Cytoplasm Of Endothelial Cells Research Articles

DEVELOPMENT OF ULTRASTRUCTURAL CHANGES IN DIAPHRAGM MUSCLE FIBERS DURING PARTIAL TRACHEAL STENOSIS IN YOUNG SEXUALLY IMMATURE RATS

Introduction. The study of congenital pathology of the respiratory tract in children is of great importance for theoretical and clinical pediatric otolaryngology. Currently, in the scientific literature there are single works concerning the study of the pathogenesis and clinic of laryngomalacia. First of all, this concerns the effect of acute and chronic hypoxia on the muscles of the respiratory system, in particular, the diaphragm. The aim of thіs work is to reveal the effect of hypoxia on the development of pronounced structural and functional changes in the diaphragm muscles of sexually immature animals when modeling partial tracheal occlusion. Materials and methods. Surgical partial occlusion of the trachea was performed on in sexually immature Wistar rats; we applied electron microscopy, morphometric and statistical research methods.
 Results. The obtained data demonstrated that the muscle fibers of the diaphragm are characterized by significant structural, functional and metabolic resistance to the development of acute or chronic hypoxia due to limited lung ventilation in rats. The density of muscle fibers did not change in any of the used terms of the experiment (7 and 21 days). On the 7th day of hypoxic exposure, a change in the state of the matrix of mitochondria of muscle fibers was observed, which was accompanied by partial ultrastructural rearrangements of the organization of these organelles. At the same time, no significant changes were observed during histological studies. Electrongrams demonstrated the ultrastructural damages were mainly limited to focal reduction of myofibrils and an increase in the mitochondria's diameter and found as especially pronounced on day 21 (a state of chronic hypoxia) on the muscular component of the respiratory system in rats. These changes are accompanied by the development of dystrophic disorders in the diaphragm myofibrils. On day 21 changes in the transendothelial transport of substance molecules were also detected. This was based on establishing the fact of a significant reduction in the number of pinocytosis vesicles in the cytoplasm of endothelial cells of diaphragm hemocapillaries in animals with partial tracheal occlusion. Disruption of the structural organization of actomyosin complexes was revealed in muscle fibers due to pathological changes in the mitochondria of myofibrils. It should be noted the development of destruction or complete loss of an important ultrastructural component of muscle fibers, Z-lines. These changes were completely absent on the 7th day following the influence of partial tracheal occlusion and the development of acute hypoxia. Moreover, on day 21, partial lysis of a significant amount of myofilaments of muscle fibers was observed. In addition, the development of the process of fiber swelling was detected against the background of which the above-mentioned ultrastructural destruction of the muscular component in the diaphragm was observed. These changes indicate the need for earlier treatment of laryngomalacia in children. The delay in surgical intervention leads to the development of severe or irreversible structural changes not only in the respiratory organs, but also in their muscular system.
 Conclusion. The muscular elements of the diaphragm have the pronounced and significant resistance to the state of hypoxia under conditions of limited ventilation of the respiratory tract. Ultrastructural and morphometric data, found on the 21st day after the modeling of partial tracheal stenosis, are not only of a compensatory and adaptive in nature, but also signs of pronounced disorders in the muscle fibers of the central part of the diaphragm in test animals.

(066) A Low Androgen State Impairs Erectile Function by Suppressing EPAC1 in Rat Corpus Cavernosum Penis

Abstract Introduction The relationship among exchange proteins activated by cAMP 1(EPAC1), androgen and erectile function is still unknown. Objective To explore whether EPAC1 expresses in penile corpus cavernosum of rats and how EPAC1 affects erectile function under low androgenic conditions. Methods Thirty 8-week-old healthy male rats were randomly divided into six groups (n=5): sham operation (sham), castrated, castrated + testosterone replacement (castrated + T), sham + EPAC1 over-expression lentivirus (sham + EPAC1), castrated + empty lentivirus vector (castrated + empty vector), and castrated + EPAC1. Four weeks after the operation, the lentivirus vectors carrying the EPAC1 gene were injected into the corpus cavernosum penis tissues of the sham + EPAC1 and castrated + EPAC1 groups (1×108TU/ml, 20 μl per rat). A week after injection, the ratio of maximum intracavernous pressure to mean arterial pressure (ICPmax/MAP) and the levels of serum testosterone (T), nitric oxide (NO), the active form of ras homolog gene family, member A (RhoA-GTP), protein kinase B (AKT), phospho-AKT (p-AKT), endothelial nitric oxide synthase (eNOS), phospho-eNOS (p-eNOS), p-AKT/AKT, p-eNOS/eNOS and EPAC1 levels were measured. Results EPAC1 is primarily located in the cytoplasm of endothelial cells and smooth muscle cells in the rat corpus cavernosum penis. In comparison to the sham group, the T, ICPmax/MAP and NO levels of the castrated group were notably reduced (p < 0.01). Meanwhile, the RhoA-GTP concentration in the castrated + EPAC1 group was reduced in comparison with the castrated + empty vector group (p < 0.01). Compared with the castrated + empty vector group, the p-AKT/AKT, EPAC1 and p-eNOS/eNOS levels in the castrated + EPAC1 group were significantly increased (p < 0.05). Conclusions Androgen deficiency can suppress EPAC1 expression in the penile corpus cavernosum of rats, while the up-regulation of which can improve the erectile function of castrated rats. Disclosure Yes, this is sponsored by industry/sponsor: This study was supported by the Research Foundation of the Department of Human Resources and Social Security of Sichuan Province of Returned Scholars grant 2019-76. Clarification: No industry support in study design or execution.

Immunohistochemical and Ultrastructural Imaging of the SARS-CoV-2 Coronavirus in Lung, Lymph Node, and Kidney Tissues of Deceased Patients

The objective: immunohistochemical and electron microscopic imaging of the SARS-CoV-2 coronavirus in lung, lymph nodes and kidney tissues of patients who died of COVID-19.Subjects and Methods. For immunohistochemical tests, specimens of sections of formalin-fixed and paraffin-embedded tissues of the lungs, lymph nodes and kidneys of patients who died from COVID-19 were used. Quantitative assessment of the SARS-CoV-2 viral load level was carried by the original RT qPCR and calculated by the formula: SARS-CoV-2 copy number/ABL1 х 100 copies, expressed as the ratio of the true number of SARS-CoV-2 cDNA copies per 100 copies cDNA of the ABL1 gene. For morphological tests, samples of native lung, lymph node and kidney tissues were taken.Results. Immunohistochemical and electron microscopic tests revealed particles of the SARS-CoV-2 coronavirus in the cytoplasm of endothelial cells of air-blood barrier of the lungs, the vascular glomerulus of the kidneys, in the cytoplasm of macrophages of the lymph node, and also in cytoplasm of lymphocytes in the lumen of lung capillaries.

Electron microscopic research of the optic nerve in an experiment with toxic neuritis

Рurpose. To identify ultrastructural changes of the optic nerve in the area of the trellis plate in rabbits after exposure to methyl alcohol. Material and methods. A model of experimental optic neuritis with the introduction of methyl alcohol into the retrobulbar space was reproduced on 8 rabbits. The animals were removed from the experiment on the 14th, 30th, 90th and 180th days after the administration of methanol. Pieces of the optic nerve (1–2 mm) were isolated from the enucleated eyeballs in the area of the lattice plate and examined using a transmission electron microscope. Results. On the 14th day after the administration of methanol, signs of pronounced cytoplasmic edema and destruction of intracellular organelles were detected in the ganglion neurons of the rabbit retina in the area of the lattice plate. In individual nerve fibers of the optic nerve, myelin sheaths were stratified and unfolded, axons swelled. Structural changes were detected in astrocytes, especially in the processes around blood vessels. Macrophages of nervous tissue — microgliocytes — became active. Morphological changes indicated a violation of microcirculation and permeability of the hematoneural barrier. The basal membranes around the walls of blood vessels were compacted. Intracellular organelles were destroyed in the cytoplasm of endothelial cells, hemostasis was determined inside the vessels. Subsequently, on the 30–90th day of the experiment, dystrophic and destructive changes in the cytoplasm of ganglion cells and their processes forming the optic nerve, as well as surrounding gliocytes progressed. There were signs of gliofibrosis. Destructive processes and gliofibrosis of the retina and nerve fibers of the optic nerve in the area of the lattice plate increased in degree of expression up to 180th days of the experiment. Conclusion. The results of electron microscopic studies of the intrabulbar part of the optic nerve in experimental animals in dynamics after the introduction of methyl alcohol indicate that the main trigger for the development of visual neuritis may be a violation of microcirculation processes in the retina and optic nerve, which leads to an increase in the permeability of the hematoneural barrier. These events entail dystrophic and destructive changes in the nervous tissue, the progression of which naturally leads, with the assistance of glial cells to gliofibrosis of the optic nerve. Keywords: toxic optic neuritis, methanol, hematoneural barrier, axon demyelination, gliofibrosis

Low testosterone state inhibits erectile function by downregulating the expression of GIT1 in rat penile corpus cavernosum.

The mechanism of erectile dysfunction (ED) caused by a low androgen level is still not clear. To explore the influence of the low testosterone state on G protein-coupled receptor kinase interactor 1 (GIT1) and its contact to erectile function. Thirty male Sprague-Dawley rats aged 8 weeks were distributed at random into 5 groups: control (sham operated), castration, testosterone supplement after castration, castration + vacant lentiviral transfection, and castration + lentiviral transfection. The testis and epididymis were removed through a scrotal incision to develop castrated rats. Four weeks after castration, a lentivirus carrying the GIT1 gene was injected into the middle of rat penile corpus cavernosum. One week after transfection, maximum intracavernous pressure/mean arterial pressure (ICPmax/MAP), serum testosterone, nitric oxide, GIT1, endothelial nitric oxide synthase (eNOS), phospho-eNOS (p-eNOS), p-eNOS/eNOS, and the interaction between eNOS and GIT1 were assessed in the rats. The levels of GIT1 in the penile cavernous tissue of castrated rats are significantly lower than that of controls. GIT1 was expressed in the cytoplasm and cell membrane of vascular endothelial cells and smooth muscle cells in rat penile tissue. In comparison with normal rats, the castrated rats showed lower levels of GIT1 expression, GIT1 and eNOS interaction, p-eNOS/eNOS, nitric oxide, and ICPmax/MAP (P < .01). Overexpression of GIT1 can intensively enhance the expression level of GIT1, the interaction between GIT1 and eNOS, p-eNOS/eNOS, nitric oxide, and ICPmax/MAP in rats (P < .01). Modulating the interaction between eNOS and GIT1 might be a novel method of treating ED caused by a low androgen level. The impact of GIT1 phosphorylation on the activity of eNOS and its possible mechanisms affecting erectile function require further study. A low testosterone state inhibits erectile function in rats by reducing the expression of GIT1 and the protein interaction between GIT1 and eNOS.

Targeting the liver to treat the eye.

Over the last two decades, gene therapy has given hope of potential cure for many rare diseases. In the simplest form, gene therapy is the transfer or editing of a genetic material to cure a disease via nonviral or viral vehicles. Gene therapy can be performed either in vivo by injecting a vector carrying the gene or tools for gene editing directly into a tissue or into the systemic circulation, or ex vivo when patient cells are genetically modified outside of the body and then introduced back into the patient (Yilmaz et al, 2022). Adeno-associated viral vectors (AAV) have been the vectors of choice for in vivo gene therapy. There has been a lot of promising research on the development of novel tissue and cell-specific serotypes in order to improve efficacy and safety for clinical applications (Kuzmin et al, 2021). In this issue of EMBO Molecular Medicine, Boffa and colleagues present a novel AAV-based liver-directed gene therapy for ornithine aminotransferase deficiency.

Features of the ultrastructure of the skin of white rats 30 days after modeling of portal hypertension

Abstract The problem of skin repair under the conditions of systemic increased pressure in microvessels has a great importance. This is due to the fact that each year the incidence of this pathology has seen an increase during different operations, including plastic surgery. As the compensatory and reparative mechanisms occurring in the skin affected by vascular hyperbaria are still unidentified, the aim of the study was to investigate the features of the ultrastructure of the skin of white rats 30 days after modeling of portal hypertension. Objects of the study were anterior abdominal wall biopsies for electron microscopy research. Our work demonstrated that the skin in intact animals (control group) was of typical structure before the beginning of the experiment and 30 days after a sham operation. Following 30 days modeling of portal hypertension, the ultrastructure of the epidermis was found to be intact, only slight thickening of the horny layer was revealed. However, in the basal epidermocytes, signs of crypts formation by cytolemma were revealed. Moreover, in the epidermocytes of the spinous and granular layers, the mitochondria, endoplasmic reticulum channels and ribosomes were almost non-evident. In addition, in the cytoplasm of the fibroblasts, a moderate amount of freely located ribosomes and a moderate number of polymorphic mitochondria were detected, while the lumens of the capillaries of the papillary layer of the dermis were narrowed. We also saw that the swelling of the cytoplasm in endothelial cells resulted in the narrowing of the microvessels lumen. What is more, the subendothelial zone was expanded – which is indicative of endothelial desquamation. Beyond the aforementioned, the nuclei of the endothelial cells were well contoured and had signs of chromatin condensation. Endothelial cells with signs of apoptosis were detected as well.

A comprehensive mouse brain acetylome-the cellular-specific distribution of acetylated brain proteins.

Nε-lysine acetylation is a reversible posttranslational modification (PTM) involved in multiple physiological functions. Genetic and animal studies have documented the critical roles of protein acetylation in brain development, functions, and various neurological disorders. However, the underlying cellular and molecular mechanism are still partially understood. Here, we profiled and characterized the mouse brain acetylome and investigated the cellular distribution of acetylated brain proteins. We identified 1,818 acetylated proteins, including 5,196 acetylation modification sites, using a modified workflow comprising filter-aided sample preparation (FSAP), acetylated peptides enrichment, and MS analysis without pre- or post-fraction. Bioinformatics analysis indicated these acetylated mouse brain proteins were mainly located in the myelin sheath, mitochondrial inner membrane, and synapse, as well as their involvement in multiple neurological disorders. Manual annotation revealed that a set of brain-specific proteins were acetylation-modified. The acetylation of three brain-specific proteins was verified, including neurofilament light polypeptide (NEFL), 2’,3’-cyclic-nucleotide 3’-phosphodiesterase (CNP), and neuromodulin (GAP43). Further immunofluorescence staining illustrated that acetylated proteins were mainly distributed in the nuclei of cortex neurons and axons of hippocampal neurons, sparsely distributed in the nuclei of microglia and astrocytes, and the lack of distribution in both cytoplasm and nuclei of cerebrovascular endothelial cells. Together, this study provided a comprehensive mouse brain acetylome and illustrated the cellular-specific distribution of acetylated proteins in the mouse brain. These data will contribute to understanding and deciphering the molecular and cellular mechanisms of protein acetylation in brain development and neurological disorders. Besides, we proposed some problems that need to be solved in future brain acetylome research.

The effect of neoadjuvant chemotherapy on the structural and functional state of tumor cells in the secondary edematous form of breast cancer

Background. Secondary edematous form of breast cancer (SЕF BC) is the most aggressive type of BC that is characterized by rapid progression, high levels of metastasis, significant resistance to chemotherapy and radiotherapy. SЕF BC is not just a combination of cancer and local inflammation, but is a rare phenomenon in which the development of the tumor is primary. The processes of edema and inflammation occur because of lymph flow blockage by the formation of emboli from tumor cells (TC), which have elevated levels of E-cadherin, properties of polyploid cells and show signs of stem cells. Trimodal therapy methods are used for the treatment of SЕF BC, the main components of which are neoadjuvant systemic chemotherapy, surgery and adjuvant radiation therapy. However, the results of treatment remain insufficient, possibly due to the fact that the features of SЕF, the role of stem cells and inflammatory factors are not taken into account. Therefore, further research is needed in various fields of oncology, molecular biology, immunology, genetics, morphology, including electron microscopy, which is an important area for establishing the characteristics of both tumor cells and their environment. Purpose. Study of the neoadjuvant polychemotherapy influence on the structural and functional state of tumor cells of different molecular subtypes in the secondary edematous form of breast cancer.&#x0D; Materials and methods. Tumors of 29 patients with normal breast cancer and 32 patients with SЕF BC were studied. Receptors to estrogen (ER), progesterone (PR) and epidermal human growth factor 2 (HER2) were immunohistochemically determined before systemic neoadjuvant polychemotherapy (PCT). According to the receptor status, tumors were divided into 4 groups: 1 – triple negative («3-neg») tumors, 2 – HER2-positive («HER2») tumors, 3 – hormonally receptor («HP») tumors, 4 – tumorswith co-expression of hormonal receptors and HER2 («HR + HER2»). For polychemotherapy (PCT) the regimen (AСx4–Рx4) was used.The ultrastructure of the tumor cells (TC) was examined using standard electron microscopy methods. In all study groups, the frequency of cases with pronounced therapeutic pathomorphosis (PTPM) was determined, as well as the frequency of tumors with luminal and non-luminal symptoms after treatment. The obtained data were calculated using non-parametric methods with the software package for PC «Biostat» application and using a non-parametric criterion of the most plausible assessment of reliability for small selections (Pmp).&#x0D; Results. It was found that PCT in patients with conventional BC causes a pronounced therapeutic pathomorphosis (РТPM) of most tumors of the receptor groups «3-neg», «HER2» and «HR+HER2», compared with the group «HR», where no case of РTPM was observed. At SЕF BC the frequency of tumors with the pronounced pathomorphosis in groups with nonluminal subtypes and co-expression of receptors decreases, that for group «3-neg» is reliable. This indicates increased chemoresistance of triple negative tumors. In the group of hormone receptor tumors at SEF, this index increases significantly. Analysis of the ultrastructure of tumors of different molecular subtypes showed that most of the processes of damage and accompanying reactions to the action of PCT are identical for both forms of BC. Thus, in response to chemotherapy, different types of cell death: necrotic, dark cell, apoptotic; processes of cell nucleus damage: presence of dinuclear PCs, cells with micronuclei; disturbance of the microvessels structure: edema and condensation of the endothelial cells cytoplasm and their exfoliation; activation of phagocytosis and immune processes are observed. Only SЕF is characterized by the presence of TCs and emboli in capillaries, as well as a significantly increased frequency of giant polyploid tumor cells. It was also found that after PCT in patients with normal BC preserved tumor cells show mainly luminal signs (72.7–100%). However, in SЕF BC such reaction occurs only for hormone-receptor subtypes and the group with co-expression of receptors, whereas for tumors of three negative and HER2-positive subtypes the frequency of luminal signs is lower than in conventional BC and is 33.3%, p ≤ 0.05 and 66.7%, respectively.&#x0D; Conclusions. Peculiarities of SЕF BC in contrast to the usual form of BC are significantly reduced sensitivity of 3-negative cancer to chemotherapy and the tendency to chemoresistance of molecular subtypes with the presence of HER2; significant survival after PCT tumors with non-luminal cells characteristic in the group of 3-negative cancer, while in other receptor subtypes most tumors have luminal features; probably increased after chemotherapy the number of tumors with giant polyploid cells. It is possible that the features of SЕF BC are associated with the presence of polyploid TCs resistant to chemotherapy, and both inflammation and chemotherapy may play a role in stimulating their formation.

Low androgen level impairs erectile function of rat by regulating the Ng/CaN/AKT/eNOS pathway in penile corpus cavernosum.

The mechanism by which low androgen status inhibit erectile function has not yet been clearly elucidated. Neurogranin (Ng) is a Ca2+ -sensitive calmodulin binding protein that is expressed in endothelial cells and regulates eNOS function. To investigate whether low androgen status inhibit erectile function by regulating the Ng/CaN/AKT/eNOS pathway in the penile cavernous tissue of rats. Thirty-six 8-week-old male Sprague-Dawley rats were randomly divided into six groups as follows (n=6): 4-week control group (4w-control), 4-week castration group (4w-cast), 4-week castration+testosterone replacement group (4w-cast+T), 8-week control group (8w-control), 8-week castration group (8w-cast), and 8-week castration+testosterone replacement group (8w-cast+T). Four weeks and eight weeks after surgery, the ratio of the maximum intracavernous pressure/mean arterial pressure (ICPmax/MAP) was examined. The level of NO and the expression of Ng, calcineurin (CaN), AKT, p-AKT(S473), eNOS, and p-eNOS(Ser1177) in the penile cavernous tissue of each group were determined. Ng and CaN were mainly expressed in the membrane and cytoplasm of endothelial cells and smooth muscle cells in the penile cavernous tissue of rats. The ICPmax/MAP and the concentration of NO in the cast group were significantly lower than those in the control group and cast+T replacement group (p<0.01). The expression of Ng and the ratios of p-AKT/AKT and p-eNOS/eNOS in the penile cavernous tissue of rats in the cast group were significantly lower than those in the control group and cast+T replacement group (p<0.01). The expression of CaN in the penile cavernous tissue of rats in the cast group was significantly increased compared with that in the control group and the cast+T replacement group (p<0.01). Inhibiting the expression of Ng and subsequently upregulating the expression of CaN in the rat penile cavernous tissue was one of the upstream mechanisms of low androgen status inhibiting erectile function by inhibiting the AKT/eNOS signaling pathway.

Low androgen status inhibits erectile function by upregulating the expression of proteins of mitochondria‐associated membranes in rat corpus cavernosum

To investigate the effect of low androgen status on mitochondria-associated membranes (MAMs) and its relationship with erectile function. A total of 36 eight-week-old male Sprague-Dawley rats were randomly divided into six groups: the control (sham-operated) group, the castration group, the castration + testosterone (cast + T) group, the control + siRNA group, the cast + siRNA group, and the cast + empty vectorgroup. Testosterone propionate (3mg/kg) was subcutaneously injected into the rats in the cast + T group every other day starting from the second day after the surgery. Four weeks later, lentiviral vectors carrying phosphofurin acidic cluster sorting protein 2 (PACS-2) gene-specific siRNA (1 × 108 TU/ml, 10 µl) were injected into the rats in the siRNA groups. At the sixth week of castration, the ratio of the maximum intracavernous pressure/the mean arterial pressure (ICPmax/MAP), the levels of nitric oxide (NO), endothelial nitric oxide synthase (eNOS), phospho-eNOS (p-eNOS), fatty acid-CoA ligase 4 (FACL-4), PACS-2, and inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) in the penile corpus cavernosum were determined. The FACL-4, PACS-2, and IP3R1 were primarily localized in the cytoplasm of endothelial cells and part of smooth muscle cells in the corpus cavernosum. The level of NO, the ratio of ICPmax/MAP, and p-eNOS/eNOS were decreased significantly in the castration group compared with the control group (p<0.01).The expressions of FACL-4, PACS-2, and IP3R1 were increased significantly in the castration group compared with the control group (p<0.01). The level of NO, the ratios of ICPmax/MAP, and the ratio of p-eNOS/eNOS were increased significantly in the cast + siRNA groupcompared with the castration group (p<0.01). The expressions of FACL-4 and PACS-2 were decreased significantly in the cast + siRNA group compared with the castration group (p<0.01). Low androgen status upregulated the expressions of patients in MAMs (FACL-4, PACS-2, and IP3R1) in the corpus cavernosumand inhibited the eNOS/NO/cGMP signaling pathway, resulting in impaired erectile function in rats. Erectile function may be improved by inhibiting the high expression of PACS-2 in the corpus cavernosum under low androgen state.

ULTRAMORPHOMETRIC CHARACTERISTICS OF ACINI AND MICROVASCULATURE OF THE PANCREAS IN THE PRESENCE OF MODERATE DEHYDRATION.

The aim: The objective of our study was to evaluate the features of ultramorphometric characteristics of exocrine parenchyma and microvasculature of the pancreas in the presence of moderate dehydration by means of an experiment in laboratory rats. Materials and methods: The experiment involved 20 mature white male rats divided into 2 groups: control and experimental (10 rats each). In the experimental group, moderate dehydration was simulated, i.e. the animals were deprived of water for 7 days, while the control rats were provided with a normal water supply during the study. Pancreatic parenchyma samples were fixed in phosphate-buffered glutaraldehyde solution and post-fixed in osmium tetroxide solution, dehydrated and embedded in a mixture of epoxy resins. Ultrastructural analysis was performed using JEOL JEM-1230 transmission electron microscope (Japan). Results: Pancreatic electron microscopy in the presence of moderate dehydration demonstrated statistically significant changes in exocrinocytes area and exocrinocyte nucleus area which increased by 8.02% (p = 0.028) and 40.28% (p < 0.001), respectively. Among the vessels of microcirculation, the largest changes occurred in the capillaries: their lumen narrowed by 22.34% (p = 0.002) as compared with the control group. The cytoplasm of endothelial cells contained a large number of vacuoles and micropinocytotic vesicles. Conclusions: Among the organelles of exocrinocytes, mitochondria appeared the most vulnerable to the effects of dehydration. They demonstrated polymorphic changes: a part of the mitochondria was hypotrophic and had partially reduced cristae, and another part was hypertrophic.

YAP/TAZ Promote Hematopoietic Regeneration Via Accelerating the Recovery of Bone Marrow Niche

Adult hematopoietic stem cells (HSCs) reside and are protected in a unique bone marrow (BM) microenvironment, termed the HSC niche, which consists mainly of vascular endothelial cells (EC) and EC-associated mesenchymal stromal cells (MSC). Myeloablative stresses, such as ionizing radiation (IR) and chemotherapy, induce not only depletion of hematopoietic cells but also disruption of HSC niche components, as exemplified by dilation and leakiness of BM vasculature and depletion and dysfunction of BM MSCs. These structural and functional changes in the HSC niche restrain efficient hematopoietic recovery, which often compromises the efficacy of HSC transplantation (HSCT) and chemotherapy. YAP/TAZ are the two transcriptional coactivators normally repressed by LATS kinases downstream of the Hippo pathway. Although cumulative evidence has established a critical role of YAP/TAZ activation in tissue regeneration of various solid organs, their role in BM regeneration remains poorly understood.Our quantitive RT-PCR revealed that YAP/TAZ are abundantly expressed in steady-state mouse ECs (CD45 -Ter119 -CD31 +Sca1 +CD105 hi) and MSCs (CD45 -Ter119 -CD31 -PDGFRα +CD51 +LepR +) but scarcely in hematopoietic cells including HSCs (Lin -cKit +Sca1 +Flk2 -CD150 +CD48 -CD34 lo), which was confirmed by reanalysis of the published single cell RNA-seq datasets (GSE128423). Immunofluorescent imaging of BM sections revealed that YAP/TAZ are distributed mainly in the cytoplasm of ECs but evenly in the cytoplasm and nuclei of MSCs, indicating their differential basal activity in these two HSC niche components. Kinetic transcriptome analysis revealed that YAP/TAZ activity is transiently activated in ECs at 24 hours and returns to a basal repressive state by day 3 after sublethal IR. This transient activation of endothelial YAP/TAZ was critical for vascular integrity, as conditional deletion of YAP/TAZ in ECs (Cdh5-Cre ERT2Yap1 f/fTaz f/f) caused 100% lethality of mice within 10 days following sublethal IR. In sharp contrast, the kinetic expression analysis of a YAP/TAZ target gene CTGF indicated their transient inhibition in MSCs after sublethal IR, and the conditional YAP/TAZ deletion in BM MSCs (Ebf3-Cre ERT2Yap1 f/fTaz f/f) led to their reduced colony forming ability when assessed by colony forming unit fibroblast (CFU-F) assay.Recently, we discovered a novel and potent LATS inhibitor GA-003 that selectively induces mouse and human YAP/TAZ activation in vitro (IC 50 against LATS1 = 1.06 ± 0.08 nM). To analyze the effect of pharmacological YAP/TAZ activation on BM regeneration in vivo, we treated mice with intraperitoneal injection of GA-003 (50 mg/kg per day, for 8 days) following sublethal IR. Remarkably, we observed an accelerated recovery of hematopoiesis, with the absolute numbers of BM cellularity, GMP (Lin -cKit +Sca1 -FcγR +CD34 +) and HSC EPCR (Lin -cKit +Sca1 +CD150 +EPCR +) on day 14 increased by 3.50-fold (p=0.0002), 6.49-fold (p=0.0022) and 11.41-fold (p=0.022), respectively in the GA-003-treated group compared to vehicle-treated group. In addition, GA-003 also promoted hematopoietic recovery after 5-FU injection (150 mg/kg) and HSCT. Nonetheless, consistent with the scarce expression of YAP/TAZ in hematopoietic stem and progenitor cells (HSPC), in vitro GA-003 treatment did not enhance HSPC growth, suggesting niche-mediated effects by GA-003. Indeed, in vitro tube formation assay indicated accelerated angiogenesis by GA-003-treated human umbilical vein ECs, and CFU-F assays revealed significant enhancement of colony formation by mouse BM-derived MSCs upon GA-003 treatment. To reveal the effect of GA-003 on the HSC niche components in vivo, we performed whole BM immunofluorescent imaging at various time points following sublethal IR and GA-003 treatment. We observed alleviated vascular dilation and leakiness and earlier restoration of vascular damage in GA-003-treated group compared to vehicle-treated group, which was associated with increased VE-Cadherin expression in ECs. These results suggest that reinforcing YAP/TAZ activity upon myelosuppression promotes HSC niche integrity and recovery and accelerates hematopoietic regeneration.Taken together, our results establish YAP/TAZ as novel regulators of HSC niche and highlight YAP/TAZ as promising therapeutic targets to boost hematopoietic recovery after myeloablative interventions such as chemotherapy and HSCT. DisclosuresAihara: Nissan Chemical Corporation: Current Employment. Iwawaki: Nissan Chemical Corporation: Current Employment. Nishino: Nissan Chemical Corporation: Current Employment. Iwama: Nissan Chemical Corporation: Research Funding.

Stillbirth and fetal capillary infection by SARS-CoV-2

We presented the case of stillbirth in a paucisymptomatic mother affected by SARS-CoV-2. At gross examination, the placenta showed a diffuse marble appearance and a focal hemorrhagic area. Multiple areas of hemorrhagic or ischemic necrosis with central and peripheral villous infarctions and thrombosis of several maternal and fetal vessels with luminal fibrin and platelet deposition were observed. All lesions seemed to be synchronous. Virus particles were identified within the cytoplasm of endothelial cells using electron microscopy, whereas SARS-CoV-2 RNA was detected in the placental tissue using real-time reverse transcription-polymerase chain reaction.Here, fetal vascular malperfusion was associated with infection; in fact, electron microscopy images showed that marked SARS-CoV-2 endotheliotropism involved the intravillous fetal capillaries. Furthermore, we confirmed that syncytiotrophoblast is the major target cell type for SARS-CoV-2 infection of the placenta. In conclusion, the possible consequences of the action of the placentotropic SARS-CoV-2 included the occurrence of vertical transmission, as reported in the literature, and/or stillbirth: the latter possibility may be triggered by a hampered maternal and/or fetal perfusion of the placenta. The diffuse thrombosis and subsequent ischemia of fetal capillaries induced by COVID-19 cannot be predicted by standard clinical surveillance.

Retraction Note to: Circular RNA circ_0003204 inhibits proliferation, migration and tube formation of endothelial cell in atherosclerosis via miR-370-3p/TGF\u03b2R2/phosph-SMAD3 axis

Circular RNAs (circRNAs) represent a class of non-coding RNAs (ncRNAs) which are widely expressed in mammals and tissue-specific, of which some could act as critical regulators in the atherogenesis of cerebrovascular disease. However, the underlying mechanisms by which circRNA regulates the ectopic phenotype of endothelial cells (ECs) in atherosclerosis remain largely elusive. CCK-8, transwell, wound healing and Matrigel assays were used to assess cell viability, migration and tube formation. QRT-qPCR and Immunoblotting were used to examine targeted gene expression in different groups. The binding sites of miR-370-3p (miR-370) with TGFβR2 or hsa_circ_0003204 (circ_0003204) were predicted using a series of bioinformatic tools, and validated using dual luciferase assay and RNA immunoprecipitation (RIP) assay. The localization of circ_0003204 and miR-370 in ECs were investigated by fluorescence in situ hybridization (FISH). Gene function and pathways were enriched through Metascape and gene set enrichment analysis (GSEA). The association of circ_0003204 and miR-370 in extracellular vesicles (EVs) with clinical characteristics of patients were investigated using multiple statistical analysis. Circ_0003204, mainly located in the cytoplasm of human aorta endothelial cells (HAECs), was upregulated in the ox-LDL-induced HAECs. Functionally, the ectopic expression of circ_0003204 inhibited proliferation, migration and tube formation of HAECs exposed to ox-LDL. Mechanically, circ_0003204 could promote protein expression of TGFβR2 and its downstream phosph-SMAD3 through sponging miR-370, and miR-370 targeted the 3′ untranslated region (UTR) of TGFβR2. Furthermore, the expression of circ_0003204 in plasma EVs was upregulated in the patients with cerebral atherosclerosis, and represented a potential biomarker for diangnosis and prognosis of cerebrovascular atherogenesis. Circ_0003204 could act as a novel stimulator for ectopic endothelial inactivation in atherosclerosis and a potential biomarker for cerebral atherosclerosis.

SARS-CoV-2 identified by transmission electron microscopy in lymphoproliferative and ischaemic intestinal lesions of COVID-19 patients with acute abdominal pain: two case reports

BackgroundSARS-CoV-2 may produce intestinal symptoms that are generally mild, with a small percentage of patients developing more severe symptoms. The involvement of SARS-CoV-2 in the physiopathology of bowel damage is poorly known. Transmission electron microscopy (TEM) is a useful tool that provides an understanding of SARS-CoV-2 invasiveness, replication and dissemination in body cells but information outside the respiratory tract is very limited. We report two cases of severe intestinal complications (intestinal lymphoma and ischaemic colitis) in which the presence of SARS-CoV-2 in intestinal tissue was confirmed by TEM. These are the first two cases reported in the literature of persistence of SARS-CoV-2 demonstrated by TEM in intestinal tissue after COVID 19 recovery and SARS-CoV-2 nasopharyngeal clearance.Case presentationDuring the first pandemic peak (1st March–30th April 2020) 932 patients were admitted in Hospital Universitari Mútua Terrassa due to COVID-19, 41 (4.4%) required cross-sectional imaging techniques to assess severe abdominal pain and six of them (0.64%) required surgical resection. SARS-CoV-2 in bowel tissue was demonstrated by TEM in two of these patients. The first case presented as an ileocaecal inflammatory mass which turned to be a B-cell lymphoma. Viral particles were found in the cytoplasm of endothelial cells of damaged mucosa. In situ hybridization was negative in tumour cells, thus ruling out an oncogenic role for the virus. SARS-CoV-2 remained in intestinal tissue 6 months after nasopharyngeal clearance, suggesting latent infection. The second patient had a severe ischaemic colitis with perforation and SARS-CoV-2 was also identified in endothelial cells.ConclusionsSevere intestinal complications associated with COVID-19 are uncommon. SARS-CoV-2 was identified by TEM in two cases, suggesting a causal role in bowel damage.

УЛЬТРАСТРУКТУРНІ ОСОБЛИВОСТІ НЕЙРО-ГЛІАЛЬНО-КАПІЛЯРНИХ ВІДНОСИН СА3 ПОЛЯ ГІПОКАМПА ЧЕРЕЗ 21 ДОБУ ЗА УМОВ ЕКСПЕРИМЕНТАЛЬНОЇ ТЕРМІЧНОЇ ТРАВМИ

Severe thermal injury leads to a number of morphofunctional changes that occur in violation of integrity or loss of skin in deep burns and endogenous intoxication on the background of which significant structural and metabolic disorders in all organs and systems of the affected organism are developed. This is manifested in a complicated symptom complex of burn disease. Changes in the central nervous system play an important role in the pathogenesis of burn injury, the nature and depth of morphofunctional changes in individual cerebral structures, in particular in the hippocampus (central organ of the limbic system, which is especially sensitive to various pathogens), after thermal lesions remain poorly studied. The aim of our study was to establish the features of submicroscopic changes in the structural components of the hippocampus after 21 days under conditions of experimental thermal injury. The experimental study was performed on white laboratory male rats. Third-degree burns were applied under thiopental-sodium anesthesia with copper plates heated in boiled water to a temperature of 97–100°C. The size of the affected area was 18–20% of the rats’ body epilated surface. Histological changes were studied on the 21st day from the beginning of the experiment. For electron microscopy, the obtained ultrathin sections were contrasted with uranyl acetate and lead citrate according to the Reynolds method. It was found that on the 21st day after the experimental thermal injury significant alternative changes in neurons, glial cells and the microcirculatory tract of the hippocampus were defined which developed on the background of excessive endogenous intoxication. Neurons are hypochromic and sharply hyperchromic, disorganized and have an altered shape; there are cell-free areas. Endoplasmic reticulum, Golgi complex, mitochondria are destructively changed. Degeneration of macroglial cells has been noticed. Their nuclei are high in electron density in which heterochromatin predominates. Destruction and lysis of organelles, lysosomes have also been found in the cytoplasm. These changes of astrocytic glia lead to impaired trophic function and the relation between astrocytes and blood capillaries. Hemocapillaries wall remodeling is manifested by the total edema, compaction of the basement membrane. The lumenal surface of the plasmolemma of endothelial cells is homogeneous, indistinctly contoured. The cytoplasm of endothelial cells is swollen, enlightened, and poor in organelles; there is a small number of micropinocytic bubbles and vesicles.

Pentraxin 3 expression in lungs and neutrophils of calves

Bacterial lung disease caused by Mannheimia haemolytica inflict significant mortality and morbidity resulting in enormous economic losses to cattle industry. The use of antibiotics is becoming more challenging because of development of anti-microbial resistance. The innate immune system plays a critical role in the initiation of immune response in the lung. Pentraxin 3 (PTX3), a pattern-recognition receptor is produced at sites of inflammation by many cell types, recognizes and binds to many pathogens, activates the complement cascade, and has a role in the clearance of apoptotic and necrotic cells. Because there are very few data on the expression of PTX3 in the lungs, we examined PTX3 expression in lungs of normal and M. haemolytica-infected calves and normal and E. coli lipopolysaccharide-treated cattle neutrophils using light and electron microscopic immunochemistry and Western blots. Immunohistology showed the presence of PTX3 in airway epithelial cells, alveolar septa and macrophages in normal and inflamed lungs of calves and the blots showed a significant increase in the expression of PTX3 in lungs from infected calves. Immuno-gold electron microscopy showed PTX3 in the nuclei, cytoplasm, and vesicular organelles of alveolar macrophages, endothelial cells and pulmonary intravascular macrophages (PIMs). Immunohistochemical staining for PTX3 in peripheral blood neutrophils shows an altered staining pattern in neutrophils stimulated with lipopolysachharide (LPS). However, western blots no significant change in PTX3 amount in LPS-treated neutrophils compared to the controls. These are the first data on the expression of PTX3 in the lungs and the neutrophils of cattle which may add to our understanding of innate immunity in cattle lungs.

Low androgen status inhibits erectile function by increasing pyroptosis in rat corpus cavernosum

The mechanism of erectile dysfunction (ED) caused by low androgen status is not fully understood. To investigate whether low androgen status inhibits erectile function of rats by inducing pyroptosis in the corpus cavernosum (CC). Thirty-six eight-weeks-old healthy male Sprague-Dawley rats were equally divided into six groups: sham-operated group (4w sham, 8w sham), castration group (4w cast, 8w cast), and castration + testosterone (T) group (4w cast + T, 8w cast + T). The rats in castration + T groups were injected with testosterone propionate subcutaneously every other day. After 4 and 8weeks, the ratio of maximum intracavernous pressure (ICPmax)/mean arterial pressure (MAP), the level of serum T, the concentration of nitric oxide (NO) and interleukin-1β (IL-1β), the expression of NOD-like receptor pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), Caspase-1 p20, gasdermin D-N (GSDMD-N), transforming growth factor β1 (TGF-β1), collagen-I, and collagen-III, the ratio of smooth muscle/collagen (SM/C), and the proportion of pyroptotic cells in the CC were analyzed. The ratio of ICPmax/MAP (3/5V) and SM/C, the level of NO and serum T was significantly decreased in castration groups when compared to other groups (p<0.01). NLRP3, ASC, Caspase-1, and GSDMD were mainly expressed in the cytoplasm of smooth muscle cells (SMCs) and endothelial cells (ECs) in the CC. The expression of NLRP3, ASC, Caspase-1p20, GSDMD-N, IL-1β, TGF-β1, collagen-I, and collagen-III was significantly increased in castration groups when compared with other groups (p<0.01). The proportion of pyroptotic cells in the CC was increased significantly in castration groups when compared with other groups (p<0.05). Low androgen status inhibits erectile function of rats by promoting CC fibrosis and reducing NO synthesis through pyroptosis of SMCs and ECs in the CC.

Exosomes derived from stem cells from apical papilla promote craniofacial soft tissue regeneration by enhancing Cdc42-mediated vascularization

BackgroundReconstruction of complex critical-size defects (CSD) in the craniofacial region is a major challenge, and soft tissue regeneration is crucial in determining the therapeutic outcomes of craniofacial CSD. Stem cells from apical papilla (SCAP) are neural crest-derived mesenchymal stem cells (MSCs) that are homologous to cells in craniofacial tissue and represent a promising source for craniofacial tissue regeneration. Exosomes, which contain compound bioactive compounds, are the key factors in stem cell paracrine action. However, the roles of exosomes derived from SCAP (SCAP-Exo) in tissue regeneration are not fully understood. Here, we explored the effects and underlying mechanisms of SCAP-Exo on CSD in maxillofacial soft tissue.MethodsSCAP-Exo were isolated and identified by transmission electron microscopy and nanoparticle tracking analysis. The effects of SCAP-Exo on wound healing and vascularization were detected by measuring the wound area and performing histological and immunofluorescence analysis on the palatal gingival CSD of mice. Real-time live-cell imaging and functional assays were used to assess the effects of SCAP-Exo on the biological functions of endothelial cells (ECs). Furthermore, the molecular mechanisms of SCAP-Exo-mediated EC angiogenesis in vitro were tested by immunofluorescence staining, Western blot, and pull-down assays. Finally, in vivo experiments were carried out to verify whether SCAP-Exo could affect vascularization and wound healing through cell division cycle 42 (Cdc42).ResultsWe found that SCAP-Exo promoted tissue regeneration of palatal gingival CSD by enhancing vascularization in the early phase in vivo and that SCAP-Exo improved the angiogenic capacity of ECs in vitro. Mechanistically, SCAP-Exo elevated cell migration by improving cytoskeletal reorganization of ECs via Cdc42 signalling. Furthermore, we revealed that SCAP-Exo transferred Cdc42 into the cytoplasm of ECs and that the Cdc42 protein could be reused directly by recipient ECs, which resulted in the activation of Cdc42-dependent filopodium formation and elevation in cell migration of ECs.ConclusionThis study demonstrated that SCAP-Exo had a superior effect on angiogenesis and effectively promoted craniofacial soft tissue regeneration. These data provide a new option for SCAP-Exo to be used in a cell-free approach to optimize tissue regeneration in the clinic.