Null mutation of exocyst complex component 3-like does not affect vascular development in mice.

Exocyst is an octameric protein complex implicated in exocytosis. The exocyst complex is highly conserved among mammalian species, but the physiological function of each subunit in exocyst remains unclear. Previously, we identified exocyst complex component 3-like (Exoc3l) as a gene abundantly expressed in embryonic endothelial cells and implicated in the process of angiogenesis in human umbilical cord endothelial cells. Here, to reveal the physiological roles of Exoc3l during development, we generated Exoc3l knockout (KO) mice by genome editing with CRISPR/Cas9. Exoc3l KO mice were viable and showed no significant phenotype in embryonic angiogenesis or postnatal retinal angiogenesis. Exoc3l KO mice also showed no significant alteration in cholesterol homeostasis or insulin secretion, although several reports suggest an association of Exoc3l with these processes. Despite the implied roles, Exoc3l KO mice exhibited no apparent phenotype in vascular development, cholesterol homeostasis, or insulin secretion.

A Single Intradermal Injection of Autologous Adipose-Tissue-Derived Stem Cells Rejuvenates Aged Skin and Sharpens Double Eyelids.

Facial skin aging is the most visible manifestation of aging in the body. In this study, we aimed to rejuvenate aging skin via a one-time intradermal injection of autologous adipose-derived stem cells (ADSCs). Eight patients were enrolled for study. Photographs of patients taken immediately before and 1, 3, 6, and 12 months after ADSC injections were comparatively evaluated for visible skin manifestations. ADSCs were cultured from the abdominal-skin-derived subcutaneous fat tissue, and 1 × 108 cultured ADSCs were injected intradermally into the facial skin. Cultured myoblasts were incubated with the supernatant derived from ADSCs, and the effect was evaluated via glucose consumption and lactic acid production in the medium. Eight cases showed the shallowing and disappearance of wrinkles, including those of the glabella, lower eyelids, crow`s feet, and forehead and nasolabial grooves, a month to several months after treatment. Double eyelids became prominent, and facial pores significantly reduced in size. These effects lasted for over one year. Myoblasts cultured in the presence of an ADSC-derived exosome were activated compared to that of ADSCs cultured without supernatant. The result supports the role of muscle in ADSC skin rejuvenation. The present study first reports that a single intradermal administration of cultured ADSCs rejuvenates aged facial skin over the course of one year. Further, patients exhibited definite double eyelids and pore shrinkage, strongly indicating the active involvement of muscle, which was supported by an in vitro study. Our study also suggested the important role of biological factors delivered from injected stem cells, although the detailed mechanism of rejuvenation effects of ADSC skin injection remains to be clarified.

Near-anoxia induces immobilization and sustains viability of sperm stored in ant queens.

After copulation, insect females store sperm in a spermatheca for some duration until fertilization. At the beginning of their adult lives, ant queens can preserve numerous viable sperm cells from copulation for over ten years. However, the key factors influencing long-term sperm storage have not been identified. Here we show that the spermathecal environment is nearly anoxic, which induces sperm immobilization. Furthermore, mitochondrial respiratory inhibitors suppress sperm motility, suggesting that sperm immobilization may be caused by a shortage of ATP generated from only glycolysis under near-anoxic conditions. Sperm immobilization is not induced by acidification via glycolytic metabolism because the spermathecal fluid is not acidic. Finally, we show that artificial anoxic conditions rather than aerobic conditions sustain viable sperm cells. Therefore, near-anoxia is a key factor influencing long-term sperm storage in ant queens. The viability of sperm cells under artificial anoxia, however, is lower than that of those dissected immediately from queens. Moreover, the immotile sperm cells under more than 4 h of anoxia do not begin swimming after aerobic exposure, unlike those under anoxic conditions for less than 2 h. This finding indicates that factors other than anoxia are also necessary for long-term sperm preservation.

Involvement of an Aberrant Vascular System in Neurodevelopmental, Neuropsychiatric, and Neuro-Degenerative Diseases.

The vascular system of the prenatal brain is crucial for the development of the central nervous system. Communication between vessels and neural cells is bidirectional, and dysfunctional communication can lead to neurodevelopmental diseases. In the present review, we introduce neurodevelopmental and neuropsychiatric diseases potentially caused by disturbances in the neurovascular system and discuss candidate genes responsible for neurovascular system impairments. In contrast to diseases that can manifest during the developing stage, we have also summarized the disturbances of the neurovascular system in neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. Furthermore, we discussed the role of abnormal vascularization and dysfunctional vessels in the development of neurovascular-related diseases.

Computational Model Exploring Characteristic Pattern Regulation in Periventricular Vessels.

The developing neocortical vasculature exhibits a distinctive pattern in each layer. In murine embryos, vessels in the cortical plate (CP) are vertically oriented, whereas those in the intermediate zone (IZ) and the subventricular zone (SVZ) form a honeycomb structure. The formation of tissue-specific vessels suggests that the behavior of endothelial cells is under a specific regulatory regime in each layer, although the mechanisms involved remain unknown. In the present study, we aimed to explore the conditions required to form these vessel patterns by conducting simulations using a computational model. We developed a novel model framework describing the collective migration of endothelial cells to represent the angiogenic process and performed a simulation using two-dimensional approximation. The attractive and repulsive guidance of tip cells was incorporated into the model based on the function and distribution of guidance molecules such as VEGF and Unc ligands. It is shown that an appropriate combination of guidance effects reproduces both the parallel straight pattern in the CP and meshwork patterns in the IZ/SVZ. Our model demonstrated how the guidance of the tip cell causes a variety of vessel patterns and predicted how tissue-specific vascular formation was regulated in the early development of neocortical vessels.

Essential Roles of Exocyst Complex Component 3-like 2 on Cardiovascular Development in Mice.

Angiogenesis is a process to generate new blood vessels from pre-existing vessels and to maintain vessels, and plays critical roles in normal development and disease. However, the molecular mechanisms underlying angiogenesis are not fully understood. This study examined the roles of exocyst complex component (Exoc) 3-like 2 (Exoc3l2) during development in mice. We found that Exoc3l1, Exoc3l2, Exoc3l3 and Exoc3l4 are expressed abundantly in endothelial cells at embryonic day 8.5. The generation of Exoc3l2 knock-out (KO) mice showed that disruption of Exoc3l2 resulted in lethal in utero. Substantial numbers of Exoc3l2 KO embryos exhibited hemorrhaging. Deletion of Exoc3l2 using Tie2-Cre transgenic mice demonstrated that Exoc3l2 in hematopoietic and endothelial lineages was responsible for the phenotype. Taken together, these findings reveal that Exoc3l2 is essential for cardiovascular and brain development in mice.

Extracellular Environment-Controlled Angiogenesis, and Potential Application for Peripheral Nerve Regeneration.

Endothelial cells acquire different phenotypes to establish functional vascular networks. Vascular endothelial growth factor (VEGF) signaling induces endothelial proliferation, migration, and survival to regulate vascular development, which leads to the construction of a vascular plexuses with a regular morphology. The spatiotemporal localization of angiogenic factors and the extracellular matrix play fundamental roles in ensuring the proper regulation of angiogenesis. This review article highlights how and what kinds of extracellular environmental molecules regulate angiogenesis. Close interactions between the vascular and neural systems involve shared molecular mechanisms to coordinate developmental and regenerative processes. This review article focuses on current knowledge about the roles of angiogenesis in peripheral nerve regeneration and the latest therapeutic strategies for the treatment of peripheral nerve injury.

Overexpression of Teashirt Homolog 2 suppresses cell proliferation and predicts the favorable survival of Lung Adenocarcinoma.

Background: Teashirt homolog 2 (TSHZ2) is essential for maintaining cellular homeostasis and regulating transcription on neoplasia development. However, the regulation of TSHZ2 in lung tumorigenesis and the underlying mechanisms remain unclear. Objective: To evaluate the relationship between TSHZ2 expression in patients' tumor tissue and prognosis in lung adenocarcinoma. Methods: TSHZ2 expression in different lung adenocarcinoma cell lines and human tissue were detected by Western blotting. The effect of TSHZ2 on cell proliferation, apoptosis and migration in lung adenocarcinoma cells was measured by CCK8, colony formation, flowcytometric analyses and wound-healing, respectively. TSHZ2 expression in different lung adenocarcinoma cell lines and human tissue from patients was detected using Western blotting and immunohistochemical staining. We also retrospectively analyzed 226 lung adenocarcinoma patients after surgical resection using immunohistochemical staining, and the association of TSHZ2 expression with the patient survival was evaluated. Results: TSHZ2 was lowly expressed in certain lung adenocarcinoma cell lines (PC9 and B203L), but other cells showed a high expression. Low expression of TSHZ2 was also observed in most lung adenocarcinoma tissues compared with adjacent tissues. Furthermore, we found that the overexpression of TSHZ2 plasmids led to the dramatic inhibition of cell proliferation, colony formation ability, migration and apoptosis induction in PC9 lung adenocarcinoma cells. In contrast, no obvious effect was found when the TSHZ2 expression was down-regulated by si-TSHZ2. An elevated TSHZ2 expression was observed in 155(68.6%) tumor tissues samples of lung adenocarcinoma patients. Notably, the lung adenocarcinoma patients with a high TSHZ2 expression tended to have EGFR mutations less frequently and a preferable prognosis to those with a lower expression. Conclusion: A high TSHZ2 expression inhabited cell proliferation and predicted a better prognosis of lung adenocarcinoma, possibly representing a useful therapeutic target for lung adenocarcinoma.

[Spatiotemporally Dependent Vascularization Regulates Neural Stem and Progenitor Cells].

Blood vessels including arteries, veins, and capillaries, are densely spread throughout the body. One round of systemic blood circulation through these blood vessels occurs approximately every minute, and blood sent by the heart transports oxygen, nutrients, and fluid to cells throughout the body. This nourishes cells, tissues, and organs and maintains homeostasis. The relatively simple structure of blood vessels consists of endothelial cells surrounded by a basal lamina and pericytes covering the outer layer. However, blood vessels patterning markedly varies among tissues. The diversity and plasticity of vascular networks are considered vital for this system to facilitate distinct functions for each tissue. Recent studies revealed that blood vessels create a tissue-specific niche, thus attracting attention as biologically active sites for tissue development. This vascular niche establishes specialized microenvironments through both direct physical contact and secreted-soluble factors. Here, we review advances in our understanding of how the vascular niche is utilized by neural stem and progenitor cells during neocortical development, and describe future perspectives regarding new treatment strategies for neural diseases utilizing this vascular niche.

Decrease in the T-box1 gene expression in embryonic brain and adult hippocampus of down syndrome mouse models.

Down syndrome (DS, Trisomy 21) is the most common genetic cause of delayed fetal brain development and postnatal intellectual disability. Although delayed fetal brain development might be involved in intellectual disability, no evidence of an association between these abnormal phenotypes has been shown. To identify molecules differentially expressed in both the prenatal forebrain and adult hippocampus of Ts1Cje mice, a mouse model of DS, we employed a transcriptomic analysis. In the present study, we conducted transcriptomic profiling of the hippocampus of adult Ts1Cje mice and compared the results with the previously obtained transcriptomic profile of the prenatal forebrain at embryonic day 14.5. Results showed that the Tbx1 mRNA expression was decreased at both life stages. In addition, the decreased expression of Tbx1 mRNA was confirmed in other DS mouse models, Dp(16)1Yey/+ and Ts1Rhr mice, which carry longer and shorter trisomic regions, respectively. Taken together, these findings suggest that Tbx1 may link the delayed fetal brain development and intellectual disability in DS.

Emerging Roles of PRDM Factors in Stem Cells and Neuronal System: Cofactor Dependent Regulation of PRDM3/16 and FOG1/2 (Novel PRDM Factors).

PRDI-BF1 (positive regulatory domain I-binding factor 1) and RIZ1 (retinoblastoma protein-interacting zinc finger gene 1) (PR) homologous domain containing (PRDM) transcription factors are expressed in neuronal and stem cell systems, and they exert multiple functions in a spatiotemporal manner. Therefore, it is believed that PRDM factors cooperate with a number of protein partners to regulate a critical set of genes required for maintenance of stem cell self-renewal and differentiation through genetic and epigenetic mechanisms. In this review, we summarize recent findings about the expression of PRDM factors and function in stem cell and neuronal systems with a focus on cofactor-dependent regulation of PRDM3/16 and FOG1/2. We put special attention on summarizing the effects of the PRDM proteins interaction with chromatin modulators (NuRD complex and CtBPs) on the stem cell characteristic and neuronal differentiation. Although PRDM factors are known to possess intrinsic enzyme activity, our literature analysis suggests that cofactor-dependent regulation of PRDM3/16 and FOG1/2 is also one of the important mechanisms to orchestrate bidirectional target gene regulation. Therefore, determining stem cell and neuronal-specific cofactors will help better understanding of PRDM3/16 and FOG1/2-controlled stem cell maintenance and neuronal differentiation. Finally, we discuss the clinical aspect of these PRDM factors in different diseases including cancer. Overall, this review will help further sharpen our knowledge of the function of the PRDM3/16 and FOG1/2 with hopes to open new research fields related to these factors in stem cell biology and neuroscience.

Aging of the Vascular System and Neural Diseases.

Vertebrates have acquired complex high-order functions facilitated by the dispersion of vascular and neural networks to every corner of the body. Blood vessels deliver oxygen and nutrients to all cells and provide essential transport systems for removing waste products. For these functions, tissue vascularization must be spatiotemporally appropriate. Recent studies revealed that blood vessels create a tissue-specific niche, thus attracting attention as biologically active sites for tissue development. Each capillary network is critical for maintaining proper brain function because age-related and disease-related impairment of cognitive function is associated with the loss or diminishment of brain capillaries. This review article highlights how structural and functional alterations in the brain vessels may change with age and neurogenerative diseases. Capillaries are also responsible for filtering toxic byproducts, providing an appropriate vascular environment for neuronal function. Accumulation of amyloid ß is a key event in Alzheimer's disease pathogenesis. Recent studies have focused on associations reported between Alzheimer's disease and vascular aging. Furthermore, the glymphatic system and meningeal lymphatic systems contribute to a functional unit for clearance of amyloid ß from the brain from the central nervous system into the cervical lymph nodes. This review article will also focus on recent advances in stem cell therapies that aim at repopulation or regeneration of a degenerating vascular system for neural diseases.

New additional scoring formula on the Pathological Features in Stage I Lung Adenocarcinoma Patients: Impact on Survival.

Background: Histological heterogeneity of lung adenocarcinoma may result in different prognosis among patients with the same TNM pathological stage. However, no objective evaluation system of lung adenocarcinoma based on pathological features has been widely accepted for assessing the prognosis. Methods: We retrospectively analyzed 179 patients with stage I lung adenocarcinoma after complete surgical resection. The pathological classification was according to the IASLC/ATS/ERS adenocarcinoma classifications, and the detailed abundance ratio using HE staining of primary tumor specimens was recorded. A new additional scoring formula on the pathological features (ASP) was established. The association of the ASP score with the patients' survival was examined. Results: The ASP scoring was significantly associated with smoking history (p=0.004), lymphatic vessel invasion (p<0.001), vascular invasion, differentiation (p<0.001) and Ki67 (p<0.001). The patients in the high-ASP-score group tended to have vascular invasion (odds ratio [OR]: 1.637, 95% confidence interval [CI]: 1.923-13.745, p=0.001) and high Ki67 expression (OR: 2.625, 95%CI: 1.328-5.190, p=0.006) by logistic regression analyses. The prognosis differed significantly in the Kaplan-Meier survival curves, and the 5-year survival rates in the low and high ASP score groups were 97.8% and 89.6%, respectively (p=0.018). Based on the univariate analysis, female (OR: 0.111, 95%CI: 0.014-0.906, p=0.040), long smoking history (OR: 7.250, 95%CI: 1.452-36.195, p=0.016), poor differentiation characteristics correlation (OR: 12.691, 95%CI: 1.557-103.453, p=0.018), and high ASP score (OR: 5.788, 95%CI: 1.138-29.423, p=0.034) were shown to be independently associated with an unfavorable prognosis. Conclusion: The ASP score can effectively screen high-risk patients for complete surgical resection of stage I lung adenocarcinoma.

Dermal Fibroblasts Internalize Phosphatidylserine-Exposed Secretory Melanosome Clusters and Apoptotic Melanocytes.

Pigmentation in the dermis is known to be caused by melanophages, defined as melanosome-laden macrophages. In this study, we show that dermal fibroblasts also have an ability to uptake melanosomes and apoptotic melanocytes. We have previously demonstrated that normal human melanocytes constantly secrete melanosome clusters from various sites of their dendrites. After adding secreted melanosome clusters collected from the culture medium of melanocytes, time-lapse imaging showed that fibroblasts actively attached to the secreted melanosome clusters and incorporated them. Annexin V staining revealed that phosphatidylserine (PtdSer), which is known as an 'eat-me' signal that triggers the internalization of apoptotic cells by macrophages, is exposed on the surface of secreted melanosome clusters. Dermal fibroblasts were able to uptake secreted melanosome clusters as did macrophages, and those fibroblasts express TIM4, a receptor for PtdSer-mediated endocytosis. Further, co-cultures of fibroblasts and melanocytes demonstrated that dermal fibroblasts internalize PtdSer-exposed apoptotic melanocytes. These results suggest that not only macrophages, but also dermal fibroblasts contribute to the collection of potentially toxic substances in the dermis, such as secreted melanosome clusters and apoptotic melanocytes, that have been occasionally observed to drop down into the dermis from the epidermis.

[Neuro-vascular Interactions during Neocortical Development-Systematic and Accurate Regulatory Mechanisms of VEGF Signaling].

Blood vessels supply oxygen and nutrients to all the cells in a living body, and provide essential transport routes for collecting waste products. For these functions, blood vessel networks should be appropriately formed in each tissue. Therefore, blood vessels are one of the earliest organs formed during the developmental process. Development of the blood vessel system promotes tissue differentiation and organ morphogenesis, allowing each organ to maintain its unique functions under changing metabolic conditions. Blood vessels have a relatively simple structure, consisting of endothelial cells covering the inner layer, and pericytes or smooth muscle cells surrounding the outside. The structure of the vascular network is extremely diverse, with blood vessels uniquely organized depending on the tissues they serve, to create tissue-specific microenvironments. How are such tissue-specific vascular environments generated? Over the years, anatomical findings have accumulated to confirm this vascular diversity. However, the molecular basis for this diversity has remained unclear. In the present article, we review the mechanisms of coordinated developmental control of the vascular and neural systems in the cerebral cortex from the viewpoint of the accurate expression control of vascular endothelial growth factor (VEGF) signaling, and describe future perspectives.

Spatiotemporally Dependent Vascularization Is Differently Utilized among Neural Progenitor Subtypes during Neocortical Development.

To facilitate efficient oxygen and nutrient delivery, blood vessels in the brain form three-dimensional patterns. However, little is known about how blood vessels develop stereographically in the neocortex and how they control the expansion and differentiation of neural progenitors during neocortical development. We show that highly vascularized and avascular regions are strictly controlled in a spatially and temporally restricted manner and are associated with distinct cell populations. Dividing basal progenitors and oligodendrocyte precursors preferentially contact honeycomb vessels, but dividing apical progenitors are localized in avascular regions without Flt1-positive endothelial cells but directly contact with sprouting neovascular tip cells. Therefore, not all blood vessels are associated equally with neural progenitors. Furthermore, a disruption of normal vascular patterning can induce abnormalities in neural development, whereas the impaired features of neural progenitors influenced angiogenesis patterning. These results indicate that close association between the nervous and vascular systems is essential for neocortex assembly.

The potential role of follicular helper T cells in idiopathic multicentric Castleman disease with and without TAFRO syndrome.

Idiopathic multicentric Castleman disease (iMCD) is a systemic inflammatory disease of unknown etiology caused by hypercytokinemia. Recently, TAFRO (thrombocytopenia, anasarca, fever, renal failure or reticulin fibrosis, and organomegaly) syndrome has been reported, which shows similar histopathological findings to iMCD and factors associated with a poor prognosis. iMCD shows no plasma cell infiltration in the germinal center (GC), but CD38-positive (CD38+)-plasma cells are observed in the interfollicular area. Our previous report revealed that atrophic change of GC, glomeruloid vascular proliferation, and abnormal proliferation of follicular dendritic cells are more prominent in iMCD with TAFRO (TAFRO+) in comparison to iMCD without TAFRO (TAFRO-). In addition, the numbers of CD38+ and immunoglobulin G4-positive (IgG4+) plasma cells were decreased in the interfollicular area. The roles of T follicular helper cells (Tfh) are well-known to assist B-cell proliferation, maturation, and differentiation．It maintains the formation of GC and is also related in the class switching of IgG isotypes, including IgG4. Thus, we immunohistochemically examined the number of Tfh in GCs in both TAFRO- and TAFRO+ iMCD. The number of Tfh was significantly decreased in TAFRO- iMCD (n = 9) and was further decreased in TAFRO+ iMCD (n = 18) in comparison to non-specific lymphadenopathy (n = 6) and IgG4-related disease (n = 4). These results suggest that decreased Tfh may be one etiology of iMCD.

Interaction of the nervous system and vascular system is required for the proper assembly of the neocortex.

Mammalian neocortical development encompasses an entire set of events that leads to the generation of excitatory and inhibitory neurons from neural progenitors in the dorsal and ventral telencephalon, including cell proliferation, production of migratory precursors and their progeny, differentiation, and integration into circuits. During these processes, the developing neocortex acquires its vasculature by angiogenesis, a process consisting of proliferation of endothelial cells in existing blood vessels or vascular plexuses, and leading to formation of new blood vessels. Recent studies have suggested that neocortical angiogenesis progresses in a spatially and temporally restricted manner to construct a specialized vascular niche that supports ongoing neurogenesis during neocortical development. Here we review that periventricular blood vessels selectively influence neocortical progenitors behavior and neurogenesis, highlighting how CNS angiogenesis is utilized to construct neocortical cytoarchitecture.