Naoki Morimoto

Adipose Tissue–Specific Regulation of Angiotensinogen in Obese Humans and Mice: Impact of Nutritional Status and Adipocyte Hypertrophy

BACKGROUND The adipose tissue renin-angiotensin system (RAS) has been implicated in the pathophysiology of obesity and dysfunction of adipose tissue. However, neither regulation of angiotensinogen (AGT) expression in adipose tissue nor secretion of adipose tissue-derived AGT has been fully elucidated in humans. METHODS Human subcutaneous abdominal adipose tissue (SAT) biopsies were performed for 46 subjects with a wide range of body mass index (BMI). Considering the mRNA level of AGT and indices of body fat mass, the amount of adipose tissue-derived AGT secretion (A-AGT-S) was estimated. Using a mouse model of obesity and weight reduction, plasma AGT levels were measured with a newly developed enzyme-linked immunosorbent assay (ELISA), and the contribution of A-AGT-S to plasma AGT levels was assessed. RESULTS A-AGT-S was substantially increased in obese humans and the value was correlated with the plasma AGT level in mice. A-AGT-S and plasma AGT were higher in obese mice, whereas lower in mice with weight reduction. However, the AGT mRNA levels in the liver, kidney, and aorta were not altered in the mouse models. In both humans and mice, the AGT mRNA levels in mature adipocytes (MAs) were comparable to those in stromal-vascular cells. Coulter Multisizer analyses revealed that AGT mRNA levels in the MAs were inversely correlated with the average size of mature adipocytes. CONCLUSIONS This study demonstrates that adipose tissue-derived AGT is substantially augmented in obese humans, which may contribute considerably to elevated levels of circulating AGT. Adipose tissue-specific regulation of AGT provides a novel insight into the clinical implications of adipose tissue RAS in human obesity.

Mammalian skull heterochrony reveals modular evolution and a link between cranial development and brain size

The multiple skeletal components of the skull originate asynchronously and their developmental schedule varies across amniotes. Here we present the embryonic ossification sequence of 134 species, covering all major groups of mammals and their close relatives. This comprehensive data set allows reconstruction of the heterochronic and modular evolution of the skull and the condition of the last common ancestor of mammals. We show that the mode of ossification (dermal or endochondral) unites bones into integrated evolutionary modules of heterochronic changes and imposes evolutionary constraints on cranial heterochrony. However, some skull-roof bones, such as the supraoccipital, exhibit evolutionary degrees of freedom in these constraints. Ossification timing of the neurocranium was considerably accelerated during the origin of mammals. Furthermore, association between developmental timing of the supraoccipital and brain size was identified among amniotes. We argue that cranial heterochrony in mammals has occurred in concert with encephalization but within a conserved modular organization.

Preparation of Collagen/Gelatin Sponge Scaffold for Sustained Release of bFGF

Artificial dermis (AD) has been used to regenerate dermis-like tissues in the treatment of full-thickness skin defects, but it takes 2 or 3 weeks to complete dermal regeneration. Our previous study demonstrated that injection of basic fibroblast growth factor (bFGF)-impregnated gelatin microspheres (MS) into the AD accelerates the regeneration of dermis-like tissue. However, injection of gelatin MS before clinical use is complicated and time consuming. This study investigated a new scaffold, in which collagen and gelatin are integrated, and which is capable of sustained bFGF release. We produced collagen/gelatin sponges with a gelatin concentration of 0wt%, 10wt%, 30wt%, and 50wt%. The mean pore size in each sponge decreased with the gelatin concentration. In an in vitro study, proliferation of fibroblasts in each sponge was not significantly different over 7 days of culture. As for in vivo sustained release of bFGF, a radioisotope study demonstrated that retention of bFGF in gelatin 10wt% and 30wt% sponges was significantly larger than that in gelatin 0wt% sponge. The collagen/gelatin sponges were grafted on full-thickness skin defects created on a rabbit ear, and we evaluated regeneration of dermis-like tissue by measuring the amount of hemoglobin and size of dermis-like tissue on histological sections. Seven days after implantation, the amount of hemoglobin in dermis-like tissue in gelatin 10wt% sponge was significantly larger than those in control and gelatin 50wt% sponge. Twenty-eight days after implantation, the area of dermis-like tissue in gelatin 10wt% sponge was significantly larger than those in the other specimens. We conclude that the collagen sponge integrated with 10wt% gelatin has the most potential for sustained release of bFGF and that the combination of collagen/gelatin 10wt% sponge and bFGF is a promising therapeutic modality for the treatment of full-thickness skin defects.

Collagen-Gelatin Scaffold Impregnated with bFGF Accelerates Palatal Wound Healing of Palatal Mucosa in Dogs

BACKGROUND We have developed a collagen-gelatin sponge (CGS) as a scaffold capable of the sustained release of bFGF to improve the healing process of the existing collagen scaffold. The aim of this study was to evaluate the efficacy of CGS impregnated with basic fibroblast growth factor (bFGF) in palatal wound healing in beagles. MATERIALS AND METHODS Four standardized 6 mm diameter full-thickness wounds were made in the palate of each dog and covered with CGS impregnated with normal saline or bFGF at concentrations of 1 μg/cm2, 7 μg/cm2 and 14 μg/cm2. One and 2 wk after surgery, the wound area, neoepithelium length, thickness, area of regenerated submucosal tissue, and the number and total area of neoformed capillaries were evaluated. RESULTS Two weeks after implantation, wounds treated with bFGF 7 μg/cm2 and 14 μg/cm2 were completely epithelized, while the length of the neoformed epithelium was significantly longer in the 7 μg/cm2 group. Groups impregnated with bFGF 7 μg/cm2 and 14 μg/cm2 showed promoted regeneration of submucosal tissue 2 wk later. The number and area of neoformed capillaries were significantly higher in the bFGF 7 μg/cm2 group than in other groups. We conclude that palatal wound healing in the bFGF 7 μg/cm2 group was promoted with good neovascularization and showed less contracture than other groups. CONCLUSIONS Our new collagen-gelatin scaffold, CGS, impregnated with bFGF, could be a promising treatment to accelerate the regeneration of palatal mucosa.

Metopic suture of Taung (Australopithecus africanus) and its implications for hominin brain evolution

The type specimen for Australopithecus africanus (Taung) includes a natural endocast that reproduces most of the external morphology of the right cerebral hemisphere and a fragment of fossilized face that articulates with the endocast. Despite the fact that Taung died between 3 and 4 y of age, the endocast reproduces a small triangular-shaped remnant of the anterior fontanelle, from which a clear metopic suture (MS) courses rostrally along the midline [Hrdlička A (1925) Am J Phys Anthropol 8:379–392]. Here we describe and interpret this feature of Taung in light of comparative fossil and actualistic data on the timing of MS closure. In great apes, the MS normally fuses shortly after birth, such that unfused MS similar to Taung’s are rare. In humans, however, MS fuses well after birth, and partially or unfused MS are frequent. In gracile fossil adult hominins that lived between ∼3.0 and 1.5 million y ago, MS are also relatively frequent, indicating that the modern human-like pattern of late MS fusion may have become adaptive during early hominin evolution. Selective pressures favoring delayed fusion might have resulted from three aspects of perinatal ontogeny: (i) the difficulty of giving birth to large-headed neonates through birth canals that were reconfigured for bipedalism (the “obstetric dilemma”), (ii) high early postnatal brain growth rates, and (iii) reorganization and expansion of the frontal neocortex. Overall, our data indicate that hominin brain evolution occurred within a complex network of fetopelvic constraints, which required modification of frontal neurocranial ossification patterns.

Platelet-rich plasma releasate promotes angiogenesis in vitro and in vivo

Platelet-rich plasma (PRP) is a plasma fraction in which several growth factors are concentrated at high levels. In recent years, the biological effects on various cells of the active soluble releasate that is isolated following platelet activation of PRP [PRP-releasate (PRPr)] have been reported. The purpose of this study was to determine the angiogenic effects of human PRPr in vitro and in vivo. PRPr was prepared from human whole blood using the double spin method and was activated with CaCl2 and autologous thrombin. PRPr stimulated proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) and in vivo angiogenesis-inducing ability in nude mice. PRPr led to the phosphorylation of Erk1/2 and Akt in HUVECs, and the induction of proliferation and migration by PRPr was suppressed by PRPr inhibitors PD98059 and LY294002. PRPr induces angiogenesis in vitro and in vivo, and the present findings suggest that the mechanism for this is activation of the ERK and phosphatidylinositol-3-kinase–Akt pathways. Our results demonstrate that PRPr is a promising autologous source for therapeutic angiogenesis in treating cardiovascular disease.

Platelet and growth factor concentrations in activated platelet-rich plasma: a comparison of seven commercial separation systems

Platelet-rich plasma (PRP) is blood plasma that has been enriched with platelets. It holds promise for clinical use in areas such as wound healing and regenerative medicine, including bone regeneration. This study characterized the composition of PRP produced by seven commercially available separation systems (JP200, GLO PRP, Magellan Autologous Platelet Separator System, KYOCERA Medical PRP Kit, SELPHYL, MyCells, and Dr. Shin’s System THROMBO KIT) to evaluate the platelet, white blood cell, red blood cell, and growth factor concentrations, as well as platelet-derived growth factor-AB (PDGF-AB), transforming growth factor beta-1 (TGF-β1), and vascular endothelial growth factor (VEGF) concentrations. PRP prepared using the Magellan Autologous Platelet Separator System and the KYOCERA Medical PRP Kit contained the highest platelet concentrations. The mean PDGF-AB concentration of activated PRP was the highest from JP200, followed by the KYOCERA Medical PRP Kit, Magellan Autologous Platelet Separator System, MyCells, and GLO PRP. TGF-β1 and VEGF concentrations varied greatly among individual samples, and there was almost no significant difference among the different systems, unlike for PDGF. The SELPHYL system produced PRP with low concentrations of both platelets and growth factors. Commercial PRP separation systems vary widely, and familiarity with their individual advantages is important to extend their clinical application to a wide variety of conditions.

Evaluation of a novel collagen–gelatin scaffold for achieving the sustained release of basic fibroblast growth factor in a diabetic mouse model

The objective of this study was to evaluate the ability of a scaffold, collagen–gelatin sponge (CGS), to release basic fibroblast growth factor (bFGF) in a sustained manner, using a pressure‐induced decubitus ulcer model involving genetically diabetic mice. We confirmed that CGSs impregnated with a bFGF concentration of up to 50 µg/cm2 were able to sustain the release of bFGF throughout their biodegradation. We prepared decubitus ulcers on diabetic mice. After debriding the ulcers, we implanted CGSs (diameter 8 mm) impregnated with normal saline solution (NSS) or bFGF solution (7, 14, 28 or 50 µg/cm2). At 1 and 2 weeks after implantation, the mice were sacrificed and tissue specimens were obtained. The wound area, neoepithelium length and numbers and total area of newly formed capillaries were evaluated. The CGSs impregnated with NSS became infected and degraded, whereas the CGSs impregnated with 7 or 14 µg/cm2 bFGF displayed accelerated dermis‐like tissue formation and the CGSs impregnated with 14 µg/cm2 bFGF produced significant improvements in the remaining wound area, neoepithelium length and numbers and total area of newly formed capillaries compared with the NSS group. No significant difference was observed between the NSS and 50 µg/cm2 bFGF groups. CGSs impregnated with 7–14 µg/cm2 bFGF accelerated wound healing, and an excess amount of bFGF did not increase the wound‐healing efficacy of the CGSs. Our CGS is a scaffold that can release positively charged growth factors such as bFGF in a sustained manner and shows promise as a scaffold for skin regeneration. Copyright

Efficacy of novel collagen/gelatin scaffold with sustained release of basic fibroblast growth factor for dermis-like tissue regeneration.

Abstract We have developed collagen/gelatin sponges (CGS) with a gelatin concentration of 10 wt% to sustain the release of basic fibroblast growth factor (bFGF). The objective of this study is to elucidate the efficacy of CGS impregnated with different concentrations of bFGF, using mouse skin defects. CGSs impregnated with normal saline solution (NSS) or bFGF solution (1, 7, 14, or 50 &mgr;g/cm2) were implanted into full-thickness skin defects on the backs of mice. The wound area, neoepithelium length, and total area of newly formed capillaries in CGS were evaluated. The group of CGS with 7-&mgr;g/cm2 bFGF was significantly superior to the NSS group in all evaluated items. CGS impregnated with the appropriate dosage of bFGF accelerates dermis-like tissue formation 2 or 3 times earlier than existing artificial dermis. The combination of CGS and bFGF could solve the problem of the existing artificial dermis and be very promising for the treatment of skin defects.

Hypoxia Enhances Proliferation of Human Adipose-Derived Stem Cells via HIF-1ɑ Activation

Background Adipose tissue-derived stem cells (ASCs) have been recently isolated from human subcutaneous adipose tissue. ASCs may be useful in regenerative medicine as an alternative to bone marrow-derived stem cells. Changes in the oxygen concentration influence physiological activities, such as stem cell proliferation. However, the effects of the oxygen concentration on ASCs remain unclear. In the present study, the effects of hypoxia on ASC proliferation were examined. Methods Normal human adipose tissue was collected from the lower abdomen, and ASCs were prepared with collagenase treatment. The ASCs were cultured in hypoxic (1%) or normoxic (20%) conditions. Cell proliferation was investigated in the presence or absence of inhibitors of various potentially important kinases. Hypoxia inducible factor (HIF)-1α expression and MAP kinase phosphorylation in the hypoxic culture were determined with western blotting. In addition, the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2 in hypoxic or normoxic conditions were determined with real-time RT-PCR. The effects of these growth factors on ASC proliferation were investigated. Chromatin immunoprecipitation (ChIP) of the HIF–1α-binding hypoxia responsive element in FGF–2 was performed. HIF–1α was knocked down by siRNA, and FGF–2 expression was investigated. Results ASC proliferation was significantly enhanced in the hypoxic culture and was inhibited by ERK and Akt inhibitors. Hypoxia for 5–15 minutes stimulated the phosphorylation of ERK1/2 among MAP kinases and induced HIF–1α expression. The levels of VEGF and FGF–2 mRNA and protein in the ASCs were significantly enhanced in hypoxia, and FGF–2 increased ASC proliferation. The ChIP assay revealed an 8-fold increase in the binding of HIF–1α to FGF–2 in hypoxia. HIF–1α knockdown by siRNA partially inhibited the FGF–2 expression of ASCs induced by hypoxia. Conclusion ASC proliferation was enhanced by hypoxia. HIF–1α activation, FGF–2 production, and the ERK1/2 and Akt pathway were involved in this regulatory mechanism.