Neurons limit angiogenesis by titrating VEGF in retina.

Vascular and nervous systems, two major networks in mammalian bodies, show a high degree of anatomical parallelism and functional crosstalk. During development, neurons guide and attract blood vessels, and consequently this parallelism is established. Here, we identified a noncanonical neurovascular interaction in eye development and disease. VEGFR2, a critical endothelial receptor for VEGF, was more abundantly expressed in retinal neurons than in endothelial cells, including endothelial tip cells. Genetic deletion of VEGFR2 in neurons caused misdirected angiogenesis toward neurons, resulting in abnormally increased vascular density around neurons. Further genetic experiments revealed that this misdirected angiogenesis was attributable to an excessive amount of VEGF protein around neurons caused by insufficient engulfment of VEGF by VEGFR2-deficient neurons. Moreover, absence of neuronal VEGFR2 caused misdirected regenerative angiogenesis in ischemic retinopathy. Thus, this study revealed neurovascular crosstalk and unprecedented cellular regulation of VEGF: retinal neurons titrate VEGF to limit neuronal vascularization. PAPERFLICK:

Preoperative photoacoustic versus indocyanine green lymphography in lymphaticovenular anastomosis outcomes for lower extremity lymphedema: A pilot study.

BACKGROUND: Identification of the proper lymphatics is important for successful lymphaticovenular anastomosis (LVA) for lymphedema; however, visualization of lymphatic vessels is challenging. Photoacoustic lymphangiography (PAL) can help visualize lymphatics more clearly than other modalities. Therefore, we investigated the usefulness of PAL and determined whether the clear and three-dimensional image of PAL affects LVA outcomes. METHODS: We recruited 22 female patients with lower extremity lymphedema. The operative time, number of incisions, number of anastomoses, lymphatic vessel detection rate (number of functional lymphatics identified during the operation/number of incisions), and limb volume changes preoperatively and 3 months postoperatively were compared retrospectively. The patients were divided according to whether PAL was performed or not, and results were compared between those undergoing PAL (PAL group; n = 10) and those who did not (near-infrared fluorescence [NIRF] group, n = 12). RESULTS: The mean age of the patients was 55.9 ± 15.1 years in the PAL group and 50.7 ± 14.9 years in the NIRF group. One patient in the PAL group and three in the NIRF group had primary lymphedema. Eighteen patients (PAL group, nine; and NIRF group, nine) had secondary lymphedema. Based on preoperative evaluation using the International Society of Lymphology (ISL) classification, eight patients were determined to be in stage 2 and two patients in late stage 2 in the PAL group. In contrast, in the NIRF group, one patient was determined to be in stage 0, three patients each in stage 1 and stage 2, and five patients in late stage 2. Lymphatic vessel detection rates were 93% (42 LVAs and 45 incisions) and 83% (50 LVAs and 60 incisions) in the groups with and without PAL, respectively (p = 0.42). Limb volume change was evaluated in five limbs of four patients and in seven limbs of five patients in the PAL and NIRF groups as 336.6 ± 203.6 mL (5.90% ± 3.27%) and 52.9 ± 260.7 mL (0.71% ± 4.27%), respectively. The PAL group showed a significant volume reduction. (p = .038). CONCLUSIONS: Detection of functional lymphatic vessels on PAL is useful for treating LVA.

Fibroblast Growth Factor 7 Suppresses Fibrosis and Promotes Epithelialization during Wound Healing in Mouse Fetuses.

Adult mammalian wounds leave visible scars, whereas skin wounds in developing mouse fetuses are scarless until a certain point in development when complete regeneration occurs, including the structure of the dermis and skin appendages. Analysis of the molecular mechanisms at this transition will provide clues for achieving scarless wound healing. The fibroblast growth factor (FGF) family is a key regulator of inflammation and fibrosis during wound healing. We aimed to determine the expression and role of FGF family members in fetal wound healing. ICR mouse fetuses were surgically wounded at embryonic day 13 (E13), E15, and E17. Expression of FGF family members and FGF receptor (FGFR) in tissue samples from these fetuses was evaluated using in situ hybridization and reverse transcription-quantitative polymerase chain reaction. Fgfr1 was downregulated in E15 and E17 wounds, and its ligand Fgf7 was upregulated in E13 and downregulated in E15 and E17. Recombinant FGF7 administration in E15 wounds suppressed fibrosis and promoted epithelialization at the wound site. Therefore, the expression level of Fgf7 may correlate with scar formation in late mouse embryos, and external administration of FGF7 may represent a therapeutic option to suppress fibrosis and reduce scarring.

Mast Cells Are Activated in the Giant Earlobe Keloids: A Case Series.

Mast cells and inflammatory cells are abundant in keloid and hypertrophic scar tissues. Even if the cause of physical injury is similar, such as piercing or scratching with hands, clinical findings show differences in the size of keloids in the same area. Hence, we performed histological studies on giant keloids larger than the earlobe, and other smaller keloids. We also examined the risk factors associated with the formation of giant lesions. No statistically significant differences in the association of the risk factors were observed. However, histological observations clearly showed a high number of degranulated or active mast cells with a trend towards a greater number of degranulated mast cells in the giant keloid tissues. Collagen production also tended to increase. Two patients with giant keloids were severely obese, suggesting that the persistent inflammatory state of obesity may also be involved in the growth of keloid lesions.

Application of Photoacoustic Imaging for Lymphedema Treatment.

BACKGROUND: Lymphatic vessels are difficult to identify using existing modalities as because of their small diameter and the transparency of the lymph fluid flowing through them. METHODS: Here, we introduce photoacoustic lymphangiography (PAL), a new modality widely used for lymphedema treatment, to observe limb lymphatic vessels. The photoacoustic imaging system used in this study can simultaneously visualize lymphatic vessels and veins with a high resolution (0.2 mm) and can also observe their three-dimensional relationship with each other. RESULTS: High-resolution images of the lymphatic vessels, detailed structure of the dermal back flow, and the three-dimensional positional relationship between the lymphatic vessels and veins were observed by PAL. CONCLUSION: The clear image provided by PAL could have a major application in pre- and postoperative use during lymphaticovenular anastomosis for lymphedema treatment.

Neuron-derived VEGF contributes to cortical and hippocampal development independently of VEGFR1/2-mediated neurotrophism.

Vascular endothelial growth factor (VEGF) is a potent mitogen critical for angiogenesis and organogenesis. Deletion or inhibition of VEGF during development not only profoundly suppresses vascular outgrowth, but significantly affects the development and function of various organs. In the brain, VEGF is thought to not only promote vascular growth, but also directly act on neurons as a neurotrophic factor by activating VEGF receptors. In the present study, we demonstrated that deletion of VEGF using hGfap-Cre line, which recombines genes specifically in cortical and hippocampal neurons, severely impaired brain organization and vascularization of these regions. The mutant mice had motor deficits, with lethality around the time of weaning. Multiple reporter lines indicated that VEGF was highly expressed in neurons, but that its cognate receptors, VEGFR1 and 2 were exclusive to endothelial cells in the brain. In accordance, mice lacking neuronal VEGFR1 and VEGFR2 did not exhibit neuronal deformities or lethality. Taken together, our data suggest that neuron-derived VEGF contributes to cortical and hippocampal development likely through angiogenesis independently of direct neurotrophic effects mediated by VEGFR1 and 2.

Implications of NAD metabolism in pathophysiology and therapeutics for neurodegenerative diseases.

Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme that mediates various redox reactions. Particularly, mitochondrial NAD plays a critical role in energy production pathways, including the tricarboxylic acid (TCA) cycle, fatty acid oxidation, and oxidative phosphorylation. NAD also serves as a substrate for ADP-ribosylation and deacetylation by poly(ADP-ribose) polymerases (PARPs) and sirtuins, respectively. Thus, NAD regulates energy metabolism, DNA damage repair, gene expression, and stress response. Numerous studies have demonstrated the involvement of NAD metabolism in neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and retinal degenerative diseases. Mitochondrial dysfunction is considered crucial pathogenesis for neurodegenerative diseases such as AD and PD. Maintaining appropriate NAD levels is important for mitochondrial function. Indeed, decreased NAD levels are observed in AD and PD, and supplementation of NAD precursors ameliorates disease phenotypes by activating mitochondrial functions. NAD metabolism also plays an important role in axonal degeneration, a characteristic feature of peripheral neuropathy and neurodegenerative diseases. In addition, dysregulated NAD metabolism is implicated in retinal degenerative diseases such as glaucoma and Leber congenital amaurosis, and NAD metabolism is considered a therapeutic target for these diseases. In this review, we summarize the involvement of NAD metabolism in axon degeneration and various neurodegenerative diseases and discuss perspectives of nutritional intervention using NAD precursors.

Placental labyrinth formation in mice requires endothelial FLRT2/UNC5B signaling.

The placental labyrinth is the interface for gas and nutrient exchange between the embryo and the mother; hence its proper development is essential for embryogenesis. However, the molecular mechanism underlying development of the placental labyrinth, particularly in terms of its endothelial organization, is not well understood. Here, we determined that fibronectin leucine-rich transmembrane protein 2 (FLRT2), a repulsive ligand of the UNC5 receptor family for neurons, is unexpectedly expressed in endothelial cells specifically in the placental labyrinth. Mice lacking FLRT2 in endothelial cells exhibited embryonic lethality at mid-gestation, with systemic congestion and hypoxia. Although they lacked apparent deformities in the embryonic vasculature and heart, the placental labyrinths of these embryos exhibited aberrant alignment of endothelial cells, which disturbed the feto-maternal circulation. Interestingly, this vascular deformity was related to endothelial repulsion through binding to the UNC5B receptor. Our results suggest that the proper organization of the placental labyrinth depends on coordinated inter-endothelial repulsion, which prevents uncontrolled layering of the endothelium.

Implications of altered NAD metabolism in metabolic disorders.

Nicotinamide adenine dinucleotide (NAD) is an important coenzyme that participates in various energy metabolism pathways, including glycolysis, ß-oxidation, and oxidative phosphorylation. Besides, it is a required cofactor for post-translational modifications such as ADP-ribosylation and deacetylation by poly (ADP-ribose) polymerases (PARPs) and sirtuins, respectively. Thus, NAD regulates energy metabolism, DNA damage repair, gene expression, and stress response through these enzymes. Numerous studies have shown that NAD levels decrease with aging and under disturbed nutrient conditions, such as obesity. Additionally, a decline in NAD levels is closely related to the development of various metabolic disorders, including diabetes and fatty liver disease. In addition, many studies have revealed that administration of NAD precursors, such as nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), efficiently increase NAD levels in various tissues and prevent such metabolic diseases. These NAD precursors are contained in natural foods, such as cow milk, vegetables, and meats. Therefore, altered NAD metabolism can be a practical target for nutritional intervention. Recently, several human clinical trials using NAD precursors have been conducted to investigate the safety, pharmacokinetics, and efficacy against metabolic disorders such as glucose intolerance. In this review, we summarize current knowledge on the implications of NAD metabolism in metabolic diseases and discuss the outcomes of recent human clinical trials.

Deletion of PHGDH in adipocytes improves glucose intolerance in diet-induced obese mice.

Serine is a nonessential amino acid and plays an important role in cellular metabolism. In mammalian serine biosynthesis, 3-phosphoglycerate dehydrogenase (PHGDH) is considered a rate-limiting enzyme and is required for normal development. Although the biological functions of PHGHD in the nervous system have been intensively studied, its function in adipose tissue is unknown. In this study, we found that PHGDH is abundantly expressed in mature adipocytes of white adipose tissue. We generated an adipocyte-specific PHGDH knockout mouse (PHGDH FKO) and used it to investigate the role of serine biosynthesis in adipose tissues. Although PHGDH FKO mice had no apparent defects in adipose tissue development, these mice ameliorated glucose intolerance upon diet-induced obesity. Additionally, we found that the serine levels increase drastically in the adipose tissues of obese wild type mice, whereas no significant rise was observed in PHGDH FKO mice. Furthermore, wild type mice fed a serine-deficient diet also exhibited better glucose tolerance. These results suggest that PHGDH-mediated serine biosynthesis has important roles in adipose tissue glucose metabolism and could be a therapeutic target for diabetes in humans.

Simultaneous measurement of NAD metabolome in aged mice tissue using liquid chromatography tandem-mass spectrometry.

Nicotinamide adenine dinucleotide (NAD) is a major co-factor that mediates multiple biological processes including redox reaction and gene expression. Recently, NAD metabolism has received considerable attention because administration of NAD precursors exhibited beneficial effects against aging-related metabolic disorders in animals. Although numerous studies have reported that NAD levels decline with aging in multiple animal tissues, the pathway and kinetics of NAD metabolism in aged organs are not completely understood. To determine the NAD metabolism upon aging, we developed targeted metabolomics based on an LC/MS/MS system. Our method is simple and applicable to crude biological samples, including culture cells and animal tissues. Unlike a conventional enzymatic cycling assay, our approach can determine NAD and NADH (reduced form of NAD) by performing a single sample preparation. Further, we validated our method using biological samples and investigated the alteration of the NAD metabolome during aging. Consistent with previous reports, the NAD levels in the liver and skeletal muscle decreased with aging. Further, we detected a significant increase in nicotinamide mononucleotide and nicotinamide riboside in the kidney upon aging. The LC/MS/MS-based NAD metabolomics that we have developed is extensively applicable to biomedical studies, and the results will present innovative ideas for the aging studies, especially for that of NAD metabolism.

Prognosis of critical limb ischemia: Major vs. minor amputation comparison.

Healthcare providers treating wounds have difficulties assessing the prognosis of patients with critical limb ischemia who had been discharged after complete healing of major amputation wounds. The word "major" in "major amputation" gives the impression of "being more severe" than "minor amputation." Therefore, even if wounds are healed after major amputation, they imagine that prognosis after major amputation would be poorer than that after minor amputation. We investigated the prognosis of diabetic nephropathy patients 2 years after amputations. Those patients underwent dialysis as well as amputation following percutaneous transluminal angioplasty for their foot wounds. They were ambulatory prior to these surgeries. Among 56 cases of minor amputation, 45 were males and 11 were females, and mortality was 41.1%. The mortality of cases with and without a coronary intervention history was 53.1% and 25.0%, respectively (p = 0.034). Among 10 cases of major amputation, 9 were males and 1 was female, and mortality was 60%. The mortality of cases with and without a coronary intervention history was 75.0% and 0%, respectively. Although we predicted poor prognosis in cases with major amputation, there was no significant difference in mortality 2 years after amputations (p = 0.267). Thus far poor prognosis has been reported for major amputation. It might be due to inclusion of the following patients: patients with wounds proximal to ankle joints, patients with extensive gangrene spreading to the lower legs, patients with septicemia from wound infection and who died around the time of operation, and patients with malnutrition. The results of our present study showed that the outcomes at 2 years postoperatively were similar between patients with major amputations and those with minor amputations, if surgical wounds were able to heal. We should not estimate the prognosis by the level of amputation, rather we should consider the effect of coronary intervention history on prognosis.

Pregnancy after hysteroscopic metroplasty under laparoscopy in a woman with complete septate uterus: a case report.

A 31-year-old nulligravid woman with a 3 year history of infertility visited our hospital. After consultation and a transvaginal ultrasound and MR imaging, her uterine anomaly was identified as complete septate uterus: class V (a) by the American Fertility Society (AFS). She had a doubled uterine cervix and a vaginal septum. Hysteroscopic metroplasty was performed with the aid of a laparoscopy. Both tubal patencies were confirmed with indigocarmine in a laparoscopic image. Laparoscopic electronic cautery was also done on the left ovarian endometrioma (stage 1 endometriosis; the revised American Society for Reproductive Medicine (rASRM) classification 4 point minimal). We distrained an intrauterine device in the uterine cavity and removed it after two cycles of menstruation. The patient subsequently became pregnant during her third menstrual cycle and the current progress of her pregnancy is favorable.

Extracellular matrix protein tenascin-C is required in the bone marrow microenvironment primed for hematopoietic regeneration.

The BM microenvironment is required for the maintenance, proliferation, and mobilization of hematopoietic stem and progenitor cells (HSPCs), both during steady-state conditions and hematopoietic recovery after myeloablation. The ECM meshwork has long been recognized as a major anatomical component of the BM microenvironment; however, the molecular signatures and functions of the ECM to support HSPCs are poorly understood. Of the many ECM proteins, the expression of tenascin-C (TN-C) was found to be dramatically up-regulated during hematopoietic recovery after myeloablation. The TN-C gene was predominantly expressed in stromal cells and endothelial cells, known as BM niche cells, supporting the function of HSPCs. Mice lacking TN-C (TN-C(-/-)) mice showed normal steady-state hematopoiesis; however, they failed to reconstitute hematopoiesis after BM ablation and showed high lethality. The capacity to support transplanted wild-type hematopoietic cells to regenerate hematopoiesis was reduced in TN-C(-/-) recipient mice. In vitro culture on a TN-C substratum promoted the proliferation of HSPCs in an integrin α9-dependent manner and up-regulated the expression of the cyclins (cyclinD1 and cyclinE1) and down-regulated the expression of the cyclin-dependent kinase inhibitors (p57(Kip2), p21(Cip1), p16(Ink4a)). These results identify TN-C as a critical component of the BM microenvironment that is required for hematopoietic regeneration.

Twist2 contributes to skin regeneration and hair follicle formation in mouse fetuses.

Unlike adult mammalian wounds, early embryonic mouse skin wounds completely regenerate and heal without scars. Analysis of the underlying molecular mechanism will provide insights into scarless wound healing. Twist2 is an important regulator of hair follicle formation and biological patterning; however, it is unclear whether it plays a role in skin or skin appendage regeneration. Here, we aimed to elucidate Twist2 expression and its role in fetal wound healing. ICR mouse fetuses were surgically wounded on embryonic day 13 (E13), E15, and E17, and Twist2 expression in tissue samples from these fetuses was evaluated via in situ hybridization, immunohistochemistry, and reverse transcription-quantitative polymerase chain reaction. Twist2 expression was upregulated in the dermis of E13 wound margins but downregulated in E15 and E17 wounds. Twist2 knockdown on E13 left visible marks at the wound site, inhibited regeneration, and resulted in defective follicle formation. Twist2-knockdown dermal fibroblasts lacked the ability to undifferentiate. Furthermore, Twist2 hetero knockout mice (Twist + /-) formed visible scars, even on E13, when all skin structures should regenerate. Thus, Twist2 expression correlated with skin texture formation and hair follicle defects in late mouse embryos. These findings may help develop a therapeutic strategy to reduce scarring and promote hair follicle regeneration.

Compound 13 Promotes Epidermal Healing in Mouse Fetuses via Activation of AMPK.

Unlike adults, early developing fetuses can completely regenerate tissue, and replicating this could lead to the development of treatments to reduce scarring. Mice epidermal structures, including wound healing patterns, are regenerated until embryonic day (E) 13, leaving visible scars thereafter. These patterns require actin cable formation at the epithelial wound margin through AMP-activated protein kinase (AMPK) activation. We aimed to investigate whether the administration of compound 13 (C13), a recently discovered AMPK activator, to the wound could reproduce this actin remodeling and skin regeneration pattern through its AMPK activating effect. The C13 administration resulted in partial formations of actin cables, which would normally result in scarring, and scar reduction during the healing of full-layer skin defects that occurred in E14 and E15 fetuses. Furthermore, C13 was found to cause AMPK activation in these embryonic mouse epidermal cells. Along with AMPK activation, Rac1 signaling, which is involved in leaflet pseudopodia formation and cell migration, was suppressed in C13-treated wounds, indicating that C13 inhibits epidermal cell migration. This suggests that actin may be mobilized by C13 for cable formation. Administration of C13 to wounds may achieve wound healing similar to regenerative wound healing patterns and may be a potential candidate for new treatments to heal scars.

Regeneration of Panniculus Carnosus Muscle in Fetal Mice Is Characterized by the Presence of Actin Cables.

Mammalian skin, including human and mouse skin, does not regenerate completely after injury; it is repaired, leaving a scar. However, it is known that skin wounds up to a certain stage of embryonic development can regenerate. The mechanism behind the transition from regeneration to scar formation is not fully understood. Panniculus carnosus muscle (PCM) is present beneath the dermal fat layer and is a very important tissue for wound contraction. In rodents, PCM is present throughout the body. In humans, on the other hand, it disappears and becomes a shallow fascia on the trunk. Fetal cutaneous wounds, including PCM made until embryonic day 13 (E13), regenerate completely, but not beyond E14. We visualized the previously uncharacterized development of PCM in the fetus and investigated the temporal and spatial changes in PCM at different developmental stages, ranging from full regeneration to non-regeneration. Furthermore, we report that E13 epidermal closure occurs through actin cables, which are bundles of actomyosin formed at wound margins. The wound healing process of PCM suggests that actin cables may also be associated with PCM. Our findings reveal that PCM regenerates through a similar mechanism.

Recruited minced skin grafting for improving the skin appearance of the donor site of a split-thickness skin graft.

OBJECTIVE: To improve skin appearance at the donor site of a split-thickness skin graft, part of the harvested skin was minced and grafted back onto the site in a process we named "recruited minced skin grafting." MATERIALS AND METHODS: Thirteen Japanese patients who needed split-thickness skin grafts were treated with recruited minced skin grafting. Five patients were used as controls, in whom donor sites were treated with the traditional method. Part of the split-thickness skin was minced using two surgical blades (number 24) to an approximate particle size of less than 0.5 mm. Minced skin was spread and transplanted onto the donor site and covered with polyurethane foam. Twelve months after the operation, donor sites were scored for hypopigmentation, hyperpigmentation, redness, and disruption of skin texture. Gross appearance was evaluated according to total score. RESULTS: Donor sites treated with recruited minced skin grafts had significantly better appearance than those of controls. Donor sites that had more than 5% of the total area treated tended to have better results. CONCLUSION: Recruited minced skin grafting is a good method of improving the appearance of the donor site.

Effect of Sonic Hedgehog on the Regeneration of Epidermal Texture Patterns.

Wounds on embryonic mouse fetuses regenerate up to embryonic day (E) 13, but after E14, the pattern is lost and a visible scar remains. We hypothesized that the sonic hedgehog (Shh), which is involved in patterning during development, is involved in the regeneration of texture. Embryos of ICR mice were surgically injured at E13, E14, and E15 and analyzed for the expression of Shh. For external Shh administration, recombinant Shh-containing slow-release beads were implanted in the wounds of mice. In contrast, cyclopamine was administered to wounds of adult mice to inhibit Shh. The expression of Shh was unaltered at E13, whereas it was upregulated in the epidermis of the wound from E14 onward. Implantation of recombinant Shh-containing beads into E13 wounds inhibited skin texture regeneration. Cyclopamine treatment inhibited epithelialization and thickening of the epidermis in the wounds of adult mice. In vitro, Shh promoted proliferation and inhibited the migration of epidermal keratinocytes through the activation of cyclin D proteins. Thus, our results suggested that the expression of Shh is involved in the regeneration of texture during wound healing, especially in epidermal keratinocyte migration and division, and could inhibit skin texture regeneration after E14.

Oral Administration of Nicotinamide Mononucleotide Is Safe and Efficiently Increases Blood Nicotinamide Adenine Dinucleotide Levels in Healthy Subjects.

Nicotinamide mononucleotide (NNM) is an orally bioavailable NAD+ precursor that has demonstrated beneficial effects against aging and aging-associated diseases in animal models. NMN is ultimately converted to NAD+, a redox cofactor that mediates many metabolic enzymes. NAD+ also serves as the substrate for poly(ADP-ribose) polymerase (PARP) and sirtuins, and regulates various biological processes, such as metabolism, DNA repair, gene expression, and stress responses. Previous mouse models showed that NMN administration can increase NAD+ in various organs and ameliorate aging-related diseases, such as obesity, diabetes, heart failure, stroke, kidney failure, and Alzheimer's disease through NAD+-mediated pathways. However, evidence of its effect on humans is still scarce. In this study, we conducted a placebo-controlled, randomized, double blind, parallel-group trial to investigate the safety of orally administered NMN and its efficacy to increase NAD+ levels in thirty healthy subjects. Healthy volunteers received 250 mg/day of NMN (n = 15) or placebo (n = 15) for 12 weeks, and physiological and laboratory tests were performed during this period. In addition, NAD+ and its related metabolites in whole blood were examined. Oral supplementation of NMN for 12 weeks caused no abnormalities in physiological and laboratory tests, and no obvious adverse effects were observed. NAD+ levels in whole blood were significantly increased after NMN administration. We also observed the significant rise in nicotinic acid mononucleotide (NAMN) levels, but not in NMN. We also found that the increased amount of NAD+ was strongly correlated with pulse rate before the administration of NMN. These results suggest that oral administration of NMN is a safe and practical strategy to boost NAD+ levels in humans. Clinical Trial Registration: JRCT [https://jrct.niph.go.jp/], identifier: [jRCTs041200034].