The development of early human lymphatic vessels as characterized by lymphatic endothelial markers.

Lymphatic vessel development studies in mice and zebrafish models have demonstrated that lymphatic endothelial cells (LECs) predominantly differentiate from venous endothelial cells via the expression of the transcription factor Prox1. However, LECs can also be generated from undifferentiated mesoderm, suggesting potential diversity in their precursor cell origins depending on the organ or anatomical location. Despite these advances, recapitulating human lymphatic malformations in animal models has been difficult, and considering lymphatic vasculature function varies widely between species, analysis of development directly in humans is needed. Here, we examined early lymphatic development in humans by analyzing the histology of 31 embryos and three 9-week-old fetuses. We found that human embryonic cardinal veins, which converged to form initial lymph sacs, produce Prox1-expressing LECs. Furthermore, we describe the lymphatic vessel development in various organs and observe organ-specific differences. These characterizations of the early development of human lymphatic vessels should help to better understand the evolution and phylogenetic relationships of lymphatic systems, and their roles in human disease.

Comprehensive phenotypic and genomic characterization of venous malformations.

Venous malformations (VMs) are the most common vascular malformations. TEK and PIK3CA are the causal genes of VMs, and may be involved in the PI3K/AKT pathway. However, the downstream mechanisms underlying the TEK or PIK3CA mutations in VMs are not completely understood. This study aimed to identify a possible association between genetic mutations and clinicopathological features. A retrospective clinical, pathological, and genetic study of 114 patients with VMs was performed. TEK, PIK3CA, and combined TEK/PIK3CA mutations were identified in 49 (43%), 13 (11.4%), and 2 (1.75%) patients, respectively. TEK-mutant VMs more commonly occurred in younger patients than TEK and PIK3CA mutation-negative VMs (other-mutant VMs), and showed more frequent skin involvement and no lymphocytic aggregates. No significant differences were observed in sex, location of occurrence, malformed vessel size, vessel density, or thickness of the vascular smooth muscle among the VM genotypes. Immunohistochemical analysis revealed that the expression levels of phosphorylated AKT (p-AKT) were higher in the TEK-mutant VMs than those in PIK3CA-mutant and other-mutant VMs. The expression levels of p-mTOR and its downstream effectors were higher in all the VM genotypes than those in normal vessels. Spatial transcriptomics revealed that the genes involved in "blood vessel development", "positive regulation of cell migration", and "extracellular matrix organization" were up-regulated in a TEK-mutant VM. Significant genotype-phenotype correlations in clinical and pathological features were observed among the VM genotypes, indicating gene-specific effects. Detailed analysis of gene-specific effects in VMs may offer insights into the underlying molecular pathways and implications for targeted therapies.

Sirt3 Regulates Proliferation and Progesterone Production in Leydig Cells via Suppression of Reactive Oxygen Species.

Sirt3 is a mitochondrial protein deacetylase functioning in energy metabolism, regulation of intracellular reactive oxygen species (ROS) levels, and aging. Although Sirt3 loss has negative effects on fertility of oocytes during in vitro fertilization and on progesterone production in granulosa cells, Sirt3's function in Leydig cells remains unclear. Therefore, we investigated Sirt3 activity in Leydig cells, focusing on androgen production. To do so, we performed immunohistochemistry to confirm Sirt3 localization in gonads and observed strong Sirt3 immunostaining in Leydig cells of human testes and of Sirt3+/+ and Sirt3+/- mouse testes, while Sirt3-/- mouse testis tissue was negative. In human ovary, hilus cells were strongly Sirt3-positive, theca cells showed weak positivity, and granulosa cells showed very weak or almost no immunostaining. Next, we used the murine Leydig tumor cell line MA-10 as a model. We overexpressed Sirt3 but observed no changes in proliferation, expression of Star, Cyp11a1 (p450scc gene), and Hsd3b, or progesterone production in MA-10 cells. Sirt3 knockdown significantly reduced proliferation, suppressed expressions of steroidogenic enzymes and of transcription factors Ad4bp (Sf-1 gene) and Gata4, and decreased progesterone production. Sirt3 knockdown in MA-10 cells also increased intracellular ROS levels based on CM-H2DCFDA fluorescence dye analysis and increased the proportion of both early and late apoptotic (necrotic) cells based on Annexin V/7AAD assays. These results indicate that Sirt3 has a potential function in androgen production in Leydig cells by regulating intracellular ROS levels.

Tenascin-C in Tissue Repair after Myocardial Infarction in Humans.

Adverse ventricular remodeling after myocardial infarction (MI) is progressive ventricular dilatation associated with heart failure for weeks or months and is currently regarded as the most critical sequela of MI. It is explained by inadequate tissue repair due to dysregulated inflammation during the acute stage; however, its pathophysiology remains unclear. Tenascin-C (TNC), an original member of the matricellular protein family, is highly up-regulated in the acute stage after MI, and a high peak in its serum level predicts an increased risk of adverse ventricular remodeling in the chronic stage. Experimental TNC-deficient or -overexpressing mouse models have suggested the diverse functions of TNC, particularly its pro-inflammatory effects on macrophages. The present study investigated the roles of TNC during human myocardial repair. We initially categorized the healing process into four phases: inflammatory, granulation, fibrogenic, and scar phases. We then immunohistochemically examined human autopsy samples at the different stages after MI and performed detailed mapping of TNC in human myocardial repair with a focus on lymphangiogenesis, the role of which has recently been attracting increasing attention as a mechanism to resolve inflammation. The direct effects of TNC on human lymphatic endothelial cells were also assessed by RNA sequencing. The results obtained support the potential roles of TNC in the regulation of macrophages, sprouting angiogenesis, the recruitment of myofibroblasts, and the early formation of collagen fibrils during the inflammatory phase to the early granulation phase of human MI. Lymphangiogenesis was observed after the expression of TNC was down-regulated. In vitro results revealed that TNC modestly down-regulated genes related to nuclear division, cell division, and cell migration in lymphatic endothelial cells, suggesting its inhibitory effects on lymphatic endothelial cells. The present results indicate that TNC induces prolonged over-inflammation by suppressing lymphangiogenesis, which may be one of the mechanisms underlying adverse post-infarct remodeling.

Structural and functional analysis of the newt lymphatic system.

Regeneration competent vertebrates such as newts and salamanders possess a weakened adaptive immune system characterized by multiple connections between the lymphatic system and the blood vascular system called lymphatic hearts. The role of lymphatic vasculature and these lymphaticovenous connections in regeneration is unknown. We used in-vivo near-infrared lymphangiography, ultra-high frequency ultrasonography, micro-CT lymphangiography, and histological serial section 3-dimentional computer reconstruction to evaluate the lymphatic territories of Cynops pyrrhogaster. We used our model and supermicrosurgery to show that lymphatic hearts are not essential for lymphatic circulation and limb regeneration. Instead, newts possess a novel intraosseous network of lymphatics inside the bone expressing VEGFR-3, LYVE-1 and CD-31. However, we were unable to show Prox-1 expression by these vessels. We demonstrate that adult newt bone marrow functions as both a lymphatic drainage organ and fat reservoir. This study reveals the fundamental anatomical differences between the immune system of urodeles and mammals and provides a model for investigating lymphatics and regeneration.

Different Types of Myocardial Injury due to the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Omicron Variant.

Coronavirus disease 2019 (COVID-19) associated myocardial injury was caused by various mechanisms. We herein describe 2 cases presenting different types of myocardial injury due to Omicron variant. In both patients, diffuse reduced left ventricular (LV) wall motion in transthoracic echocardiography, electrocardiographic abnormality, and elevated myocardial enzymes were demonstrated. In addition, cardiovascular magnetic resonance (CMR) findings fulfilled the 2018 Lake Louise Criteria (LLC) for myocarditis. However, histological findings in 1 patient showed inflammatory cell infiltration with myocyte degeneration, while those in the other showed interstitial edema without inflammatory cell infiltration. Histological findings were crucial for a differential diagnosis of myocardial injury due to Omicron variant.

Histopathological findings of pericarditis in a patient with multisystem inflammatory syndrome in children associated with COVID-19: A case report.

Multisystem inflammatory syndrome in children (MIS-C), which is associated with the novel coronavirus disease 2019 (COVID-19), has been described as an inflammatory complication of exposure to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It carries a risk of serious and lethal complications, including cardiogenic shock. Here, we report the pathological findings of the pericardium in a 10-year-old child with MIS-C, who developed pericarditis-induced cardiac tamponade. In the patient's pericardium, the numbers of infiltrating CD68+ macrophages; CD3+ , CD4+ , and CD8+ T cells; and myeloperoxidase+ granulocytes were increased, although the number of CD20+ B cells was not. These findings provide a clue to understanding the pathophysiology of MIS-C.

The cardiopharyngeal mesoderm contributes to lymphatic vessel development in mouse.

Lymphatic vessels are crucial for tissue homeostasis and immune responses in vertebrates. Recent studies have demonstrated that lymphatic endothelial cells (LECs) arise from both venous sprouting (lymphangiogenesis) and de novo production from non-venous origins (lymphvasculogenesis), which is similar to blood vessel formation through angiogenesis and vasculogenesis. However, the contribution of LECs from non-venous origins to lymphatic networks is considered to be relatively small. Here, we identify the Islet1 (Isl1)-expressing cardiopharyngeal mesoderm (CPM) as a non-venous origin of craniofacial and cardiac LECs. Genetic lineage tracing with Isl1Cre/+ and Isl1CreERT2/+ mice suggested that a subset of CPM cells gives rise to LECs. These CPM-derived LECs are distinct from venous-derived LECs in terms of their developmental processes and anatomical locations. Later, they form the craniofacial and cardiac lymphatic vascular networks in collaboration with venous-derived LECs. Collectively, our results demonstrate that there are two major sources of LECs, the cardinal vein and the CPM. As the CPM is evolutionarily conserved, these findings may improve our understanding of the evolution of lymphatic vessel development across species. Most importantly, our findings may provide clues to the pathogenesis of lymphatic malformations, which most often develop in the craniofacial and mediastinal regions.

The Pathogenesis of Cardiac Fibrosis: A Review of Recent Progress.

Fibrosis is defined as the excessive deposition of extracellular matrix (ECM) proteins in the interstitium. It is an essential pathological response to chronic inflammation. ECM protein deposition is initially protective and is critical for wound healing and tissue regeneration. However, pathological cardiac remodeling in excessive and continuous tissue damage with subsequent ECM deposition results in a distorted organ architecture and significantly impacts cardiac function. In this review, we summarized and discussed the histologic features of cardiac fibrosis with the signaling factors that control it. We evaluated the origin and characteristic markers of cardiac fibroblasts. We also discussed lymphatic vessels, which have become more important in recent years to improve cardiac fibrosis.

Surgical protocol for permanent ligation of the left anterior descending coronary artery in mice to generate a model of myocardial infarction.

Myocardial infarction (MI) is one of the most common causes of death worldwide. Animal models for MI are useful for studying the pathophysiology and developing therapies. Here, we describe a surgical protocol for permanent ligation of the left anterior descending coronary artery in mice, which mimics human acute coronary syndrome. This protocol includes descriptive step-by-step surgical procedures and high-quality surgical videos, which are useful for performing stable and highly reproducible operations. For complete details on the use and execution of this protocol, please refer to Maruyama et al. (2021).

Semaphorin3E-PlexinD1 signaling in coronary artery and lymphatic vessel development with clinical implications in myocardial recovery.

Blood and lymphatic vessels surrounding the heart develop through orchestrated processes from cells of different origins. In particular, cells around the outflow tract which constitute a primordial transient vasculature, referred to as aortic subepicardial vessels, are crucial for the establishment of coronary artery stems and cardiac lymphatic vessels. Here, we revealed that the epicardium and pericardium-derived Semaphorin 3E (Sema3E) and its receptor, PlexinD1, play a role in the development of the coronary stem, as well as cardiac lymphatic vessels. In vitro analyses demonstrated that Sema3E may demarcate areas to repel PlexinD1-expressing lymphatic endothelial cells, resulting in proper coronary and lymphatic vessel formation. Furthermore, inactivation of Sema3E-PlexinD1 signaling improved the recovery of cardiac function by increasing reactive lymphangiogenesis in an adult mouse model of myocardial infarction. These findings may lead to therapeutic strategies that target Sema3E-PlexinD1 signaling in coronary artery diseases.

PERK participates in cardiac valve development via fatty acid oxidation and endocardial-mesenchymal transformation.

Protein kinase R-like endoplasmic reticulum kinase (PERK) is one of the endoplasmic reticulum (ER) stress sensors. PERK loss-of-function mutations are known to cause Wolcott-Rallison syndrome. This disease is characterized by early-onset diabetes mellitus, skeletal dysplasia, and cardiac valve malformation. To understand the role of PERK in valve formation in vivo, we used an endothelial-specific PERK conditional knockout mice as well as in vitro PERK inhibition assays. We used ProteoStat dyes to visualize the accumulation of misfolded proteins in the endocardial cushion and valve mesenchymal cells (VMCs). Then, VMCs were isolated from E12.5 fetal mice, by fluorescence assisted cell sorting. Proteomic analysis of PERK-deleted VMCs identified the suppression of proteins related to fatty acid oxidation (FAO), especially carnitine palmitoyltransferase II (CPT2). CPT2 is a critical regulator of endocardial-mesenchymal transformation (EndoMT); however how TGF-ß downstream signaling controls CPT2 expression remains unclear. Here, we showed that PERK inhibition suppressed, not only EndoMT but also CPT2 protein expression in human umbilical vein endothelial cells (HUVECs) under TGF-ß1 stimulation. As a result, PERK inhibition suppressed mitochondrial metabolic activity. Taken together, these results demonstrate that PERK signaling is required for cardiac valve formation via FAO and EndoMT.

Comprehensive analysis of a novel mouse model of the 22q11.2 deletion syndrome: a model with the most common 3.0-Mb deletion at the human 22q11.2 locus.

The 22q11.2 deletion syndrome (22q11.2DS) is associated with an increased risk for psychiatric disorders. Although most of the 22q11.2DS patients have a 3.0-Mb deletion, existing mouse models only mimic a minor mutation of 22q11.2DS, a 1.5-Mb deletion. The role of the genes existing outside the 1.5-Mb deletion in psychiatric symptoms of 22q11.2DS is unclear. In this study, we generated a mouse model that reproduced the 3.0-Mb deletion of the 22q11.2DS (Del(3.0 Mb)/ +) using the CRISPR/Cas9 system. Ethological and physiological phenotypes of adult male mutants were comprehensively evaluated by visual-evoked potentials, circadian behavioral rhythm, and a series of behavioral tests, such as measurement of locomotor activity, prepulse inhibition, fear-conditioning memory, and visual discrimination learning. As a result, Del(3.0 Mb)/ + mice showed reduction of auditory prepulse inhibition and attenuated cue-dependent fear memory, which is consistent with the phenotypes of existing 22q11.2DS models. In addition, Del(3.0 Mb)/ + mice displayed an impaired early visual processing that is commonly seen in patients with schizophrenia. Meanwhile, unlike the existing models, Del(3.0 Mb)/ + mice exhibited hypoactivity over several behavioral tests, possibly reflecting the fatigability of 22q11.2DS patients. Lastly, Del(3.0 Mb)/ + mice displayed a faster adaptation to experimental jet lag as compared with wild-type mice. Our results support the validity of Del(3.0 Mb)/ + mice as a schizophrenia animal model and suggest that our mouse model is a useful resource to understand pathogenic mechanisms of schizophrenia and other psychiatric disorders associated with 22q11.2DS.

Isl1-expressing non-venous cell lineage contributes to cardiac lymphatic vessel development.

The origin of the mammalian lymphatic vasculature has been studied for more than a century; however, details regarding organ-specific lymphatic development remain unknown. A recent study reported that cardiac lymphatic endothelial cells (LECs) stem from venous and non-venous origins in mice. Here, we identified Isl1-expressing progenitors as a potential non-venous origin of cardiac LECs. Genetic lineage tracing with Isl1-Cre reporter mice suggested a possible contribution from the Isl1-expressing pharyngeal mesoderm constituting the second heart field to lymphatic vessels around the cardiac outflow tract as well as to those in the facial skin and the lymph sac. Isl1+ lineage-specific deletion of Prox1 resulted in disrupted LYVE1+ vessel structures, indicating a Prox1-dependent mechanism in this contribution. Tracing back to earlier embryonic stages revealed the presence of VEGFR3+ and/or Prox1+ cells that overlapped with the Isl1+ pharyngeal core mesoderm. These data may provide insights into the developmental basis of heart diseases involving lymphatic vasculature and improve our understanding of organ-based lymphangiogenesis.

Postotic and preotic cranial neural crest cells differently contribute to thyroid development.

Thyroid development and formation vary among species, but in most species the thyroid morphogenesis consists of five stages: specification, budding, descent, bilobation and folliculogenesis. The detailed mechanisms of these stages have not been fully clarified. During early development, the cranial neural crest (CNC) contributes to the thyroid gland. The removal of the postotic CNC (corresponding to rhombomeres 6, 7 and 8, also known as the cardiac neural crest) results in abnormalities of the cardiovascular system, thymus, parathyroid glands, and thyroid gland. To investigate the influence of the CNC on thyroid bilobation process, we divided the CNC into two regions, the postotic CNC and the preotic CNC (from the mesencephalon to rhombomere 5) regions and examined. We found that preotic CNC-ablated embryos had a unilateral thyroid lobe, and confirmed the presence of a single lobe or the absence of lobes in postotic CNC-ablated chick embryos. The thyroid anlage in each region-ablated embryos was of a normal size at the descent stage, but at a later stage, the thyroid in preotic CNC-ablated embryos was of a normal size, conflicting with a previous report in which the thyroid was reduced in size in the postotic CNC-ablated embryos. The postotic CNC cells differentiated into connective tissues of the thyroid in quail-to-chick chimeras. In contrast, the preotic CNC cells did not differentiate into connective tissues of the thyroid. We found that preotic CNC cells encompassed the thyroid anlage from the specification stage to the descent stage. Finally, we found that endothelin-1 and endothelin type A receptor-knockout mice and bosentan (endothelin receptor antagonist)-treated chick embryos showed bilobation anomalies that included single-lobe formation. Therefore, not only the postotic CNC, but also the preotic CNC plays an important role in thyroid morphogenesis.

One-pot enzymatic synthesis of docosahexaenoic acid-rich triacylglycerols at the sn-1(3) position using by-product from selective hydrolysis of tuna oil.

Docosahexaenoic acid (DHA)-rich oil has been industrially produced by selective hydrolysis of tuna oil with a lipase that acts weakly on DHA. The free fatty acids (FFAs) generated in this process as by-products contain a high DHA concentration (46wt%) but are treated as industrial waste. This study attempted to reuse these by-product FFAs using a one-pot process, and succeeded in producing triacylglycerols (TAGs) through the esterification of the by-product FFAs with glycerol using immobilized Rhizomucor miehei lipase. Regiospecific analysis of the resulting TAGs showed that the content of DHA at the sn-1(3) position (51.7mol%) was higher than the content of DHA at the sn-2 position (17.3mol%). The DHA distribution in TAGs synthesized in this study was similar to the DHA distribution in TAGs from seal oil.

Effects of supplemented diacylglycerol rich in docosahexaenoic acid on serum triacylglycerol in a diet-induced hyperlipidemic model of rats are essentially equivalent to those of triacylglycerol rich in docosahexaenoic acid.

Effects of supplemented docosahexaenoic acid (DHA), given as diacylglycerol (DG) rich in DHA (DHA-DG), triacylglycerol (TG) rich in DHA (DHA-TG) or fish oil concentrate (DHA-70), on the serum concentration of TG and its bioavailability in the rats with diet-induced hyperlipidemia were studied. Hypertriglyceridemia was induced by feeding male Wistar rats a semi-purified diet that contained 5% corn oil and 50% sucrose by weight. In addition to the feeding of dietary corn oil, the rats received DHA intragastrically at a dose of 500 mg/kg body weight once a day for 28 d and the control rats were given olive oil. The serum concentration of TG in the rats that received DHA-DG was significantly lower than in the control rats. However, there were no significant differences in diet intake, energy intake, body weight gain, visceral fat mass or fecal excretion of total fatty acids among the four groups. The amounts of DHA excreted into the feces of the three groups of rats that received DHA were approximately 0.4% of the DHA administered. The extent of the decreases induced by DHA-DG in the serum level of TG was almost the same as those induced by DHA-TG and DHA-70. The administration of DHA, regardless of the differences in molecular structure, did not affect the hepatic contents of TG or phospholipid. The administration of DHA-DG considerably increased the proportions of DHA and eicosapentaenoic acid (EPA) while decreasing the proportion of arachidonic acid in hepatic lipids, and as a result in the lipids in serum and erythrocytes, to the same extents as did DHA-TG and DHA-70. These results suggest that the hypotriglyceridemic effects and bioavailability of DHA when supplemented in the form of DG are essentially equivalent to those of DHA-TG and DHA-70.

Autoxidation kinetic analysis of docosahexaenoic acid ethyl ester and docosahexaenoic triglyceride with oxygen sensor.

The application of omega-3 polyunsaturated fatty acids (PUFAs) as food additives is restricted by their chemically quite reactive properties. However, quantitative analyses of the oxidative kinetics of PUFAs are very few compared to other studies on food chemistry. In this study, the autoxidation kinetics of ethyl docosahexaenoate (DHAEE), docosahexaenoic triglyceride (DHA oil), and emulsified DHA oil were investigated with an oxygen sensor. The autocatalytic reaction rate constants for DHAEE, DHA oil, and the emulsified DHA oil with 20% (w/v) GA, 20% SSPS, or 20% SSPS containing 5% soy protein were obtained at 35, 50, and 70 degrees C. A plot of the natural logarithm of the frequency factor, In ka0, vs. the activation energy, Ea, demonstrated that In ka0 against Ea fitted well with a single straight line both for the data from this study and for other reported results. This implies that the chemical compensation relationship holds between ka0 and Ea for PUFA and emulsified DHA oil.