The Influence of Adiponectin on Transport of Low-Density Lipoproteins through Human Endothelial Cell Monolayer In Vitro.

The influence of adiponectin, a protein secreted by adipocytes, on the activation of transendothelial LDL transport, the initial event of atherogenesis, was studied. The addition of adiponectin to the cultured endothelial hybridoma EA.hy926 cells did not affect both basal and TNF-stimulated transendothelial transport of LDL. In addition, adiponectin affects neither expression levels of CAV1, SCARB1, and ACVRL1 genes encoding proteins involved in transendothelial LDL transport, nor the MMP secretion by the EA.hy926cells. At the same time, adiponectin suppressed the TNF-stimulated IL-8 production and expression of the adhesion molecule gene ICAM1 in these cells. Thus, adiponectin reduces proinflammatory activation of EA.hy926 cells, which is not accompanied by changes in the transendothelial LDL transport. We speculate that anti-inflammatory action of adiponectin is the base for the influence of this adipokine on atherogenesis.

Effect of Concentration of Collagen Gel on Functional Activity of Bone Marrow Mesenchymal Stromal Cells.

Collagen I gels with protein concentrations of 1, 2, and 3.5 mg/ml were prepared and embedded in a porous polylactide scaffold to reduce their contraction. Concentration of the gel did not affect its degradation. Collagen gels promoted the formation of cell networks. The cells in the collagen gel with a concentration of 1 mg/ml embedded in polylactide scaffold had elongated spindle-like shape, in contrast to flattened cells in collagen gel of the same concentration not embedded in the scaffold. Stabilization of the collagen gel in the polylactide scaffold promoted active synthesis of laminin and fibronectin by cells as soon as on day 5 of culturing in comparison with that in free collagen substrate.

STABILITY OF THE GELS BASED ON TYPE I COLLAGEN AND HYALURONIC ACID.

Development of hyaluronic acid (HA) supplemented biomaterials for regenerative medicine is complicated with easy water solubility of HA. It results in decreased stability of HA-based scaffolds that, consequently, initiated search for crosslinking techniques intended to retain HA within a scaffold. In this study, gels composed of type I collagen and native high molecular weight HA were prepared and their stability in buffer solutions has been evaluated. Fibronectin and human dermal fibroblasts were incorporated into gels as biological linkages for HA instead of chemical agents. Gel stability was estimated based on HA diffusion toward a buffer solution. In addition, HA and HA-supplemented gels resistance to fetal calf serum (FSC) as a part of cultural media for fibroblasts has been studied and no degradation of HA by serum hyaluronidase has been detected under the experimental conditions. The results showed that HA diffuses from collagen gels within 1­3 days. Neither fibronectin nor fibroblasts added separately prevent HA diffusion. But if gels were inoculated with fibroblasts together with fibronectin, more HA was left in the gels according to alcian blue staining. Agarose gel electrophoresis has shown that conditioned media collected from such gels are enriched with HA that differs from the initial one in that it contains a new portion of HA with higher molecular weight. It is concluded that HA-supplemented collagen gels are not stable in buffer solutions but fibroblasts incorporated into such gels synthesize significant amount of HA under the influence of fibronectin and partly compensate the loss of HA due to diffusion.

Matrix Metalloproteinases in Primary Culture of Cardiomyocytes.

The highly organized contractile apparatus of cardiomyocytes in heart tissue allows for their continuous contractility, whereas extracellular matrix components are synthesized and spatially organized by fibroblasts and endothelial cells. However, reorganization of the cardiomyocyte contractile apparatus occurs upon their 2D cultivation, which is accompanied by transient loss of their contractility and acquired capability of extracellular matrix synthesis (Bildyug, N. B., and Pinaev, G. P. (2013) Tsitologiya, 55, 713-724). In this study, matrix metalloproteinases were investigated at different times of cardiomyocyte 2D cultivation and 3D cultivation in collagen gels. It was found that cardiomyocytes in 2D culture synthesize matrix metalloproteinases MMP-2 and MMP-9, wherein their amount varies with the cultivation time. The peak MMP-9 amount is at early cultivation time, when the reorganization of cardiomyocyte contractile apparatus occurs, and the MMP-2 peak precedes the recovery of the initial organization of their contractile apparatus. Upon cardiomyocyte cultivation in 3D collagen gels, in which case their contractile apparatus does not rearrange, a steady small amount of MMP-2 and MMP-9 is observed. These data indicate that the cardiomyocyte contractile apparatus reorganization in culture is associated with synthesis and spatial organization of their own extracellular matrix.

[Activity of matrix metalloproteinases of transformed fibroblasts under the antioxidant action].

We have shown that antioxidant N-acetylcysteine (NAC, 2-10 mM) quickly (for 2 hours) and completely inactivates the activity of matrix metalloproteinases (gelatinases MMP-2 and MMP-9, and collagenases MMP-1 and MMP-8) secreted by transformed mouse fibroblasts 3T3-SV40 into the medium. The same MMP inhibition took place in the cell-free conditioned medium of HT-1080 fibroblasts, which suggests a direct chemical interaction between NAC and MMP resulting in the loss of MMP activity. Besides inhibitory effect, NAC decreased MMP-1 and MMP-9 (but not MMP-2) production in the cell medium. However, the level of MMP-1 and MMP-9 inhibitor (TIMP-1) remained normal, indicating a shift in the balance between the enzyme and inhibitor. The correlation between MMP-2 level and tissue enzyme inhibitor TIMP-2 was similar in control and NAC treated cells. At the same time, reorganization of type I collagen at the cell surface occurred. All together permits the conclusion that NAC action results in the extracellular matrix remodeling and changing in cellular functions.

[Migration rate of rabbit bone marrow stromal cells and rabbit dermal fibroblasts in different gels and activity of their MMPs].

This paper presents the results of the study of rabbit bone marrow stromal cells (BMSC) and rabbit dermal fibroblasts (DF) migration rates to collagen type I and fibrin gels. It has been shown that DF exhibit greater migration activity in collagen gel, whereas BMSC show a higher migration activity in fibrin gels. By studying the activity of matrix metalloproteinases (MMPs) synthesized by cells during cultivation in gels, it has been found for both cell types that the activity of MMP-9 is increased in fibrin gels and activity of MMP-2 is increased in collagen gels. Different speed of the migration of cells may be due to the properties of the cells, the activity of MMP synthesized by the cells and the influence of the microenvironment (collagen or fibrin) on the process of synthesis.

[Functional properties of smooth muscle cells in ascending aortic aneurysm].

Thoracic aortic aneurism (TAA) develops as a result of complex series of events that dynamically alter the structure and composition of the aortic vascular extracellular matrix (ECM). The main elements that alter the composition of aortic wall are smooth muscle cells (SMC). The purpose of the present work was to study alteration of smooth muscle cell functions derived from the patients with TAA and from healthy donors. As it is supposed that TAA associated with bicuspid aortic valve (BAV) and with tricuspid aortic valve (TAV) differ in their pathogenesis, we compared the SMC and tissues samples from BAV-, TAV-patients and healthy donors. We compared TAA patients' derived tissues and SMC to healthy donors' ones in several parameters: SMC growth, migration and apoptotic dynamics; metalloproteinase MMP2 and MMP9 activity (zymography) and elastin, collagen and fibrillin content (Western blot) in both tissue samples and cultured SMC. Proliferation ability of both BAV and TAV SMC was decreased comparing to donors cells; migration ability in scratch tests was increased in TAV-derived SMC comparing to donor cells. BAV-cells migration ability was not changed comparing to donor-SMC. Elastin content was decreased in TAA SMC comparing to donor cells whereas the content of fibrillin and collagen was not altered. At the same time elastin and collagen protein level was significantly higher in tissue samples of TAA patients comparing to donor-derived samples. SMS proliferation and migration ability is differently affected in TAV and BAV-associated TAA that supports the idea of different nature of these two groups of TAA. Also our data show that SMC functional properties are altered in TAA patients and these alterations could play a significant role in the disease pathogenesis.

[Developing of a new feeder-free system and characterization of human embryonic stem cell sublines derived in this system under autogenic and allogenic culturing].

A new feeder-free culture system for human embryonic stem cells (hESC) was developed. It consist of extracellular matrix proteins synthesized by feeder cells--mesenchymal stem cell line SC5-MSC, which was derived from initial hESC line SC5. The major ECM proteins--fibronectin and laminin--that maintain hESC growth in feeder-free system were identified. An essential component of this system is a SC5-MSC-conditioned medium. Two hESC sublines were derived. The subline SC5-FF was cultured in autogenic and subline SC7-FF in allogenic system. Sublines SC5-FF and SC7-FF passed through more than 300 and 115 cell population doublings, retained normal diploid karyotype and an ability of in vitro differentiation into derivates of three germ layers. These sublines express markers of undifferentiated hESC: alkaline phosphatase, Oct-4, SSEA-4, TRA-1-81 and multidrug resistance transporter--ABCG2. The RT-PCR analysis revealed that undifferentiated cells SC5-FF subline, like cells of initial feeder-maintained hESC line SC5, expressed genes OCT4 and NANOG, and germ line specific genes such as DPPA3/STELLA and DAZL. An expression of OCT4, NANOG, DPPA3/STELLA ans DAZL was down-regulated during embryonic bodies differentiation, whereas expression of somatic lineages specific genes like GATA4 and AFP (extra embryonic and embryonic endoderm), PAX6 (neuroectoderm) and BRY (mesoderm) was up-regulated. The comparative analysis of some typical features (karyotype structure, the average population doubling time and the number of undifferentiated cells in populations) did not reveal essential differences between initial SC5 and SC7 lines and their sublines SC5-FF and SC7-FF. This shows that feeder-free culture systems, which are much more stable than any feeder systems, do not break main hESC features during long cultivation and can be recommended for fundamental, biomedicine and pharmacological investigations, using hESCs.

[Dynamics of matrix metalloproteinases activity of primary murine embryonic fibroblasts during cultivation].

Embryonic cells regulate the expression of matrix metalloproteinases (MMP) providing remodulation of extracellular matrix, which in turn provides the changes in cell adhesion and migration during the cell development and differentiation. In present work we studied the changes of gelatinases (MMP-2 and MMP-9) and collagenases (MMP-1 and MMP-8) activities in the process of cultivating the primary murine embryonic fibroblasts (MEF). Cultivation was continued for 6 passages, after that the culture died in time. According to gelatin and collagen zymography results, drastic changes of all MMPs activities occurred during the third passage of cell cultivation. The MMP-1 and MMP-9 activity appears in the middle of cultivation and then disappeared at the end. The most important event MEF cultivation is appearance and subsequent reservation of collagenase MMP-8 and active form of gelatinase MMP-2.

[Possibility of prediction of rat wound epithelization by changes of matrix metalloproteinases levels in wound fluid].

The engraftment of a skin sheet together with transplantation of dermal equivalent was studied on rats. The skin sheet was taken as a source of epithelization material in unhealing wound. The process of wound healing was evaluated by changes of matrix metalloproteinases (MMP) activity levels in wound fluid. It was shown that results of skin sheet transplantation could be predicted by monitoring of wound fluid MMP-2 and MMP-9 activity levels. It was determined that skin sheet engraftment appeared at medium values of wound fluid MMP-2 and MMP-9, and transplanted skin sheet was lysed at high and low values.

[Expression of osteoprotegerin and soluble ligand of receptor of kappa-B transcription factor activator in the calcification of aortic valve]. / Ékspressiia osteoprotegerina i rastvorimogo liganda retseptora aktivatora faktora transkriptsii kappa-B pri kal'tsifikatsii aortal'nogo klapana.

The mechanism of valve calcification that is the main cause of aortic stenosis formation and progression is not yet clear. In recent years, the role of the OPG/RANKL/RANK system is considered as one of possible variants of pathogenesis of valve calcification. In presented work the differences in OPG and sRANKL levels involved in the calcification processes in tissues of patients with severe aortic stenosis have been examined. The study was performed using three groups of patients: group 1 - patients with aortic stenosis, group 2 - patients with aortic aneurysm, and group 3 - patients with aortic stenosis and aortic dilatation. In patients with aortic stenosis, the level of RANKL was significantly higher, and the level of RANKL was higher in valve than in tissue. The negative correlation between aortic dilatation and RANKL level indicated the lack of RANKL influence on pathogenesis of aortic dilatation. The obtained data confirm the increased expression of RANKL in patients with aortic valve calcification. The results of this study confirm importance of the OPG/RANKL/RANK system in calcification in patients with aortic stenosis. Athough patients of all groups had comparable values of OPG (including patients with aortic dilatation), the RANKL level increased only in patients with aortic stenosis. This suggest involvement of some additional mechanisms influencing the increase of RANKL expression.

[Changes in matrix metalloproteinases activities in normal and transformed mouse fibroblasts under effect of antioxidants].

The effect of two antioxidants on the activities of matrix metalloproteinases (MMP) secreted by normal (3T3) and transformed (3T3-SV40) mouse fibroblasts was examined. We compared the effect of N-acetylcystein (NAC) and alpha-lipoic acid (ALA) on two gelatinases, MMP-2 and MMP-9. Gel zymography demonstrated that activity of MMP-2 was higher in normal 3T3 cells, and MMP-9 activity was higher in transformed 3T3-SV40 cells. NAC action for 2-6 hours completely inhibited MMP-2 and MMP-9 activity in both cell lines. The inhibitory effect almost did not depend on NAC concentration at the range of 1-10 mM. ALA (1.2 mM) affected the cells not so dramatically. ALA decreased the MMP-2 activity in both cellular types. As to MMP-9 activity, it decreased in 3T3 cells and slightly increased in 3T3-SV40 cells in the presence of ALA. The activity of membrane bound and intracellular MMP was not changed under the same conditions. In conclusion, an altered activity of MMP in the presence of an antioxidant may influence the intracellular signalling and cell functions.

[The dependence of MMP-2 and MMP-9 activity in wound fluid on the wound tissue state at initial stages of wound healing process].

One of the problems in wound treatment optimization is the necessity of an effective and objective method of laboratory wound process monitoring. In present study the current wound process was estimated by changes in MMP-2 and MMP-9 activities in the wound fluid. An original model was used in this work to study correlation of morphological structure of the wound with the activities of MMP-2 and MMP-9 in wound fluid at various types of wound process. Protein fractions of the coelomic liquid from regenerating sea star Asterias rubens were used as the substances changing the wound process. The correlation of wound process with MMP-2 and MMP-9 activities in wound fluid was revealed on the basis of correlation analysis.

[Isolation of human basal keratinocytes by selective adhesion to extracellular matrix proteins].

Epidermal human cells (keratinocytes) differently interact with extracellular matrix proteins of the skin basal membrane depending on the stages of their differentiation. The pool of basal keratinocytes commonly includes stem cells and transient amplifying cells. They directly attach to the skin basal membrane. Keratinocytes change their adhesive properties during differentiation, lose direct interaction with the basal membrane and move to suprabasal epidermal strata. From this, it is suggested that basal and primarily stem cells can be isolated from a heterogenous keratinocyte population due to their selective adhesion to the extracellular matrix proteins. In the current study, we analysed the specificity of interaction between primary keratinocytes and extracellular matrix proteins (collagens of I and IV types, laminin-2/4, fibronectin and matrigel). We have demonstrated that the basal keratinocytes extracted from the skin have different adhesive abilities. The rapidly spreading cells usually interacted with collagen and fibronectin rather that with laminin-2/4 or matrigel. The majority of these cells being represented by basal keratinocytes. Our data demonstrate that the applied method of keratinocyte selection may be directed for precise isolation of skin stem from a common cell population.

Prognostication criterion of potential and practical use of analytical reagents in titrimetria of individual substances.

Various possible criterion in titrimetric methods have been discussed in this paper and more informative, visual and universal criterion-the degree of proceeding of individual reaction at the equivalence point have been chosen. The equation for the degree of proceeding of individual analytical reaction suited for any chemical reactions under real conditions with an allowance made for both component and titrant concentration have been deduced. This criterion allows us to make a prognosis of any parametres of a titrimetric procedure of an individual substance determination and the procedure as a whole, not having carried out the experiment.

[Effect of burn exudate on functional activity of Balb/3T3 cell line and the role of matrix metalloproteinases in this process]. / Vliianie ozhogovogo ékssudata na funktsional'nuiu aktivnost' kletok linii Balb/3T3 i uchastie v étom protsesse matriksnykh metalloproteinaz.

The influence of burn fluid and its matrix metalloproteinases (MMPs) on Balb/3T3 cells was studied. The influence of burn fluid was assessed by morphology and specific functional activities of cells characteristic of the healing process--proliferation, monolayer contraction and migration of cells in wound model. The presence of burn fluid in cultivating medium accelerated cell proliferation by 2.5 times compared to normal conditions, promoted fibroblast monolayer contraction, and accelerated cell migration on the wound surface, thus stimulating cell functions necessary for successful heating. This effect is partly due to MMPs. The burn fluid contains, presumably, some additional factors not inhibited by specific MMP inhibitors EDTA and 1,10-phenantrolin. These factors may stimulate migration and proliferation of cells. The presence of 1-2% burn fluid is sufficient for enhancing cell proliferation.

[Features of formation of phases of dual-phase polymeric system in the presence of collagen I, laminin I, and their mixtures]. / Osobennosti formirovaniia faz dvukhfaznoi polimernoi sistemy v prisutstvii kollagena I, lamnina I i ikh smesi.

A study was made of the phase formation kinetics of a two-phase aqueous polymer system, consisting of 7.8% (w/w) dextran-500 and 4.6% (w/w) polyethylene glycol-6000, in the presence of various concentrations of collagen (0.07-0.20 mg/ml), laminin I (0.01-0.03 mg/ml) and their mixture. The phase formation was evaluated by registration of its optical density on a spectrophotometer. The obtained two-phase polymer system optical density curves and also the partitioning coefficients for the studied objects depend on surface properties of these objects. It has been shown that the surface properties of collagen I and laminin I differ according to differences in their molecules conformations, and that the phase formation kinetics points to their interaction during a mutual partitioning of these proteins in the system. The authors made a conclusion that collagen I and laminin I in the two-phase polymer system conditions could make complexes.

[The "air pouch" model in mice and a study of the wound fluid proteolytic activity]. / Model' "vozdushnogo puzyria" u myshei i izuchenie proteoliticheskoi aktivnosti ranevogo ékssudata.

The experimental model of a cutaneous wound in mice has been offered, aimed to study the proteolytic activity of the wound fluid produced at early stages of wound healing, and to examine the continuous inflammatory state. The presence of metalloproteinases MMP-2 and MMP-9 in the wound fluid matrix was found to correlate with the existence of neutrophils and macrophages in the tissue.

[Matrix metalloproteinases MMP-2 and MMP-9 of wound and burn exudates and their effect on extracellular matrix proteins]. / Matriksnye metalloproteinazy MMP-2 i MMP-9 ranevykh i ozhogovykh ékssudatov i ikh deistvie na belki vnekletochnogo matriksa.

The dynamics of matrix metalloproteinases (MMP), as well as of fibronectin concentration in wound and burn fluids was traced. The wound fluid proteolytic activity was studied by gelatin zymography method. The data on degradation of fibronectin and various laminin isoforms by wound fluid proteases show that laminin-1, laminin-2/4 and fibronectin were degraded by wound fluid into small fragments. Remodelling of extracellular matrix proteins occurs. Dynamics of MMP-2 and MMP-9 content in wound or burn fluids as well as that of adhesive protein fibronectin content could be used as a base for development of method of controlling the extracellular matrix remodelling process.

[Complex evaluation of wound healing process by a deep wound rat model with implanted polychlorvinyl camera]. / Kompleksnaia otsenka techeniia ranevogo protsessa na modeli glubokoi rany u krys s implantatsiei polikhlorvinilovoi kamery.

A simultaneous study of wound proteolytic activity and morphological picture of the first stages of wound healing on rat deep wound model has been shown. The process of wound healing can be evaluated by dynamics of matrix metalloproteinase activities in wound fluid. Changes in activities of different matrix metalloproteinases correlate with different stages of healing. Implantation of polychlorvinyl camera in the wound makes it possible to obtain the volume of wound fluid sufficient for a complex evaluation of healing at the initial stages of wound process.