evaluated in comparison with 2D-cultured islets in advanced DMEM/F12 medium. Relatively short-term (approximately 14 days)-cultured porcine islet organoids were enlarged and proliferated, but had an attenuated insulin-releasing function. Long-term (over a month)-cultured islet organoids could be passaged and cryopreserved. However, they showed pancreatic duct characteristics, including cystic induction, strong expression of Sox9, loss of PDX1 expression, and no insulin-releasing function. These findings were seen in long-term-cultured porcine islets. In conclusion, our porcine islet organoids showed the characteristics of pancreatic ducts. Further study is necessary for producing porcine islet-derived organoids having characteristics as islets.

Efficacy of Neonatal Porcine Bone Marrow-Derived Mesenchymal Stem Cell Xenotransplantation for the Therapy of Hind Limb Lymphedema in Mice.

Lymphedema is an intractable disease with few effective therapeutic options. Autologous mesenchymal stem cell (MSC) transplantation is a promising therapy for this disease. However, its use is limited by the cost and time for preparation. Recently, xenotransplantation of porcine MSCs has emerged as an alternative to autologous MSC transplantation. In this study, we aimed to clarify the usefulness of neonatal porcine bone marrow-derived MSC (NpBM-MSC) xenotransplantation for the treatment of lymphedema. One million NpBM-MSCs were xenotransplanted into the hind limbs of mice with severe lymphedema (MSC transplantation group). The therapeutic effects were assessed by measuring the femoral circumference, the volume of the hind limb, the number and diameter of lymphatic vessels in the hind limb, and lymphatic flow using a near-infrared fluorescence (NIRF) imaging system. We compared the effects using mice with lymphedema that did not undergo NpBM-MSC transplantation (negative control group). The condition of the transplanted NpBM-MSCs was also evaluated histologically. The femoral circumference and volume of the hind limb had been normalized by postoperative day (POD) 14 in the MSC transplantation group, but not in the negative control group (P = 0.041). NIRF imaging revealed that lymphatic flow had recovered in the MSC transplantation group by POD 14, as shown by an increase in luminance in the hind limb. Histological assessment also showed that the xenotransplantation of NpBM-MSC increased the proliferation of lymphatic vessels, but they had been rejected by POD 14. The xenotransplantation of NpBM-MSCs is an effective treatment for lymphedema, and this is mediated through the promotion of lymphangiogenesis.

Safety and Potential Effect of Intrauterine Infusion of Autologous Adipose Tissue-Derived Regenerative Cells in Patients With Implantation Failure: A Pilot Study.

BACKGROUND: Implantation failure due to thin endometrium has emerged as a major cause of infertility. In this study, we aimed to assess the safety and preliminary efficacy of adipose tissue-derived regenerative cells (ADRCs), a source of adipose-derived stem cells, in infertility patients with implantation failure. METHODS: Five infertile women with implantation failure despite artificial reproductive technology were enrolled in this study and treated with ADRCs via the intrauterine route. The primary outcome was the incidence of adverse events. Additional outcomes were endometrial thickness after ADRC treatment and pregnancy success after embryo transfer. RESULTS: There were no adverse events in any patient. There was no elevation of white blood cell count, C-reactive protein, or D-dimer levels. There was a significant difference in endometrial thickness in the secretory phase before versus after intrauterine transplantation of ADRCs (3.8 ± 1.3 mm versus 8.8 ± 2.8 mm, respectively; p<0.05). A gestational sac and fetal heartbeat were detected on transvaginal ultrasound in two of five patients. CONCLUSION: Intrauterine infusion of autologous ADRCs is a simple and safe procedure that may ameliorate the endometrial microenvironment in infertile women with implantation failure.

Vascular endothelial growth factor-C derived from CD11b+ cells induces therapeutic improvements in a murine model of hind limb ischemia.

OBJECTIVE: The use of bone marrow cells (BMCs) in therapeutic angiogenesis has been studied extensively. However, the critical paracrine effects of this treatment are still unclear. Therefore, we studied autotransfusable cells that produce vascular endothelial growth factor (VEGF), especially VEGF-C. METHODS: Male C57BL/6 mice with hind limb ischemia were administered intramuscular injections of phosphate-buffered saline as controls, or unsorted BMCs, sorted CD11b(+), or CD11b(-) cells from BMCs, and recombinant VEGF-C. To evaluate the treatments, perfusion was measured by laser Doppler scanning performed on days 0, 1, 3, 7, 14, 21, and 28. A functional assay was performed in parallel, with mice traversing an enclosed walkway. Capillary density was determined by directly counting vessels stained positive with von Willebrand factor at individual time points. Lymphangiogenesis was assessed by LYVE-1 positive cells. RESULTS: Postischemic recovery of hind limb perfusion significantly improved in BMC, CD11b(+), and VEGF-C treatment groups compared with the control groups, as assessed by laser Doppler scanning. On early operative days 1 and 3, the blood flow recovery ratio was higher in the CD11b(+)-treated group compared with BMC and VEGF-C treatment groups. In the functional assay, the VEGF-C group dramatically recovered compared with the control group. The capillary/myofiber ratio in the thigh muscle and number of LYVE-1 positive cells was higher in the CD11b(+) and VEGF-C groups than in controls. Furthermore, expression of VEGF-A, VEGF-C, and VEGF receptor messenger ribonucleic acid and protein was observed in CD11b(+) cells. CONCLUSIONS: The VEGF-C derived from CD11b(+) cells play a critical role in angiogenesis and lymphangiogenesis in a murine model of hind limb ischemia. Consequently, treatment with self-CD11b(+) cells accelerated recovery from ischemia and may be a promising therapeutic strategy for peripheral arterial disease patients.

Procedure of Adult Porcine Islet Isolation.

Islet transplantation is a useful therapeutic choice for severe diabetes mellitus; however, limited donor supplies have interfered with the use of this treatment. Therefore, the establishment of alternative donor sources and engineered tissue, which enables to produce appropriate insulin for controlling blood glucose, is an important challenge. The adult pig is a promising and feasible donor source and materials for the engineered tissue for the clinical setting among various candidates. The recent progress of gene-editing technology contributes to possible clinical porcine xenotransplantation, including porcine islet xenotransplantation. For the success of future clinical porcine islet xenotransplantation, establishing an islet isolation technique for acquiring adequate, good-quality porcine islets is equally important to use a gene-edited pig. However, the characteristics of porcine islets are different from other species; therefore, establishing a suitable technique for porcine islets is challenging. Impact statement Recent technological progress promotes the feasibility of xenotransplantation, including islet xenotransplantation, for clinical setting. Adult pig is a promising and feasible donor source for islet xenotransplantation and engineered tissue, which enables to control blood glucose in recipients. It is important to acquire porcine islets in good qualities for the promotion, however, establishing a technique for adult porcine islet isolation is important but challenging because of the vulnerability of adult porcine islets. Deciding the proper timing of stopping pancreatic digestion is one of the important factors for obtaining adult porcine islets in good quality.

Optimal temperature for the long-term culture of adult porcine islets for xenotransplantation.

Porcine islet xenotransplantation represents a promising therapy for severe diabetes mellitus. Long-term culture of porcine islets is a crucial challenge to permit the on-demand provision of islets. We aimed to identify the optimal temperature for the long-term culture of adult porcine islets for xenotransplantation. We evaluated the factors potentially influencing successful 28-day culture of islets at 24°C and 37°C, and found that culture at 37°C contributed to the stability of the morphology of the islets, the proliferation of islet cells, and the recovery of endocrine function, indicated by the expression of genes involved in pancreatic development, hormone production, and glucose-stimulated insulin secretion. These advantages may be provided by islet-derived CD146-positive stellate cells. The efficacy of xenotransplantation using islets cultured for a long time at 37°C was similar to that of overnight-cultured islets. In conclusion, 37°C might be a suitable temperature for the long-term culture of porcine islets, but further modifications will be required for successful xenotransplantation in a clinical setting.

Rescue of murine hind limb ischemia via angiogenesis and lymphangiogenesis promoted by cellular communication network factor 2.

Critical limb ischemia (CLI) is caused by severe arterial blockage with reduction of blood flow. The aim of this study was to determine whether therapeutic angiogenesis using cellular communication network factor 2 (CCN2) would be useful for treating CLI in an animal model. Recombinant CCN2 was administered intramuscularly to male C57BL/6J mice with hind limb ischemia. The therapeutic effect was evaluated by monitoring blood flow in the ischemic hind limb. In an in vivo assay, CCN2 restored blood flow in the ischemic hind limb by promoting both angiogenesis and lymphangiogenesis. VEGF-A and VEGF-C expression levels increased in the ischemic limb after treatment with CCN2. In an in vitro assay, CCN2 promoted proliferation of vascular and lymphatic endothelial cells, and it upregulated expression of Tgfb1 followed by expression of Vegfc and Vegfr3 in lymphatic endothelial cells under hypoxia. Suppression of Tgfb1 did not affect the activity of CCN2, activation of the TGF-ß/SMAD signaling pathway, or expression of Vegfr3 in lymphatic endothelial cells. In summary, treatment using recombinant CCN2 could be a promising therapeutic strategy for CLI.

Establishment of a Simple, Reproducible, and Long-lasting Hind Limb Animal Model of Lymphedema.

Background: Lymphedema is an intractable disease for which there is currently no established curative therapy. A reliable and long-lasting lymphedema model is essential for development of better treatments. In this study, we aimed to establish a simple, reproducible and long-lasting mouse model of lymphedema. Methods: Our model is characterized by a combination of a circumferential skin incision in the femoral region, complete dissection of regional lymph nodes, and ablation of the inguinal route in the femoral region. The characteristics of the lymphedema were evaluated and compared with those of two other models. One of these models involved dissection of the subiliac, popliteal, and sciatic lymph nodes (model A) and the other excision of the subiliac, popliteal, and sciatic lymph nodes with cauterization of lymphatic vessels and closure without a skin excision (model B). Results: Although the lymphedema in models A and B resolved spontaneously, that in the new model lasted for a month with increases in femoral circumference and hind limb volume, thickening of the skin, especially subcutaneous tissue, and congestion of peripheral lymphatic vessels. Furthermore, this model could be used for assessing the therapeutic effects of syngeneic mesenchymal stem cell transplantation. The average operation time for the new model was 14.4 ±â€…1.3 minutes. Conclusion: Long-lasting lymphedema can be achieved by our new model, making it suitable for assessing therapies for lymphedema.

Elevation in white blood cell count and development of hyper LDL cholesterolemia.

To investigate the relationship between white blood cell (WBC) count and incidence of hyper-low-density lipoprotein (LDL) cholesterolemia in a population-based longitudinal study. This is a retrospective study using data of annual health check-ups for residents of Iki City, Japan. A total of 3312 residents (≥ 30 years) without hyper-LDL cholesterolemia at baseline were included in this analysis. Primary outcome was incidence of hyper-LDL cholesterolemia (LDL cholesterol levels ≥ 3.62 mmol/L and/or use of lipid lowering drugs). During follow-up (average 4.6 years), 698 participants development of hyper-LDL cholesterolemia (incidence 46.8 per 1000 person-years). Higher incidence of hyper-LDL cholesterolemia was observed among participants with higher leukocyte count (1st quartile group: 38.5, 2nd quartile group: 47.7, 3rd quartile group: 47.3, and 4th quartile group: 52.4 per 1,000 person-years, P = 0.012 for trend). Statistically significant relation was observed even after adjustment for age, gender, smoking, alcohol intake, leisure-time exercise, obesity, hypertension and diabetes: hazard ratio 1.24 (95% confidence interval 0.99 to 1.54) for 2nd quartile group, 1.29 (1.03-1.62) for 3rd quartile group and 1.39 (1.10-1.75) for 4th quartile group, compared with 1st quartile group (P for trend = 0.006). Increased WBC count was related to incidence of hyper-LDL cholesterolemia in general Japanese population.

Serum triglyceride levels and incidence of hypertension in a general Japanese population: ISSA-CKD study.

The aim of this study was to clarify the relationship between fasting and nonfasting serum triglyceride (TG) levels and the incidence of hypertension in a general Japanese population. We conducted a population-based retrospective cohort study using annual health check-up data of residents of Iki City, Nagasaki Prefecture, Japan. A total of 3202 participants without hypertension at baseline were included in the present analysis. TG levels were classified as quartile 1 (<0.82 mmol/L), quartile 2 (0.83-1.13 mmol/L), quartile 3 (1.14-1.70 mmol/L) and quartile 4 (≥1.71 mmol/L) for men, and as quartile 1 (<0.70 mmol/L), quartile 2 (0.71-0.96 mmol/L), quartile 3 (0.97-1.34 mmol/L) and quartile 4 (≥1.35 mmol/L) for women. The outcome was incident hypertension. During an average follow-up of 4.4 years, 983 participants developed hypertension, according to the Cox proportional hazards model. The annual incidence of hypertension increased with an elevation in TG levels for men (5.88% in quartile 1, 8.30% in quartile 2, 7.62% in quartile 3, and 9.82% in quartile 4). This association was significant, even after adjustment for other risk factors: hazard ratio 1.41 [95% CI 1.07-1.85] for quartile 2, 1.30 [0.99-1.71] for quartile 3, and 1.59 [1.22-2.08] for quartile 4 compared with quartile 1 (p = 0.041 for trend). In contrast, there was no clear association between serum TG levels and the incidence of hypertension after adjustment for confounding factors among women (p = 0.240 for trend). High levels of serum TG were associated with the future incidence of hypertension in a general population of Japanese men but were not associated with that in women. Casual serum triglyceride levels and incidence of hypertension in a general Japanese population: ISSA-CKD study.