DHCR7 links cholesterol synthesis with neuronal development and axonal integrity.

The DHCR7 enzyme converts 7-DHC into cholesterol. Mutations in DHCR7 can block cholesterol production, leading to abnormal accumulation of 7-DHC and causing Smith-Lemli-Opitz syndrome (SLOS). SLOS is an autosomal recessive disorder characterized by multiple malformations, including microcephaly, intellectual disability, behavior reminiscent of autism, sleep disturbances, and attention-deficit/hyperactivity disorder (ADHD)-like hyperactivity. Although 7-DHC affects neuronal differentiation in ex vivo experiments, the precise mechanism of SLOS remains unclear. We generated Dhcr7 deficient (dhcr7-/-) zebrafish that exhibited key features of SLOS, including microcephaly, decreased neural stem cell pools, and behavioral phenotypes similar to those of ADHD-like hyperactivity. These zebrafish demonstrated compromised myelination, synaptic anomalies, and neurotransmitter imbalances. The axons of the dhcr7-/- zebrafish showed increased lysosomes and attenuated autophagy, suggesting that autophagy-related neuronal homeostasis is disrupted.

Microvascular system of hip joint constituents with special reference to ultrastructural findings and early arteriosclerosis.

This study aimed to examine the ultrastructure of the arteries of the synovium, acetabular labrum, and ligamentum teres of the hip joint using light, scanning electron, and transmission electron microscopes to identify features of early arteriosclerosis. Tissues collected from three patients (under 40 years of age) with osteonecrosis of the femoral head were immersed in 8 N HCl at 60 °C for 20 min to digest collagen fibers for scanning electron microscopy. Tortuous arterioles and arteries were noted in the joint components, including the synovium, acetabular labrum, and ligamentum teres. The ultrastructure of the arterioles appeared normal; however, intimal thickening was found in most arteries. The thickened intima had abundant elastic fibers and many smooth muscle cells (which were of a synthetic phenotype because they had a few actin filaments and well-developed rough endoplasmic reticulum). This study illustrates that arteriosclerotic changes are present in tortuous arteries in the synovium, acetabular labrum, and ligamentum teres of the hip joint even from a relatively young age and suggests that meandering blood vessels may be the preferred foci of arteriosclerosis.

Exosc2 deficiency leads to developmental disorders by causing a nucleotide pool imbalance in zebrafish.

Exosc2 is one of the components of the exosome complex involved in RNA 3' end processing and degradation of various RNAs. Recently, EXOSC2 mutation has been reported in German families presenting short stature, hearing loss, retinitis pigmentosa, and premature aging. However, the in vivo function of EXOSC2 has been elusive. Herein, we generated Exosc2 knockout (exosc2-/-) zebrafish that showed larval lethality 13 days post fertilization, with microcephaly, loss of spinal motor neurons, myelin deficiency, and retinitis pigmentosa. Mechanistically, Exosc2 deficiency caused impaired mRNA turnover, resulting in a nucleotide pool imbalance. Rapamycin, which modulated mRNA turnover by inhibiting the mTOR pathway, improved nucleotide pool imbalance in exosc2-/- zebrafish, resulting in prolonged survival and partial rescue of neuronal defects. Taken together, our findings offer new insights into the disease pathogenesis caused by Exosc2 deficiency, and might help explain fundamental molecular mechanisms in neuronal diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, and spinal muscular atrophy.

Histological evaluation of the acetabular labrum after bipolar hip hemiarthroplasty: a case report.

We report herein the microstructure of the acetabular labrum obtained from a patient with stem loosening but without bipolar cup migration who had undergone hemiarthroplasty for femoral neck fracture 18 years ago. We used light and scanning electron microscopy to investigate the influence of bipolar cup on acetabular labrum in vivo. Deparaffinized blocks were treated with 2 N NaOH to digest the cell matrix, allowing the collagen fibers, constituting the acetabular labrum, to be observed under scanning electron microscopy. Although chondrocyte atrophy was seen, the basic structure was not different from the normal tissue images of the elderly. However, in the deep part of the acetabular labrum, there was an area that was not stained with Alcian blue observed with light microscopy, and there was an amorphous tissue without type II collagen fibrils observed with scanning electron microscopy. These findings proved that the acetabular labrum has partially degenerated over the long term after bipolar hemiarthroplasty, and that the acetabular labrum can survive in vivo in such a condition. Given that hemiarthroplasty has a possibility to preserve the face-to-face tissue in the long term in vivo, it may be one of the valuable options for modern or future joint reconstruction surgery.

Three-dimensional architecture of the acetabular labrum in the human hip joint.

The acetabular labrum is frequently damaged with advancing age. As collagen fibers are the main sources of strength, knowledge of their ultrastructure is important to determine the cause of age-induced changes. We aimed to investigate the ultrastructure of collagen fibers constituting the acetabular labrum using scanning electron microscopy (SEM). Acetabular labrum samples obtained during total hip arthroplasty were studied. The samples were specially prepared to observe the steric construction of collagen fibrils constituting the acetabular labrum under light microscopy followed by SEM. The acetabular labrum was mostly composed of cartilage tissue, consisting of chondrocytes and collagen type II, with a layer of collagen type I. In adults, chondrocytes with a rich cytoplasm were surrounded by a dense network of fine type II collagen fibrils, and small bundles of type I collagen fibrils were interposed in the cartilage layer. In elderly individuals, the chondrocytes atrophied and both type I and II collagen fibrils were sparse. We suggest that cartilage has three to five layers, consisting of type I and type II collagen fibrils with a solid cartilage substrate. In elderly individuals, the density of chondrocytes decreases and the cellular shape and architecture of collagen fibrils also changes.

Ultrastructure and three-dimensional architecture of the anterior cruciate ligament in the knee joints of young and old monkeys.

We examined the ultrastructure of the anterior cruciate ligament and assessed age-related changes by comparing the ligaments of young and old monkeys. Ultrathin sections of the anterior cruciate ligament were observed by transmission electron microscopy. The three-dimensional architecture of collagen fibers in the ligament was examined by scanning electron microscopy after tissue specimens were treated with 2 N NaOH to digest the extracellular matrix. At the surface layer of the cruciate ligament in young monkeys, fusiform-shaped fibroblasts actively produced collagen fibrils. The ligament consisted of parallel bundles of dense collagen fibrils of approximately 200 nm in diameter. Collagen fibrils appeared to run linearly. Ligament fibrocytes in the deep layer had a stellate form. Ligament fibrocytes decreased in number and showed marked atrophy in old age. Collagen fibrils had a looser configuration in older monkeys. Despite atrophy of fibroblasts in the deep layer of the anterior cruciate ligament, the area with atrophic fibroblasts in the ligament expands with age, which can likely cause deterioration of and a reduction in collagen fibers. This information can be applied in studies on the cause of the low repair ability of and aging-related changes in the anterior cruciate ligament in humans.

Immuno-histochemistry and three-dimensional architecture of the intermediate filaments in Purkinje cells in mammalian hearts.

In mammalian hearts, Purkinje cells varied greatly in morphological appearance in different species, and were divided into three groups. Bovine Purkinje cells corresponding to group I were a large size, and had a few myofibrils and abundant intermediate filaments throughout the cytoplasm. The aim of the present study was to clarify the more detailed distribution and three-dimensional architecture of intermediate filaments in Purkinje cells. The hearts in various mammals including humans were investigated by both immuno-histochemistry and scanning electron microscopy (SEM).Immuno-histochemical studies demonstrated that sheep Purkinje cells in group I had a great number of intermediate filaments of 10 nm positive for desmin antibody. Purkinje cells in group II (humans, monkeys and dogs) and group III (mice) were somewhat larger or smaller in size than myocardial cells, but also showed a strong positive reaction for desmin antibody. The saponin or NaOH treatment of cardiac tissues in sheep and humans enabled us to view intermediate filaments by SEM three-dimensionally. Intermediate filaments in sheep Purkinje cells formed a considerably delicate network, and were distributed throughout the cytoplasm. In contrast, those in human Purkinje cells were lower in density, and were present around the nucleus and between myofibrils. It was concluded that a delicate network of intermediate filaments in Purkinje cells of mammalian hearts acted as the cytoskeleton to maintain intercellular stability.

Effect of nonsense-mediated mRNA decay factor SMG9 deficiency on premature aging in zebrafish.

SMG9 is an essential component of the nonsense-mediated mRNA decay (NMD) machinery, a quality control mechanism that selectively degrades aberrant transcripts. Mutations in SMG9 are associated with heart and brain malformation syndrome (HBMS). However, the molecular mechanism underlying HBMS remains unclear. We generated smg9 mutant zebrafish (smg9oi7/oi7) that have a lifespan of approximately 6 months or longer, allowing for analysis of the in vivo function of Smg9 in adults in more detail. smg9oi7/oi7 zebrafish display congenital brain abnormalities and reduced cardiac contraction. Additionally, smg9oi7/oi7 zebrafish exhibit a premature aging phenotype. Analysis of NMD target mRNAs shows a trend toward increased mRNA levels in smg9oi7/oi7 zebrafish. Spermidine oxidase (Smox) is increased in smg9oi7/oi7 zebrafish, resulting in the accumulation of byproducts, reactive oxygen species, and acrolein. The accumulation of smox mRNA due to NMD dysregulation caused by Smg9 deficiency leads to increased oxidative stress, resulting in premature aging.

Morphological characteristics of the human skin over posterior aspect of heel in the context of pressure ulcer development.

The posterior aspect of heel is known as a particularly vulnerable site for pressure ulcer development, however, it is not well understood why this is so. This study was undertaken to identify the morphological characteristics of the skin over posterior aspect of heel in the context of pressure ulcer development. Human skin tissues were obtained from four different sites of the body of 4 aged subjects postmortem: posterior aspect of heel, plantar aspect of heel, sacrum and centre of gluteus maximus. The skin samples were processed for the examination using light microscopy and scanning electron microscopy. The posterior aspect of heel was characterized by a thicker epidermis, denser distribution and larger diameter of capillaries in the papillary layer, 3-dimensional architecture of collagen fibre meshwork in the reticular layer and elliptic adipose tissues situated perpendicularly to the skin surface being surrounded by thick collagen and elastic fibre septa compared to the sacrum. Given our observations in the papillary layer of the posterior aspect of heel, we assume that the tissue of this area may be less tolerant to ischaemia since the tissue has high metabolic demand to provide oxygen and nutrients to the epidermis which protects underlying tissue from external force. In addition, elliptic configuration of adipose tissues in the posterior aspect of heel situated perpendicularly to the skin surface may result in deep lesion if the forces applied exceed the tolerable level since the forces will be concentrated within the elliptic compartments.

Association between interatrial septum adiposity and atrial fibrillation: transesophageal echocardiography imaging and autopsy study.

Recent clinical evidence has suggested that interatrial septal (IAS) adiposity contributes to atrial fibrillation (AF). The present study aimed to confirm the usefulness of transesophageal echocardiography (TEE) to estimate IAS adiposity in patients with AF. The histological IAS analysis based on autopsy samples sought to clarify characteristics that underlie the contribution of IAS adiposity to AF. The imaging study analyzed the TEE results in patients with AF (n = 184) in comparison with transthoracic echocardiography (TTE) and computed tomography (CT) results. The autopsy study histologically analyzed IAS in subjects with (n = 5) and without (n = 5) history of AF. In the imaging study, the ratio of interatrial septum adipose tissue (IAS-AT) volume per epicardial adipose tissue (EpAT) volume was greater in patients with persistent AF compared (PerAF) to those with paroxysmal AF (PAF). Multivariable analysis revealed that both TEE-assessed IAS thickness and TTE-assessed left atrial dimension were predicted by CT-assessed IAS-AT volume. In the autopsy study, the histologically-assessed IAS section thickness was greater in the AF group than that in the non-AF group and was positively correlated with the IAS-AT area percentage. In addition, the size of adipocytes in IAS-AT was smaller, compared to EpAT and subcutaneous adipose tissue (SAT). IAS-AT infiltrated into the IAS myocardium, as if adipose tissue split the myocardium (designated as myocardial splitting by IAS-AT). The number of island-like myocardium pieces as a result of myocardial splitting by IAS-AT was greater in the AF group than in the non-AF group and was positively correlated with the IAS-AT area percentage. The present imaging study confirmed the usefulness of TEE to estimate IAS adiposity in patients with AF without radiation exposure. The autopsy study suggested that the myocardial splitting by IAS-AT may contribute to atrial cardiomyopathy leading to AF.

Dermatopontin interacts with fibronectin, promotes fibronectin fibril formation, and enhances cell adhesion.

We report that dermatopontin (DP), an abundant dermal extracellular matrix protein, is found in the fibrin clot and in the wound fluid, which comprise the provisional matrix at the initial stage of wound healing. DP was also found in the serum but at a lower concentration than that in wound fluid. DP co-localized with both fibrin and fibronectin on fibrin fibers and interacted with both proteins. Both normal fibroblast and HT1080 cell adhesion to the fibrin-fibronectin matrix were dose-dependently enhanced by DP, and the adhesion was mediated by α5ß1 integrin. The cytoskeleton was more organized in the cells that adhered to the fibrin-fibronectin-DP complex. When incubated with DP, fibronectin formed an insoluble complex of fibronectin fibrils as visualized by electron microscopy. The interacting sites of fibronectin with DP were the first, thirteenth, and fourteenth type III repeats (III(1), III(13), and III(14)), with III(13) and III(14) assumed to be the major sites. The interaction between III(2-3) and III(12-14) was inhibited by DP, whereas the interaction between I(1-5) and III(12-14) was specifically and strongly enhanced by DP. Because the interaction between III(2-3) and III(12-14) is involved in forming a globular conformation of fibronectin, and that between I(1-5) and III(12-14) is required for forming fibronectin fibrils, DP promotes fibronectin fibril formation probably by changing the fibronectin conformation. These results suggest that DP has an accelerating role in fibroblast cell adhesion to the provisional matrix in the initial stage of wound healing.

Morphology of the epithelium of the lower rectum and the anal canal in the adult human.

The anal canal is an important body part clinically. However, there is no agreement about the epithelium of the anal canal, the anal transitional zone (ATZ) epithelium in particular. The aim of this study is to clarify the structure of the epithelium of the human lower rectum and anal canal. Intact rectum and anus obtained from patients who underwent surgery for rectal carcinoma were examined by light and scanning electron microscopy (LM and SEM). By LM, three types of epithelium were observed in the anal canal: simple columnar epithelium, stratified squamous epithelium, and stratified columnar epithelium. The lower rectum was composed of simple columnar epithelium. SEM findings showed stratified squamous epithelium that consisted of squamous cells with microridges, changing to simple columnar epithelium consisting of columnar cells with short microvilli at the anorectal line. LM and SEM observations in a one-to-one ratio revealed that the area of stratified columnar epithelium based on LM corresponded to the anal crypt and sinus. In conclusion, the epithelium of the human anal canal was fundamentally composed of simple columnar epithelium and stratified squamous epithelium. We found no evidence of the ATZ.

Cytoarchitecture of the lamina muscularis mucosae and distribution of the lymphatic vessels in the human stomach.

The aim of the present study was to clarify the anatomical structure of the lamina muscularis mucosae (LMM) in the human stomach and to correlate it with the lymphatic spread of gastric cancer cells. Human stomachs taken at operation or autopsy were used. The specimens derived from these stomachs were examined by light microscopy immunohistochemistry and scanning electron microscopy (SEM). In the cardia and pyloric wall, bundles of smooth muscle cells of the LMM were relatively loose and thin and formed a reticular configuration. Small lymphatic capillaries (approximately 10-30 µm in diameter) were present directly above the LMM, and relatively large lymphatics (approximately 80-100 µm in diameter) were observed in the submucosal layer and within the LMM. In contrast, the LMM in the fundus, body, and antral wall was composed of tight, thick bundles of smooth muscle cells that ran straight. Large lymphatics were found directly beneath the LMM, but they were few in the lamina propria mucosae. In addition, lymphatics adjacent to veins were also found in the submucosa of the fundus. Structural differences in the LMM of the stomach wall might depend on physiological function. In this study, the relationship between the cytoarchitecture of the LMM or the distribution of lymphatic vessels and cancer invasion is discussed.

Long-term administration of the fungus toxin, sterigmatocystin, induces intestinal metaplasia and increases the proliferative activity of PCNA, p53, and MDM2 in the gastric mucosa of aged Mongolian gerbils.

OBJECTIVE: The causal agents of gastric cancer could include fungus toxins. Sterigmatocystin (ST), a fungus toxin, is a risk factor of gastric cancer. We investigated the effects of ST on the stomach tissues of Mongolian gerbils. METHODS: Seventy-five-week-old male Mongolian gerbils received ST ad libitum at a concentration of 0 ppb (non-treated, n = 11), 100 ppb (n = 7), or 1000 ppb (n = 13) dissolved in drinking water for a period of 24 weeks. After administration, we tested the histopathological changes and immunostaining for proliferating cell nuclear antigen (PCNA), p53, and MDM2 expression. RESULTS: We investigated the histopathological changes and determined the incidence of histopathological changes in animals with various gastric diseases after ST administration at a dose of 0 ppb (non-treated control), 100, or 1,000 ppb as follows: firstly, indices for gastritis were 18.2, 100, and 100%, those for erosion events were 9.1, 100, and 92.3%, and those for polyps were 0, 71.4, and 61.5%, respectively. These incidences in the ST-administered groups (100 or 1000 ppb) showed significant increases compared with those in the non-treated control group. And, lastly, indices for intestinal metaplasia were 0, 100, and 15.4%, respectively. Furthermore, immunostaining for PCNA, p53, and MDM2 expression showed significantly greater rates in the ST-administered groups (100 or 1000 ppb) than in the non-treated control group. CONCLUSION: The histopathological and immunohistopathological findings of this study indicate that ST exerts a marked influence on gastric mucus and gland cells, showing dominant gastritis, erosion events, polyps, and intestinal metaplasia in these animals.

Effect of age and sex on femoral curvature in the Japanese population: three-dimensional computed tomography findings.

Age- or sex-related differences in femoral curvature affect the onset of trauma or degenerative diseases of the hip joints. This study aimed to investigate femoral curvature in detail in Japanese individuals using three-dimensional computed tomography, evaluate its effects on the position of proximal femur, and assess differences in femoral curvature according to age and sex. We measured sagittal and coronal femoral bowing in 40 elderly (mean age 85.2 years) and 40 adult (mean age 30.1 years) Japanese individuals using computed tomography. In adult individuals, the radii of the femoral curvatures of the distal end and shaft in the coronal planes were significantly smaller in women than in men. In contrast, no significant difference was observed in femoral curvature between the sexes in the elderly group. Furthermore, the radius of femoral curvature was significantly smaller in elderly individuals than in adult individuals, regardless of the sex and location of the measurement. The highest point of the greater trochanter of the femoral head center was 7.3 ± 5.6 mm in the elderly group and 2.2 ± 4.6 mm in the adult group (p < 0.05). Thus, the femoral curvature varies with age and sex in Japanese individuals. In addition, the femoral curvature could cause positional changes in the proximal femur, such as the highest point of the greater trochanter. Therefore, further studies investigating the biomechanical effects of these morphological changes are warranted.

Detection of fibrotic remodeling of epicardial adipose tissue in patients with atrial fibrillation: Imaging approach based on histological observation.

BACKGROUND: Fibrotic remodeling of epicardial adipose tissue (EAT) is crucial for proinflammatory atrial myocardial fibrosis, which leads to atrial fibrillation (AF). OBJECTIVES: We tested the hypothesis that the ratio of central to marginal adipocyte diameter in EAT represents its fibrotic remodeling. Based on a similar concept, we also tested whether the percent (%) change in EAT fat attenuation determined using computed tomographic (CT) images can detect this remodeling. METHODS: Left atrial appendages were obtained from 76 consecutive AF patients during cardiovascular surgery. EAT in the central area (central EAT: C-EAT) and that adjacent to the atrial myocardium (Marginal EAT: M-EAT) were evaluated histologically. CT images for all of the 76 patients were also analyzed. RESULTS: The adipocyte diameter was smaller, fibrotic remodeling of EAT (EAT fibrosis) was more severe, and infiltration of macrophages and myofibroblasts was more extensive in M-EAT than in C-EAT. EAT fibrosis was positively correlated with adipocyte diameter in C-EAT and negatively correlated in M-EAT, resulting in a positive correlation between EAT fibrosis and the ratio of central to marginal adipocyte diameter (C/M diameter ratio; r = 0.73, P < .01). The C/M diameter ratio was greater in patients with persistent AF than in those with paroxysmal AF. CT images demonstrated that the %change in EAT fat attenuation was positively correlated with EAT fibrosis. CONCLUSION: Our results suggest that the central-to-marginal adipocyte diameter ratio is tightly associated with fibrotic remodeling of EAT. In addition, the %change in EAT fat attenuation determined using CT imaging can detect remodeling noninvasively.

Role of angiopoietin-like protein 2 in atrial fibrosis induced by human epicardial adipose tissue: Analysis using an organo-culture system.

BACKGROUND: We have recently reported that peri-left atrial epicardial adipose tissue (EAT) is associated with atrial myocardial fibrosis, in which angiopoietin-like protein 2 (Angptl2) protein content in EAT is associated with atrial myocardial fibrosis. OBJECTIVE: This study aimed to examine whether Angptl2 contained in peri-left atrial EAT can induce atrial myocardial fibrosis. METHODS: Human peri-left atrial EAT and abdominal subcutaneous adipose tissue (SAT) were collected from 9 autopsy cases. EAT- or SAT-conditioned medium was dropped onto the rat left atrial epicardial surface using an organo-culture system. Conditioned medium, recombinant Angptl2, and its antibody effects on organo-cultured rat atrial myocardial fibrosis were evaluated. Angptl2 effects on cultured neonatal rat fibroblasts were also investigated. RESULTS: EAT-conditioned medium induced atrial fibrosis in organo-cultured rat atrium with a progressive increase in the number of myofibroblasts. The profibrotic effect of EAT was greater than that of SAT. EAT in patients with atrial fibrillation induced a more significant atrial fibrosis than in those without. Treatment with human recombinant Angptl2 induced fibrosis in organo-cultured rat atrium, which was suppressed by the concomitant treatment with Angptl2 antibody. In cultured fibroblasts, Angptl2 upregulated the expression of α-smooth muscle actin, transforming growth factor-ß1, phospho-extracellular signal-regulated kinase,phospho-inhibitor of κBα, and phospho-p38 mitogen-activated protein kinase. CONCLUSION: This study demonstrated that Angptl2 contained in EAT played a crucial role in EAT-induced inflammatory atrial fibrosis. The results also suggested that antagonizing the expression of Angptl2 in EAT can be a novel therapeutic approach to prevent atrial fibrillation.

Preclinical efficacy and safety of 1-deoxygalactonojirimycin in mice for Fabry disease.

Fabry disease is an inborn error of glycosphingolipid metabolism caused by deficiency of alpha-galactosidase A (alpha-Gal A) activity. It has been shown that protein misfolding is primarily responsible for the enzyme deficiency in a large proportion of mutations identified in Fabry patients with residual enzyme activity, and 1-deoxygalactonojirimycin (DGJ) can effectively increase the residual enzyme activity in cultured patient's cells. Herein, we demonstrate the preclinical efficacy and safety of DGJ in transgenic mice that express human mutant alpha-Gal A activity. alpha-Gal A activity in heart, kidney, spleen, and liver was increased dose- and time-dependently. The mutant alpha-Gal A was increased in cardiomyocytes and distal convoluted tubules of the transgenic mice in a null background after 2 weeks of DGJ treatment. Globotriaosylceramide storage was remarkably reduced in kidney of mice after a 4-week treatment at a dosage of approximately 3 mg/kg body weight/day. The half-life of DGJ was less than 1 day in all major issues and that of the enzyme synthesized during the DGJ treatment period was approximately 4 days. No abnormality of blood chemistry and pathological tissue damage was found in mice treated with DGJ at approximately 30 mg/kg body weight/day for 9 weeks. Furthermore, no change was observed in appearance, growth, fertility, and life span in mice during a 2-year period of continuous administration of DGJ at the effective dosage. These preclinical results indicate that DGJ is effective in restoring mutant enzyme activity in tissues and reversing substrate storage in kidney and is well tolerated in mice.