STIMULATION OF RAT SKELETAL MUSCLE ANGIOGENESIS BY DIRECT AND CELL-MEDIATED ADMINISTRATION OF RECOMBINANT ANGIOGENIN GENE

Морфология ◽

10.34922/ae.2020.157.1.006 ◽

2020 ◽

pp. 35-40

Author(s):

И. В. Саматошенков

Keyword(s):

Skeletal Muscle ◽

Gastrocnemius Muscle ◽

Mononuclear Cells ◽

Muscle Fibers ◽

Control Group ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord ◽

Ischemic Area ◽

Serotype 5 ◽

Genetic Constructs

Цель - оценить эффективность реваскуляризации икроножной мышцы крысы в условиях прямой и опосредованной мононуклеарными клетками крови пуповины человека (МККП) доставки в область ишемии рекомбинантного гена ангиогенина (Ang) человека при помощи аденовирусного вектора 5-го серотипа (Ad5). Материал и методы. Исследования проведены на 30 крысах линии Wistar. Через 14 сут после иссечения фрагмента бедренной артерии животным инъецировали в ишемизированную икроножную мышцу генетическую конструкцию (группа Ad5-Ang, n=15). Крысам другой группы (МККП+Ad5-Ang, n=15) в тот же срок трансген доставляли в мышцу при помощи МККП. В группе контроля животным (n=15) вводили в мышцу 0,9 % NaCl в тех же условиях. Через 14 и 28 сут в области ишемии оценивали отношение капилляры/мышечные волокна, количество мышечных волокон и количество мышечных волокон с центральным расположением ядер (МЦЯ). Капилляры идентифицировали по локализации эндотелиальных клеток, выявляемых при помощи иммуногистохимической реакции с антителами против CD31. Результаты. На 14-е сутки после введения МККП+Ad5-Ang показатель отношения количества капилляров к количеству мышечных волокон в области ишемии увеличивается на 57 % (p<0,05). На 28-е сутки в группе МККП+Ad5-Ang и в группе Ad5-Ang значимые различия по данному показателю при сравнении с контрольной группой не выявлены. Количество мышечных волокон на 14-е сутки в группе Ad5-Ang не изменяется, а в группе МККП+Ad5-Ang - уменьшается на 58,4 % (p<0,05). К 28-м суткам этот показатель в группе МККП+Ad5-Ang уменьшается на 95,9 % (p<0,05), а в группе Ad5-Ang - на 197,8 % (p<0,05). Количество МЦЯ существенно увеличивается в обеих экспериментальных группах с применением генетических конструкций на 14-е сутки. Выводы. Введение рекомбинантного гена Ang в область ишемии скелетной мышцы или его доставка в эту область при помощи мононуклеарных клеток крови пуповины стимулирует ангиогенез и постишемическую регенерацию мышечных волокон. Objective - to evaluate the effectiveness of revascularization of the rat gastrocnemius muscle following direct and human umbilical cord blood mononuclear cells (MNCs)-mediated delivery of human recombinant angiogenin (Ang) gene to the ischemic area using adenovirus serotype 5 vector (Ad5). Materials and methods. The study was carried out on 30 Wistar rats. Fourteen days after the excision of the femoral artery fragment, the genetic construct was injected into the animals’ ischemic gastrocnemius muscle (AD5-Ang group, n=15). In the other group (mccp+Ad5-Ang, n=15), the transgene was delivered to the muscle with the help of MNCs within the same time limit. In the control group (n=15) 0,9 % NaCl was injected into the muscle of animals under the same conditions. Fourteen and twenty-eight days after the injection, the ratio of capillaries/muscle fibers, the number of muscle fibers and the number of muscle fibers with a central location of nuclei (MCN) were evaluated in the ischemic area. Capillaries were identified by localization of endothelial cells detected by immunohistochemical reaction with antibodies against CD31. Results. On the 14th day after administration of MNCs+Ad5Ang, the ratio of capillaries to the number of muscle fibers in the ischemic area increased by 57 % (p<0,05). On the 28th day in the MNCs+Ad5-Ang group and in the Ad5-Ang group, no significant differences in this indicator were found compared with the control group. The number of muscle fibers on the 14 day in the Ad5-Ang group did not change, and in the MNCs+Ad5-Ang group, it decreased by 58,4 % (p<0,05). By the 28th day, this indicator in the MNCs+Ad5-Ang group decreased by 95,9 % (p<0,05), and in the Ad5-Ang group - by 197,8 % (p<0,05). The number of MCN on the 14 day significantly increased in both experimental groups, in which the genetic constructs were used. Conclusion. The introduction of recombinant ang gene into the area of skeletal muscle ischemia or its delivery to this area with the help of MCNs stimulates angiogenesis and post-ischemic regeneration of muscle fibers.

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Evaluation of Direct and Cell-Mediated Lactoferrin Gene Therapy for the Maxillofacial Area Abscesses in Rats

Pharmaceutics ◽

10.3390/pharmaceutics13010058 ◽

2021 ◽

Vol 13 (1) ◽

pp. 58

Author(s):

Elima Agatieva ◽

Said Ksembaev ◽

Mikhail Sokolov ◽

Vage Markosyan ◽

Ilnaz Gazizov ◽

...

Keyword(s):

Gene Therapy ◽

Wound Healing ◽

Mononuclear Cells ◽

Fluorescent Protein ◽

Viral Vector ◽

Control Group ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord ◽

Gene Encoding ◽

Cervical Lymph Nodes

Resistance to antibacterial therapy requires the discovery of new methods for the treatment of infectious diseases. Lactoferrin (LTF) is a well-known naïve first-line defense protein. In the present study, we suggested the use of an adenoviral vector (Ad5) carrying the human gene encoding LTF for direct and cell-mediated gene therapy of maxillofacial area phlegmon in rats. Abscesses were developed by injection of the purulent peritoneal exudate in the molar region of the medial surface of the mandible. At 3–4 days after phlegmon maturation, all rats received ceftriaxone and afterward were subcutaneously injected around the phlegmon with: (1) Ad5 carrying reporter gfp gene encoding green fluorescent protein (Ad5-GFP control group), (2) Ad5 carrying LTF gene (Ad5-LTF group), (3) human umbilical cord blood mononuclear cells (UCBC) transduced with Ad5-GFP (UCBC + Ad5-GFP group), and (4) UCBC transduced with Ad5-LTF (UCBC + Ad5-LTF group). Control rats developed symptoms considered to be related to systemic inflammation and were euthanized at 4–5 days from the beginning of the treatment. Rats from therapeutic groups demonstrated wound healing and recovery from the fifth to seventh day based on the type of therapy. Histological investigation of cervical lymph nodes revealed purulent lymphadenitis in control rats and activated lymphatic tissue in rats from the UCBC + Ad5-LTF group. Our results propose that both approaches of LTF gene delivery are efficient for maxillofacial area phlegmon recovery in rats. However, earlier wound healing and better outcomes in cervical lymph node remodeling in the UCBC + Ad5-LTF group, as well as the lack of direct exposure of the viral vector to the organism, which may cause toxic and immunogenic effects, suggest the benefit of cell-mediated gene therapy.

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Structure of the cortical cytoskeleton in fibers of postural muscles and cardiomyocytes of mice after 30-day 2-g centrifugation

Journal of Applied Physiology ◽

10.1152/japplphysiol.00812.2014 ◽

2015 ◽

Vol 118 (5) ◽

pp. 613-623 ◽

Cited By ~ 14

Author(s):

Irina V. Ogneva ◽

V. Gnyubkin ◽

N. Laroche ◽

M. V. Maximova ◽

I. M. Larina ◽

...

Keyword(s):

Skeletal Muscle ◽

Tibialis Anterior ◽

Mechanical Load ◽

Tibialis Anterior Muscle ◽

Muscle Fibers ◽

Control Group ◽

Negative Effects ◽

Transverse Stiffness ◽

G 12 ◽

Cortical Cytoskeleton

Altered external mechanical loading during spaceflights causes negative effects on muscular and cardiovascular systems. The aim of the study was estimation of the cortical cytoskeleton statement of the skeletal muscle cells and cardiomyocytes. The state of the cortical cytoskeleton in C57BL6J mice soleus, tibialis anterior muscle fibers, and left ventricle cardiomyocytes was investigated after 30-day 2- g centrifugation (“2- g” group) and within 12 h after its completion (“2- g + 12-h” group). We used atomic force microscopy for estimating cell's transverse stiffness, Western blotting for measuring protein content, and RT-PCR for estimating their expression level. The transverse stiffness significantly decreased in cardiomyocytes (by 16%) and increased in skeletal muscles fibers (by 35% for soleus and by 29% for tibialis anterior muscle fibers) in animals of the 2-g group (compared with the control group). For cardiomyocytes, we found that, in the 2- g + 12-h group, α-actinin-1 content decreased in the membranous fraction (by 27%) and increased in cytoplasmic fraction (by 28%) of proteins (compared with the levels in the 2- g group). But for skeletal muscle fibers, similar changes were noted for α-actinin-4, but not for α-actinin-1. In conclusion, we showed that the different isoforms of α-actinins dissociate from cortical cytoskeleton under increased/decreased of mechanical load.

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Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells (huMSCs) Carrying MicroRNA-342 Relieve Protect Severe Acute Pancreatitis Injury (SAP)

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2021.2744 ◽

2021 ◽

Vol 11 (9) ◽

pp. 1838-1843

Author(s):

Xiaohong Zhou ◽

Xuzhong Hao ◽

Feifei He

Keyword(s):

Stem Cells ◽

Acute Pancreatitis ◽

Mesenchymal Stem Cells ◽

Umbilical Cord ◽

Severe Acute Pancreatitis ◽

Pancreatic Tissue ◽

Inflammatory Factors ◽

Control Group ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord

To investigate whether exosomes (exo) derived from human umbilical cord mesenchymal stem cells (huMSCs) and microRNA (miRNA)-342 have a protective effect on severe acute pancreatitis (SAP). Human umbilical cord blood was collected to extract huMSC-exo. With sham-operated mice as control group (n = 10), the other mice were induced to SAP model (n = 20), while 10 of the SAP mice received treatment with huMSC-exo. ELISA was performed to determine amylase and TAP level as well as inflammatory factors and HE staining to evaluate pathological changes of pancreatic tissue. The expression of miR-342 and Shh, Ptchl, and Smo in the Hh signal pathway was detected using RT-qPCR. The expression of miR-342 and the mRNA expression of Shh, Ptchl, and Smo was higher than that in model group (p < 0.05). The level of serum amylase, trypsinogen, and IFN-γ,Fasl, and IL-6 was upregulated in pancreas tissues of SAP mice relative to healthy mice, but their levels were decreased upon treatment with huMSC-exo and slightly higher than those of the control group, just not significantly. Collectively, the huMSC-exo may activate the Hh signaling pathway by regulating the expression of miR-342 increasing the expression of Shh, Ptchl, and Smo, and thereby healing of damaged pancreatic tissues in SAP.

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Effect of Experimentally Induced Chronic Kidney disease on Skeletal, Cardiac and Smooth Muscle fibers of albino rat

QJM ◽

10.1093/qjmed/hcab099.006 ◽

2021 ◽

Vol 114 (Supplement_1) ◽

Author(s):

Ghada Lotfy ◽

Amel Soliman ◽

Nevine Bahaa ◽

Mohammed Hegazi

Keyword(s):

Skeletal Muscle ◽

Chronic Kidney Disease ◽

Smooth Muscle ◽

Kidney Disease ◽

Intramuscular Injection ◽

Muscle Fibers ◽

Lateral Wall ◽

Control Group ◽

Once Daily ◽

Group Ii

Abstract Background Chronic kidney disease (CKD), or chronic renal failure (CRF) as it was historically termed, includes all degrees of decreased renal function, starting from mild, and moderate, to severe chronic kidney failure. Skeletal muscle atrophy frequently complicates the course of CKD and is associated with excess morbidity and mortality. Cardiovascular diseases have been reported to be the leading causes of death in CKD patients. Chronic Kidney Disease was also reported to be associated with an increased incidence of acid-related gastrointestinal disorders. Aim of the work The aim of this study was to investigate the effect of chronic kidney disease experimentally induced by gentamicin intramuscular injection on the histological structure of gastrocnemius skeletal muscle, left ventricular cardiac muscle and smooth muscle fibers of lower esophagus. Materials and methods Twenty male adult Wistar albino rats were randomly and equally divided into two groups. Group I (control group) received physiological saline intramuscular injection, once daily for 28 consecutive days, in a dose equivalent to that taken in group II. Group II (Gentamicin-treated group) were given Gentamicin intramuscular injection for induction of CKD. Gentamicin was given as Gentamycin sulfate, 40 mg/ml (Sandoz, Switzerland), once daily, in a dose of 80 mg/kg/day for 28 days to induce CKD. After 28 days of the first injection of gentamicin, rats were anaesthetized and blood samples were collected to measure the level of serum urea and creatinine. The left kidneys, the middle third of left gastrocnemius muscle, the lateral wall of left ventricle (LV) and the gastroesophageal junction of all rats of both groups (I and II) were processed for light microscopic study. The middle third of left gastrocnemius muscle, the lateral wall of left ventricle (LV) were further processed for transmission electron microscopic study. Histomorphometrical and statistical analysis were also done. Results The LM examination revealed moderate obliteration of glomeruli, dilatation in some renal tubules and collapse in others, mainly in distal convoluted tubules, with significant fibrosis of renal parenchyma. Serum urea and creatinine levels were increased significantly. The skeletal muscle fibers of the rats in group II (CKD) showed focal areas of myofibers degeneration with siginificant fibrosis. The cardiac muscle fibers of the rats in the group II (CKD) showed focal areas of cardiomyocytes degeneration and other areas of significantly hypertrophied fibers. The smooth muscle fibers of the lower esophageal sphincter of the rats in group II (CKD) showed no significant structural changes compared with the control group, however, the myenetric plexus showed multiple pyknotic and karyolitic nuclei with vacuolated cytoplasm. In addition, insignificant increase in the amount of collagen fibers was observed in almost all layers. Conclusion CKD produced moderate atrophy of skeletal muscle fibers, significant increase in the cardiomyocyte size and no significant structural effect of smooth muscle fibers of the lower esophageal sphincter.

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Establishment of an adherent cell layer from human umbilical cord blood

Genetics and Molecular Biology ◽

10.1590/s1415-47572000000300002 ◽

2000 ◽

Vol 23 (3) ◽

pp. 519-522 ◽

Cited By ~ 2

Author(s):

Zeni Z.C. Alfonso ◽

Eduardo D. Forneck ◽

Waldir F. Allebrandt ◽

Nance B. Nardi

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Umbilical Cord Blood ◽

Mononuclear Cells ◽

Adherent Cell ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord ◽

Adherent Cells ◽

Bone Marrow Cultures ◽

Marrow Cultures

In addition to bone marrow and peripheral blood, stem cells also occur in human umbilical cord blood (HUCB), and there is an increasing interest in the use of this material as an alternative source for bone marrow transplantation and gene therapy. In vitro hematopoiesis has been maintained for up to 16 weeks in HUCB cultures, but the establishment of an adherent, stromal layer has consistently failed. Adherent cell precursors among mononuclear cells from HUCB were sought for in long-term cultures. Mononuclear cells obtained from cord blood after full term, normal deliveries were cultivated at different concentrations in Iscove's modified Dulbecco's medium (IMDM) with weekly feeding. An adherent layer was detected in 16 of 30 cultures, 12 of which were plated at cell concentrations higher than 2 x 10(6) cells/ml. In contrast to bone marrow cultures, in which the stroma is detected early, in most (10/16) positive cultures from HUCB the adherent layer was identified only after the fourth week of culture. The cells never reached confluence and detached from the plate approximately four weeks after detection. May-Grünwald-Giemsa staining of positive cultures revealed fibroblast- or endothelial-like adherent cells in an arrangement different from that of bone marrow stroma in 13 samples. In two of these, the adherent cells were organized into characteristic, delimited cords of cells. Unlike bone marrow cultures, fat cells were never observed in the adherent layers. A rapid development of large myeloid cells in the first week of culture was characteristic of negative cultures and these cells were maintained for up to 12 weeks. HUCB contains adherent cell precursors which occur in lower numbers than in bone marrow and may be at a different (possibly less mature) stage of differentiation.

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