[Peculiarities of angiogenesis in clear cell renal cancer]. / Osobennosti angiogeneza pri svetlokletochnom rake pochki.

A literature search was conducted to review papers on the results of studies of clear cell renal cancer (CCRC) vascularization. Numerous data on the relationship between tumor pathogenesis and its vascularization have been revealed, which indicates the multifactorial nature of CCRC development and the significant role of angiogenesis in this process. It should be taken into account that patients with CCRC may have impaired vessel formation even before tumor development. To evaluate normal and pathologic angiogenesis, a pathohistologic study using immunohistochemistry is certainly necessary. Due to the significant role of angiogenesis in the development and course of CCRC, the use of drugs that suppress the formation of the vascular network in the tumor is relevant and advisable. To date, many drugs have been developed and introduced into clinical practice to inhibit angiogenesis. However, such drugs have not lived up to the expectations placed due to the frequent and rapidly developing drug resistance. Timely detection of pre-tumor and tumor processes, as well as effective treatment of cancer, including CCRC, is possible only with close cooperation between pathomorphologists and oncologists.

Possible Kidney Complications after Application of Cell Technologies for the Repair of the Resected Liver.

The state of rat kidneys after injection of bone marrow multipotent stromal cells (MSC) labeled with Vybrant CM-Dil into intact or resected liver was studied by fluorescence microscopy. The main structural changes in the kidneys after MSC injection into intact and partially resected liver manifested as granular dystrophy and necrobiotic/necrotic changes in single epithelial cells of the distal tubules and collecting ducts, thrombosis of some vessels, progression of an ascending urinary tract infection (detection of dust-like fluorescent objects), which can be due to the immunomodulating or even immunosuppressive influence of MSC and their detritus. MSC injected into the intact or resected liver, as well as the products of their degradation were not detected in the kidneys at all terms of observation. After injection of MSC into partially resected liver, manifestations of bacterial contamination of the renal medulla appeared later. The injection of MSC into the liver can be complicated by thrombosis of the renal vessels, which should be taken into account when using this administration route in the cell therapy.

Preservation of Mesenchymal Stem Cell-Derived Extracellular Vesicles after Abdominal Delivery in the Experiment.

Light luminescent microscopy was used to study the distribution of extracellular microvesicles with PKH26-stained membranes secreted by placenta-derived mesenchymal stromal cells in the uterine tissues at different terms after injections to intact rats and after abdominal delivery (a model of cesarian section). Microvesicles migrated through the uterine tissues and were detected for at least 8 days after injection. In some cases, microvesicles were more numerous in the uterus after cesarian section modeling, which can be related to blockade of microcirculation and lymph flow due to inflammation accompanying surgical intervention. The content of microvesicles in the uterine tissues gradually declined due to macrophage phagocytosis and, probably, due to their migration into the vascular bed. Despite their size, properly stained extracellular microvesicles can be detected by light microscopy in tissues after injections.

Injection of Multipotent Mesenchymal Stromal Cells as a Cause of Hemorrhages in the Regional Lymph Nodes: Experimental Study.

Hemorrhagic changes after subcutaneous injection of autologous bone marrow multipotent mesenchymal cells with transfected GFP gene and additionally stained cell membranes to WAG rats in the projection of ligated femoral vein were studied by fluorescent microscopy. Hemorrhages in tissues with experimental acute local venous occlusion were caused by a combination of venous hypertension with inflammation around the foreign body - the ligature used for ligation of the vein. Fibrin found in tissues together with erythrocytes in the hemorrhages could stimulate the formation of granulations and new vessels instead of damaged or thrombosed ones. Multipotent mesenchymal stromal cells and their detritus getting into the regional lymph nodes initiated immune reactions morphologically confirmed by stubborn hypertrophy and hyperplasia of the lymphoid nodules, hemorrhages, and manifest diapedesis of erythrocytes to the organ parenchyma and sinus system.

Some Peculiarities of Local Distribution of Multipotent Mesenchymal Stromal Cells after Their Injection into Intact Muscle Tissue in Experiment.

Changes in the muscular tissue after subcutaneous injection of autologous bone marrow multipotent mesenchymal stromal cells transfected with GFP gene and additionally stained with cell membrane dye Vybrant CM-Dil in the projection of ligated femoral vein were studied by light microscopy with luminescence. Stromal cells injected through the skin can appear not only in the damaged tissue where acceleration of regeneration processes is required, but also in intact structures located in superficial or deeper layers. In intact muscular tissue, stromal cells spreading in the perivascular tissue initiate inflammation and migration of macrophages, activate and even trigger sclerotic processes due to differentiation into connective tissue cells (fibroblasts) and stimulation of proliferation and collagen synthesis by host fibroblasts. Injected multipotent mesenchymal stromal cells are gradually phagocytized by macrophages.

Hypolipidemic effect of mannans from C. albicans serotypes a and B in acute hyperlipidemia in mice.

Mannans, which are biological macromolecules of polysaccharide origin and function as immunomodulators, have been shown to stimulate macrophages in vivo by interaction with the mannose receptor. Thus, they can be used to stimulate macrophages in order to effectively remove circulating atherogenic lipoproteins. Our primary aim was to evaluate the hypolipidemic potential of mannans from C. albicans serotype A (mannan A) and serotype B (mannan B) in a murine model of hyperlipidemia. Mannan A and mannan B were shown to significantly (p<0.05) stimulate both the proliferation (p <0.05) and nitric oxide production of murine peritoneal macrophages in vitro. Pre-treatment of CBA/Lac mice with mannan A prior to induction of hyperlipidemia significantly (p<0.001) reduced serum atherogenic LDL-cholesterol, total cholesterol, and triglycerides. Mannan B exhibited a similar, but more potent, hypolipidemic effect. Electron microscopic analysis of liver revealed a significant (p<0.001) decrease in the volume of lipid droplets when hyperlipidemic mice were pretreated by both mannans. In conclusion, our findings would suggest that both polysaccharide-based biological macromolecules evaluated in the present study, specifically, the natural immunomodulators (mannans A and B), appeared to function as effective lipid-lowering macromolecules, which could potentially serve as adjunct therapy to more conventional hypolipidemic medications such as a statin drug.

Results of Experimental Ligation of the Main Vein with the Use of Cell Technologies.

Autologous multipotent mesenchymal stromal cells (MMSC) of bone marrow origin with transfected GFP gene and additionally stained cell membranes were injected to rats through the skin in the projection of ligated femoral vein. The results were evaluated by fluorescent microscopy. No signs of MMSC incorporation into the wall of ligated vessel or reorganized collaterals were detected. Angiogenesis processes involving MMSC were detected in experimental rats within just 4 days and progressed until week 2 postinjection, mainly in granulations at the site of surgical intervention and the cicatrix forming there. Injected MMSC completely formed all tunics of the new vessels and incorporated in the vessels forming from the recipient cells. MMSC and the objects created from them were gradually eliminated with participation of macrophages and replaced by structures formed from the recipient cells.

Possibility of Aggravation of Tissue Sclerosis after Injection of Multipotent Mesenchymal Stromal Cells Near the Forming Cicatrix in the Experiment.

The peculiarities of tissue sclerosis after injection of autologous bone marrow multipotent mesenchymal stromal cells transfected with GFP gene and stained with Vybrant CM-Dil cell membrane dye were studied by light microscopy with luminescence. The surgical intervention consisting in ligation of the great vein was followed by tissue sclerotic transformation caused by direct damage and chronic inflammation caused by the presence of slowly resorbed ligature. Injection of stromal cells after this intervention led to formation of more extensive scar. This can attest to the possibility of stromal cells differentiation into connective tissue cells, fibroblasts, and stimulation of proliferation and collagen synthesis by host fibroblasts. A decrease in the volume of dense fibrous connective tissue due to scar reorganization at latter terms cannot not excluded.

Features of Blood Supply to Axillary Lymph Nodes in Breast Cancer Patients.

Numerical density of vessels in axillary lymph node structures in breast cancer patients was studied by light microscopy with antibodies to CD34. The correlation between the stage of breast cancer and vascularization of lymph nodes was analyzed. The development of breast cancer was followed by rapid and significant increase in the number of blood vessel in axillary lymph nodes in general and in all their specific areas, especially in the paracortex and medullary substance. Strong or very strong positive correlation between the stage of tumor process and parameters of vascularization was observed in various areas of the axillary lymph nodes. No significant differences in node vascularization were observed between women without breast cancer and women with stage I breast cancer; the differences were found only starting from stage II. Considerable vascularization of lymph nodes can be a symptom of malignant tumor growth in the area of lymph collection. The duration of the tumor process and the size of the tumor can be indirectly evaluated by the increase in vessel number in lymph nodes.

Initial Stages of Angiogenesis after Acute Experimental Local Venous Outflow Disturbances and Application of Cell Technologies.

The initial stages of angiogenesis in rats after transcutaneous injection of autologous bone marrow multipotent mesenchymal stromal cells transfected with GFP gene and stained cell membranes in the projection of ligated femoral vein were studied by fluorescent light and confocal laser microscopy. Large clusters of brightly fluorescing elongated fibroblast-like cells were seen in the paravasal tissue and in the postoperative scar and signs of angiogenesis were noted as soon as in 4 days. The injected cells not only formed new vessels, but also integrated into vessels formed by host cells. Some injected cells were phagocytizied by macrophages and the latter started to fluoresce due to the presence of the membrane dye. These macrophages within the specified period appeared in the regional inguinal lymph nodes where they formed clusters in the lymphoid parenchyma of the cortical substance.

Possibility of Using Mesenchymal Stromal Cells to Restore Lymph Flow in Experimental Phlebothrombosis.

The possibility of formation of lymphatic vessels after introduction of autologous bone marrow-derived multipotent mesenchymal stromal cells transfected with GFP gene into thrombosed femoral vein was studied by fluorescent microscopy. Vascular thrombosis caused by ligation of the great vein with subsequent injection of thrombin solution was accompanied by blockade of regional lymph flow. The cells injected into thrombosed vein directly participate in the formation of new lymphatic vessels in the paravasal tissue surrounding the vein, its tissue region, and around regional lymph nodes. This is seen from bright specific fluorescence of individual cells in the walls of lymphatic vessels and all vascular layers and valves in UV light.

[SOME REACTIONS OF THE REGIONAL LYMPH NODES OF RATS AFTER IMPLANTATION OF MULTIPOTENT STROMAL CELLS ADSORBED ON POLYHYDROXYALKANOATE INTO A BONE TISSUE DEFECT].

The reactions of the regional lymph nodes, caused by implantation of the autologous multipotent stromal cells of bone marrow origin (AMSCBMO) to accelerate the healing of mandibular bone defect were studied by fluorescent microscopy in inbred male Wag rats aged 6 months (n=62). After the introduction of polyhydroxyalkanoate transplant containing adsorbed AMSCBMO with a transfected Green Fluorescent Protein (GFP) gene into a damaged bone area, the lymphoid nodules in submandibular lymph nodes demonstrated the appearance of numerous large macrophages containing multiple oval fluorescent inclusions in the cytoplasm. The number of these macrophages increased within 2 weeks after surgery and then began to decline. Apparently, AMSCBMO introduced in this way, were partially absorbed by macrophages. After destruction of the structures formed from AMSCBMO, the debris was also phagocytized by macrophages. In either case, these macrophages appeared in the germinal centers of lymphoid nodules in lymph nodes, where the induction of immune responses against DNA and GFP protein was probable.

[The age changes of lymphoid organs structure: review of literature].

Numerous and often contradictory research results specify that the mechanism of increased susceptibility to diseases in old age has no satisfactory explanation so far. Together with it in literature almost completely there are no comparative data on features of surgery and injury influence on a structure and functions of lymphoid organs at patients of different age groups. However, lymph nodes are a marker of activity of inflammatory process in the region, on their changes it is possible to estimate precisely productivity of different medical actions, to predict development of many complications, and, thus, successfully to take actions for their prophylaxis. All this is especially important and actual because of constant increase in average age, in life expectancy of human population and strengthening of surgical activity at patients of advanced and old age.

The features of lymph node reactions to tissue damage of lymph collecting region at old rats.

The features of submandibular lymph node (LN) reactions after lower jaw tissue damage at old rats were studied by method of light microscopy. In LN of old rats for all terms of observation, including intact control, the volume of connective tissue is considerable increased. Besides, old animals in LN have of more change duration of a cortical plateau volume, less - of paracortex and lymphoid nodules without light centers. Features of cellular element number consisted in considerable decrease in quantity of lymphocytes, mitotic figures, immunoblasts and plasmablasts against increase in keeping of macrophages, cells with destruction, stromal elements and erythrocytes.

[ANGIOGENESIS IN THE TISSUES AFTER THE INJECTION OF STROMAL STEM CELLS OF BONE MARROW ORIGIN CLOSE TO THROMBOSED VEIN IN AN EXPERIMENT].

The effects of the injection of autologous multipotent stromal stem cells of bone marrow origin (MSSCBM) (mesenchymal stem cells) with green fluorescent protein gene, additionally marked with DAPI nuclear stain, close to a thrombosed hindlimb vein, were studied by fluorescent microscopy in adult male Wag rats (n = 214). The control groups consisted of intact rats (n = 12), animals with venous thrombosis without the injection of MSSCBM (n = 71) and rats that received paravasal injection of MSSCBM, but without preliminary modeling of venous thrombosis (n = 72). It was found that MSSCBM participated in the development of granulation tissue at the site of surgical intervention performed during the modeling of thrombosis. The rapid development of granulation tissue at the site of surgical trauma may contribute to faster wound clearance from detritus, nonviable tissue and antigenic substances, early onset of tissue repair processes and rapid healing. Restoration of blood flow in the tissue region of a thrombosed vein began later than after the intravenous injection of MSSCBM.

Morphological Changes in Rat Uterine Tissues and Possibility of Spontaneous Labor as a Result of Injection of Multipotent Mesenchymal Stromal Cells against the Background of Hydrometra.

The possibility of pregnancy and labor was evaluated and tissue changes after injection of autologous bone marrow multipotent mesenchymal stromal cells with transfected GFP gene were studied in rats with experimental hydrometra. Injection of stromal cells to the uterine cicatrix increased the number of vessels (vascular walls or their elements) formed de novo with participation of injected cells. The animals produced progeny 2 estrous cycles earlier, the percentage of "puerperal" rats in this group was higher, their progeny was more numerous and they had the maximum numbers of little rats. The maternal mortality was lower after injection of stromal cells. Injection of stromal cells led to development of a trend to more rapid reparative processes in the uterus in animals with cicatricial stenosis of its lumen.

Acceleration of Angiogenesis after Paravasal Injection of Mesenchymal Stem Cells at the Site of Modeled Venous Thrombosis.

The results of transplantation of autologous bone marrow multipotent mesenchymal stem cells carrying GFP gene and labeled with cell nucleus-specific dye DAPI near the thrombosed vein in rat hind limb were studied by methods of luminescent microscopy. It was demonstrated that autologous multipotent mesenchymal stem cells participate in the formation of granulations at the site of surgery. The blood fl ow in the thrombosed great vein was always restored through thrombolysis. We observed no signs of incorporation of the transplanted cells into the wall of the great vessel, clot recanalization, or formation of collaterals. Small branches of the great vein in the affected region were also thrombosed. The blood fl ow in these branches was always restored with participation of the transplanted cells or through clot recanalization or through obliteration of the thrombosed vessels and formation of new vessels. The transplanted cells and structures formed by them were gradually replaced by the recipient cells.

[MORPHOLOGICAL CHANGES AFTER THE USE OF MULTIPOTENT STROMAL CELLS OF BONE MARROW ORIGIN TO RESTORE THE LYMPH FLOW IN THE REGION OF THROMBOSED VEIN].

In the male Wag rats aged 6 months with the body mass of 180-200 g the luminescent microscopy was used to examine the possibility of lymphatic vessel formation after injection into thrombosed vein of the thigh of autologous multipotent stromal cells of bone marrow origin (AMSCBMO) transfected with green fluorescent protein gene. Animals were sacrificed 4 days and 1, 2, 3, 4 and 5 weeks after the injection of AMSCBMO. The control group consisted of intact rats, animals with venous thrombosis receiving no injection of AMSCBMO and those injected with AMSCBMO but without the prior modelling of venous thrombosis. In each experimental and control groups at each time point 11-12 animals were used (total number equal to 226). After the main vein ligation with the subsequent injection of thrombin solution, in addition to the thrombosis of the blood vessels, morphological signs of thrombosis of the lymphatic bed and lymphostasis were present: the dilation of lymphatic vessel lumen, thinning of their layers, intense staining of their luminal heterogeneous content. AMSCBMO, injected into thrombosed vein, were found to directly participate in lymphangiogenesis in the connective tissue around vein, its tissue region and in the area of regional lymph nodes. This is indicated by bright specific luminescence of both individual cells in the wall of the lymphatic vessels, and all their tunics together with the valves, when exposed to UV light.

Local tissue reaction to implantation of biodegradable suture materials.

Morphological changes in tissues adjacent to the implant were studied 1, 2, 6, and 12 months after implantation of biodegradable suture materials (catgut thread, DemeTECH polyfilament thread, and Surgilactin monofilament thread) into subcutaneous fat of rats. Tissue reaction to implantation of different suture materials developed as usual in response to a wound process and to a foreign body. By the end of month 1. the stage of traumatic perifocal inflammation was replaced in all groups by the proliferative phase with formation of new vessels and connective tissue. A trend to reduction of the productive inflammation activity from month 1 to month 12 was noted. Complete degeneration of the material after 12 months with complete recovery of the histoarchitecture of the adjacent tissues (restitution) was detected only after implantation of DemeTECH polyfilament thread.

[Peculiarities of tissue reactions to implantation of non-degradable surgical materials].

The method of light microscopy was used to study the reactions of the surrounding tissues to the implantation of non-degradable surgical materials (prolene 3/0 thread made from polypropelene, the composite grid made from polypropelene or polyester with polylactic acid) in the subcutaneous adipose tissue of rats 1, 2, 6 and 12 months after grafting. No less than 6 rats were examined in every time point. It was shown that the tissue reactions to implantation of different non-degradable surgical materials follows the general pattern, characteristic of wound process and foreign body reaction. By the first month of observation in both groups of animals the stage of traumatic perifocal inflammation was replaced by the proliferative phase with the formation of new blood vessel and connective tissue. The tendency to reduction of the activity of productive inflammation was observed from the 1st to the 12th month together with the increased fibrous scar transformation of the granulation tissue.