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Adipose-derived regenerative cell (ADRC) is a promising alternative source of autologous somatic stem cells for the repair of damaged tissue. This study aimed to assess the safety and feasibility of autologous ADRC implantation for therapeutic angiogenesis in patients with critical limb ischaemia (CLI). A clinical pilot study—Therapeutic Angiogenesis by Cell Transplantation using ADRCs (TACT-ADRC) study—was initiated in Japan. Adipose tissue was obtained by ordinary liposuction method. Isolated ADRCs were injected into the ischaemic limb. We performed TACT-ADRC procedure in five patients with CLI. At 6 months, no adverse events related to the TACT-ADRC were observed. No patients required major limb amputation, and ischaemic ulcers were partly or completely healed during the 6-month follow-up. In all cases, significant clinical improvements were seen in terms of rest pain and 6-min walking distance. Numbers of circulating CD34+ and CD133+ cells markers of progenitor cell persistently increased after ADRC implantation. The ratio of VEGF-A165b (an anti-angiogenic isoform of VEGF) to total VEGF-A in plasma significantly decreased after ADRC implantation. In vitro experiments, cultured with ADRC-conditioned media (CM) resulted in increased total VEGF-A and decreased VEGF-A165b in C2C12 cells, but not in macrophages. ADRC-CM also increased CD206+ cells expression and decreased TNF-α in macrophages. Autologous ADRC implantation was safe and effective in patients with CLI and could repair damaged tissue via its ability to promote angiogenesis and suppress tissue inflammation.

Background Circadian rhythm disorders, often seen in modern lifestyles, are a major social health concern. The aim of this study was to examine whether circadian rhythm disorders would influence angiogenesis and blood perfusion recovery in a mouse model of hind limb ischemia. Methods and Results A jet‐lag model was established in C57BL/6J mice using a light‐controlled isolation box. Control mice were kept at a light/dark 12:12 (12‐hour light and 12‐hour dark) condition. Concentrations of plasma vascular endothelial growth factor and circulating endothelial progenitor cells in control mice formed a circadian rhythm, which was diminished in the jet‐lag model ( P P P Clock , Bmal1 , and Cry ) in local tissues was upregulated after ischemic injury, the expression levels of cryptochrome (Cry) 1 and Cry2 were inhibited by the jet‐lag condition. Next, Cry1 and Cry2 double‐knockout mice were examined for blood perfusion recoveries and a reparative angiogenesis. Cry1 and Cry2 double‐knockout mice revealed suppressed capillary density ( P P CRY1/2 in human umbilical vein endothelial cells was accompanied by increased expression of WEE1 and decreased expression of HOXC5 . This was associated with decreased proliferative capacity, migration ability, and tube formation ability of human umbilical vein endothelial cells, respectively, leading to impairment of angiogenesis. Conclusions Our data suggest that circadian rhythm disorder deteriorates reparative ischemia‐induced angiogenesis and that maintenance of circadian rhythm plays an important role in angiogenesis.

The number of patients with ischemic cardiovascular diseases is significantly increasing as populations age. Therapeutic angiogenesis has been developed as a new treatment strategy for such patients. In recent years, the presence of mesenchymal stem cells in adipose tissues was reported, and regenerative medicine using these cells has attracted attention worldwide. In this review, we describe how the transplantation of adipose-derived regenerative cells enhances angiogenesis and tissue regeneration because of their multilineage potential and cytokine secretion. Then, the current status of therapeutic angiogenesis using adipose-derived regenerative cells in the field of cardiovascular medicine was also described. These cells present great advantages over bone marrow mononuclear cells, as these need easier, shorter, and less invasive preparations as well as less ethical concerns and immunological problems. The efficacy of adipose-derived regenerative cell transplantation in the treatment of various diseases was examined in several clinical trials with favorable results. Currently, a multicenter study of therapeutic angiogenesis using these cells is being conducted in patients with critical limb ischemia. In conclusion, we expect that this method will soon be established as a treatment for cardiovascular diseases that have been refractory to conventional treatments.

AimsSecreted factors produced by adipose tissue are involved in the pathogenesis of cardiovascular disease. We previously identified adipolin, also known as C1q/TNF-related protein 12, as an insulin-sensitizing adipokine. However, the role of adipolin in vascular disease remains unknown. Here, we investigated whether adipolin modulates pathological vascular remodelling.Methods and resultsAdipolin-knockout (APL-KO) and wild-type (WT) mice were subjected to wire-induced injury of the femoral artery. APL-KO mice showed increased neointimal thickening after vascular injury compared with WT mice, which was accompanied by an enhanced inflammatory response and vascular cell proliferation in injured arteries. Adipolin deficiency also led to a reduction in transforming growth factor-β (TGF-β) 1 protein levels in injured arteries. Treatment of cultured macrophages with adipolin protein led to a reduction in lipopolysaccharide-stimulated expression of inflammatory mediators, including tumour necrosis factor (TNF)-α, interleukin (IL) 6, and monocyte chemotactic protein (MCP)-1. These effects were reversed by inhibition of TGF-β receptor II (TGF-βRII)/Smad2 signalling. Adipolin also reduced platelet-derived growth factor (PDGF)-BB-stimulated proliferation of vascular smooth muscle cells (VSMCs) through a TGF-βRII/Smad2-dependent pathway. Furthermore, adipolin treatment significantly increased TGF-β1 concentration in media from cultured VSMCs and macrophages.ConclusionThese data indicate that adipolin protects against the development of pathological vascular remodelling by attenuating macrophage inflammatory responses and VSMC proliferation.

Introduction: Circadian rhythm disorder seen in shift-worker or jet-lag is major social health concerns in advanced industrialized countries. The aim of this study was to examine if circadian rhythm disorders would influence on angiogenesis and blood perfusion recovery in a mouse model of hind limb ischemia (HLI). Methods and Results: First, we established a jet-lag model in C57BL/6J (wild type; WT) mice (8-10 weeks old, N=10 for each) using a light controlled isolation box. Mice were exposed to advanced 8-hr light phase once every 4 days in a jet-lag group as previously described. Conversely, control mice were kept a regular condition of LD 12:12 (12-hr light and 12-hr dark). Then, we surgically induced HLI in each group. The results showed that the condition jet-lag deteriorated capillary formation detected by CD31-immunohistochemistry at post-operative day (POD) 28 and tissue blood perfusion recovery demonstrated by laser Doppler perfusion imaging (LDPI) in HLI. The expression of clock genes (i.e. Clock, Bmal1, Per2, Cry1 and 2 ) in ischemic muscles were regulated by jet-lag condition at POD7.Next, we examined whether inhibition of clock gene had any effects on angiogenesis. For this study, we focused on Cryptochrome ( Cry ), which is well known as one of the core-loop forming clock genes producing circadian rhythm in mammals. Our loss-of-function study revealed that the abilities of proliferation, migration and tube formation were significantly inhibited by CRY1 and CRY2 double knockdown in HUVECs. Interestingly, although the knockdown of CRY1 and CRY2 changed the mRNA expression of PERIOD2 , it did not affect those of BMAL1 and CLOCK in HUVECs. Finally, we tested if Cry1 and Cry2 double knockout ( Cry1/2 -DKO) mice of HLI models displayed worse blood perfusion recoveries with deterioration of angiogenesis. Cry1/2 -DKO mice were reported to display circadian rhythm disorder in previous reports. As results, compared with control WT mice, Cry1/2 -DKO mice revealed suppressed capillary density and tissue blood perfusion recovery in HLI model. Conclusion: Our data suggest that a maintenance of circadian rhythm plays an important role in reparative angiogenesis of the tissue ischemia model.

Introduction: Implantation of adipose-derived regenerative cells (ADRC) is a promising novel strategy to augment angiogenesis and blood perfusion recovery in ischemic diseases with no other therapeutic option. However, there is a clinical concern underlying therapeutic angiogenesis that implantation of ADRC may promote tumor growth and metastasis via remote angiogenesis. Accordingly, we tested whether therapeutic angiogenesis with ADRC against hindlimb ischemia (HLI) would affect remote tumor growth and angiogenesis in a tumor-bearing mouse ischemic hindlimb model. Methods and results: B16F10-Luc (murine melanoma cells expressing luciferase, 1x106 cells/animal) were implanted to C57BL/6J mice’s (male, 8-10 weeks old, n=10) back. Mice were subjected to unilateral HLI surgery one day after tumor implantation. Then, mice were randomly assigned to the control group or the ADRC group (n=5 for each). ADRC (1x106 cells/animal) or PBS were implanted/injected into ischemic hindlimb muscles one day after the surgery. Blood perfusion recovery in HLI by laser Doppler perfusion imaging system and tumor size by a caliper were measured every week up to 21 days after surgery. At POD 21, tumor weight and luciferase activity in primary tumors obtained by in vivo bioluminescence imaging system were also evaluated. Immunohistochemistry by CD31 or LYVE1 staining was performed to detect feeder arteries or outflow lymphatic vessels in tumors. The results demonstrated that better blood perfusion recovery and more capillary density in HLI was observed in the ADRC group than in the control group (p Conclusions: Our data indicated that therapeutic angiogenesis with ADRC implantation against HLI did not promote remote tumor growth, angiogenesis and metastasis.

Therapeutic angiogenesis with autologous stem/progenitor cells is a promising novel strategy for treatment of severe ischemic diseases. Human clinical trials utilizing autologous adipose-derived regenerative cells (ADRCs) have not reported treatment-related critical adverse effects thus far. However, there is still a large knowledge gap regarding whether treatment of ischemic diseases with angiogenic therapy using ADRCs would promote unfavorable angiogenesis associated with tumors in vivo. Herein, we addressed this clinical question using a mouse hindlimb ischemia (HLI) and simultaneous remote tumor implantation model. C57BL/6J background wild-type mice were injected with murine B16F10 melanoma cells on their back, 1 day before ischemic surgery. These mice were subjected to surgical unilateral hindlimb ischemia, followed by ADRC implantation or PBS injection into the hindlimb ischemic muscles on the next day. Intramuscular implantation of ADRCs enhanced tissue capillary density and blood flow examined by a laser Doppler blood perfusion analysis in hind limb. However, this therapeutic regimen for ischemic limb using ADRCs did not affect remote melanoma growth nor the density of its feeder artery, angiogenesis, and lymphatic vessels compared with the PBS group. In addition, no distant metastases were detected in any of the mice regardless of the group. In conclusion, local implantation of ADRCs promotes angiogenesis in response to tissue ischemia in the hindlimb without promoting remote tumor growth and related angio/lymphangiogenesis. Therapeutic angiogenesis to the ischemic hindlimb using ADRCs seems to be safe regarding remote tumor growth.

Strategies to promote revascularization are valuable for ischemic cardiovascular disease. Although C1q/TNF-related protein (CTRP) 9 is an adiponectin paralog with protective properties against cardiometabolic disorders, the role of endogenous CTRP9 in endothelial function is largely unknown. This study aimed to investigate the effects of CTRP9 on revascularization processes and dissected the potential mechanisms. CTRP9-knockout (KO) and wild-type (WT) mice were subjected to unilateral hindlimb ischemic surgery. CTRP9-KO mice exhibited impaired blood flow recovery and decreased capillary density in the ischemic limb compared with WT mice. In both CTRP9-KO and WT mice, systemic delivery of an adenoviral vector expressing CTRP9 (Ad-CTRP9) accelerated blood flow recovery. Treatment with recombinant CTRP9 protein increased network formation and migration of cultured human umbilical vein endothelial cells (HUVECs). CTRP9 promoted the phosphorylation of AMP-activated kinase (AMPK), Akt, and endothelial nitric oxide synthase (eNOS) in HUVECs. CTRP9-KO mice also showed reduced phosphorylation levels of AMPK, Akt, and eNOS in the ischemic limbs compared with WT mice. Furthermore, blockade of AMPK or Akt signaling pathway reversed the CTRP9-stimulated eNOS phosphorylation in HUVECs. Treatment with the NOS inhibitor significantly reduced CTRP9-stimulated network formation and migration of HUVECs. Of note, Ad-CTRP9 had no effects on blood flow of the ischemic limb in eNOS-KO mice. These results indicated that CTRP9 promotes endothelial cell function and ischemia-induced revascularization through the eNOS-dependent mechanism, suggesting that CTRP9 represents a target molecule for treatment of ischemic vascular diseases.

Background and purpose Fluorine-18-flurodeoxyglucose positron emission computed tomography (18FDG-PET CT) is the sole but time-consuming and expensive examination assessing active inflammatory myocardium noninvasively in patients with cardiac sarcoidosis (CS). Though immunosuppression is used to treat CS, little is known about the cardiac biomarkers for determining therapeutic effect to immunosuppression therapy other than 18FDG-PET. Methods From Aug 2016, we prospectively enrolled 50 sarcoidosis patients with positively accumulated of FDG in the heart. The initial dose of prednisolone was 30mg/day, wherefrom the dose was tapered down 5mg/month until 6 months. After 6 months, follow-up 18FDG-PET was performed. Using 18FDG-PET images, we calculated total lesion glycolysis (TLG; SUVmeam x metabolic volume) and compared it to various cardiac markers. Non-responder was defined as TLG reduction rate Results In 50 CS patients, 41 patients had serial 18FDG-PET before and 6 months after immunosuppression therapy. 18FDG-PET images were acquired following 7 day's carbohydrate limitation and after at least 18-h fasting (mean free fatty acid level right before 18FDG-PET acquisition was 1.03 mEq/L). The mean age and plasma brain natriuretic peptide (BNP) level were 61.9 years old, 184 pg/mL at baseline, respectively. Because of immunosuppression therapy, TLG was significantly reduced from 195.3 (38.6–339.6) to 1.1 (0.01–7.37), p Figure 1 Conclusions 1) By immunosuppression therapy using prednisolone for CS, about 85% patients showed significant reduction of increased myocardial FDG accumulation. 2) The sustained high level of serum TnT at post 6 months therapy indicates persistent inflammation of sarcoidosis in the heart, which could be an expedient marker for determining therapeutic effect.

Transcription factors and epigenetic modulators are involved in the maintenance of self-renewal in embryonic stem (ES) cells. Here, we demonstrate the existence of a regulatory loop in ES cells between Sox2, an indispensable transcription factor for self-renewal, and embryonic ectoderm development (Eed), an epigenetic modulator regulating histone methylation. We found that Sox2 and Eed positively regulate each other's expression. Interestingly, Sox2 overexpression suppressed the induction of differentiation-associated genes in Eed-deficient ES cells without restoring histone methylation. This Sox2-mediated suppression was prevented by knockdown of the histone acetyltransferase (HAT), Tip60 or Elp3, and Sox2 stimulated expression of these HATs. Furthermore, forced expression of either HAT resulted in repression of differentiation-associated genes in Eed-deficient cells. These results suggest that Sox2 overcame the phenotype of Eed-deficient ES cells by promoting histone acetylation. We also found that knockout of Eed and knockdown of these HATs synergistically enhanced the upregulation of differentiation-associated genes in ES cells. Taken together, our results suggest that the Eed/Sox2 regulatory loop contributes to the maintenance of self-renewal in ES cells by controlling histone methylation and acetylation.

Transcription factors and epigenetic modulators are involved in the maintenance of self-renewal in embryonic stem (ES) cells. Here, we demonstrate the existence of a regulatory loop in ES cells between Sox2, an indispensable transcription factor for self-renewal, and embryonic ectoderm development (Eed), an epigenetic modulator regulating histone methylation. We found that Sox2 and Eed positively regulate each other's expression. Interestingly, Sox2 overexpression suppressed the induction of differentiation-associated genes in Eed-deficient ES cells without restoring histone methylation. This Sox2-mediated suppression was prevented by knockdown of the histone acetyltransferase (HAT), Tip60 or Elp3, and Sox2 stimulated expression of these HATs. Furthermore, forced expression of either HAT resulted in repression of differentiation-associated genes in Eed-deficient cells. These results suggest that Sox2 overcame the phenotype of Eed-deficient ES cells by promoting histone acetylation. We also found that knockout of Eed and knockdown of these HATs synergistically enhanced the upregulation of differentiation-associated genes in ES cells. Taken together, our results suggest that the Eed/Sox2 regulatory loop contributes to the maintenance of self-renewal in ES cells by controlling histone methylation and acetylation.