Blood Vessel Remodeling Research Articles

NOK/STYK1 promotes the genesis and remodeling of blood and lymphatic vessels during tumor progression

Previous studies have indicated that the overexpression of NOK, also named STYK1, led to tumorigenesis and metastasis. Here, we provide evidence that increased expression of NOK/STYK1 caused marked alterations in the overall and inner structures of tumors and substantially facilitates the genesis and remodeling of the blood and lymphatic vessels during tumor progression. In particular, NOK-expressed HeLa stable cells (HeLa-K) significantly enhanced tumor growth and metastasis in xenografted nude mice. Hematoxylin and eosin (HE) staining demonstrated that the tumor tissues generated by HeLa-K cells were much more ichorous and had more interspaces than those generated by control HeLa cells (HeLa-C). The fluorescent areas stained with cluster of differentiation 31 (CD31), a marker protein for blood vessels, appeared to be in different patterns. The total blood vessels, especially the ring patterns, within the tumors of the HeLa-K group were highly enriched compared with those in the HeLa-C group. NOK-HA was demonstrated to be well colocalized with CD31 in the wall of the tubular structures within tumor tissues. Interestingly, antibody staining of the lymphatic vessel endothelial hyaluronan receptor (LYVE-1) further revealed the increase in ring (oratretic strip-like) lymphatic vessels in either the peritumoral or intratumoral areas in the HeLa-K group compared with the HeLa-C group. Consistently, the analysis of human cancerous tissue also showed that NOK was highly expressed in the walls of tubular structures. Thus, our results reveal a novel tumorigenic function of NOK to mediate the genesis and remodeling of blood and lymphatic vessels during tumor progression.

Physiological cerebrovascular remodeling in response to chronic mild hypoxia: A role for activated protein C

Activated protein C (APC) is a serine protease that promotes favorable changes in vascular barrier integrity and post-ischemic angiogenic remodeling in animal models of ischemic stroke, and its efficacy is currently being investigated in clinical ischemic stroke trials. Interestingly, application of sub-clinical chronic mild hypoxia (CMH) (8% O2) also promotes angiogenic remodeling and increased tight junction protein expression, suggestive of enhanced blood-brain barrier (BBB) integrity, though the role of APC in mediating the influence of CMH has not been investigated. To examine this potential link, we studied CMH-induced cerebrovascular remodeling after treating mice with two different reagents: (i) a function-blocking antibody that neutralizes APC activity, and (ii) exogenous recombinant murine APC. While CMH promoted endothelial proliferation, increased vascular density, and upregulated the angiogenic endothelial integrins α5β1 and αvβ3, these events were almost completely abolished by functional blockade of APC. Consistent with these findings, addition of exogenous recombinant APC enhanced CMH-induced endothelial proliferation, expansion of total vascular area and further enhanced the CMH-induced right-shift in vessel size distribution. Taken together, our findings support a key role for APC in mediating physiological remodeling of cerebral blood vessels in response to CMH.

Upregulation of MicroRNA-214 Contributes to the Development of Vascular Remodeling in Hypoxia-induced Pulmonary Hypertension Via Targeting CCNL2.

Hypoxia-induced pulmonary hypertension (PH), which is characterized by vascular remodeling of blood vessels, is a significant complication of chronic obstructive pulmonary disease (COPD). In this study, we screened 13 candidate miRNAs in pulmonary artery smooth muscle cells (PASMCs) harvested from COPD patients with PH (n = 18) and normal controls (n = 15) and found that the expression of miR-214 was differentially expressed between these two groups. Additionally, cyclin L2 (CCNL2) was validated as a target of miR-214 in PASMCs using a luciferase assay. Based on real-time PCR, immunohistochemistry and western blot, the expression of CCNL2 was substantially downregulated in PASMCs from COPD patients with PH compared with those from normal controls. Moreover, the relationship between miRNA and mRNA expression was confirmed using real-time PCR and western blot in PASMCs transfected with miR-214 mimics. Furthermore, the introduction of miR-214 significantly promoted the proliferation of PASMCs by suppressing cell apoptosis, and this effect was mediated by the downregulation of CCNL2. Exposure of PASMCs to hypoxia significantly increased the expression of miR-214, decreased the expression of CCNL2, and promoted cell proliferation. However, these effects were significantly attenuated by the introduction of miR-214 inhibitors, which significantly downregulated miR-214 expression and upregulated CCNL2 expression.

External Volume Expansion Modulates Vascular Growth and Functional Maturation in a Swine Model.

Despite increasing application of the pre-grafting expansion during autologous fat transplantation in breast reconstruction, little is known about its mechanism of action. To address that, ventral skins of miniature pigs were treated over a 10-day or 21-day period, with continuous suction at −50 mm Hg via a 7-cm diameter rubber-lined suction-cup device. Soft tissue thickness increased immediately after this external volume expansion (EVE) treatment, such increase completely disappeared by the next day. In the dermis and subcutaneous fat, the EVE treated groups showed significant increases in blood vessel density evident by CD31 staining as well as in vascular networks layered with smooth muscle cells when compared with the control group. This finding was corroborated by the increased percentage of endothelial cells present in the treatment groups. There was no significant difference in the percentages of proliferating basal keratinocytes or adipocytes, nor in epidermal thickness. Moreover, the EVE had no effect on proliferation or differentiation potential of adipose stem cells. Taken together, the major effects of EVE appeared to be vascular remodeling and maturation of functional blood vessels. This understanding may help clinicians optimize the vascularity of the recipient bed to further improve fat graft survival.

Changes in miRNA expression patterns and vascular aging phenotypes in mice with developmental liver‐specific knockdown of IGF‐1

The endocrine changes that occur during development are highly conserved across mammalian species and include dramatic increases in the anabolic hormone IGF‐1 during adolescence. There is increasing experimental and clinical evidence that alterations in IGF‐1 levels during development regulate multiple aspects of the aging process and affect the incidence of multiple age‐related diseases, including cardiovascular and cerebrovascular diseases. The present study was designed to characterize the effect of developmental IGF‐1 deficiency on the vascular aging phenotype. To achieve that goal early‐onset, isolated endocrine IGF‐1 deficiency was induced mice by developmental knockdown of IGF‐1 specifically in the liver using Cre‐lox technology (Igf1f/f mice crossed with mice expressing albumin‐driven Cre recombinase). The animals were studied at an age representing ~75% of maximal lifespan potential, which corresponds to the biological age of a ~67 year old human. To assess vascular health endothelium‐dependent vasorelaxation and vascular ROS production were tested. Due to the emergence of miRNAs as important regulators of vascular aging phenotype, miRNA expression profile in the aorta of mice with developmental IGF‐1 deficiency was also tested. We found that aging was associated with endothelial dysfunction, which was unaffected by developmental IGF‐1 deficiency. We identified 19 miRNAs that were differentially expressed in the aortas of aged mice and 11 miRNAs that were differentially expressed miRNAs in aortas of mice with developmental IGF‐1 deficiency. GO terms enriched among predicted targets for miRNAs differentially expressed with age in the aorta include cytokine production, chromatin silencing, intracellular protein transport and regulation of apoptotic cell death. GO terms enriched among predicted targets for miRNAs differentially expressed with developmental IGF‐1 deficiency in the aorta include regulation of apoptotic cell death, blood vessel remodeling and intracellular protein transport. Collectively, developmental IGF‐1 deficiency is associated with a vascular miRNA expression profile later in life that likely plays a role in development of age‐related vascular pathologies.Support or Funding InformationThis work was supported by grants from the American Heart Association and the National Institute on Aging

Pro-lymphangiogenic properties of IFN-γ-activated human dendritic cells

Dendritic cells (DCs) play a crucial role in the initiation of adaptive immune responses. In addition, through the release of pro- and anti-angiogenic mediators, DCs are key regulators of blood vessel remodeling, a process that characterizes inflammation. Less information is available on the role of DCs in lymphangiogenesis. This study reports that human DCs produce VEGF-C, a cytokine with potent pro-lymphangiogenic activity when stimulated with IFN-γ. DC-derived VEGF-C was biologically active, being able to promote tube-like structure formation in cultures of human lymphatic endothelial cells (LECs). DCs co-cultured with IL-15-activated NK cells produced high levels of VEGF-C, suggesting a role for NK-DC cross-talk in peripheral lymphoid and non-lymphoid tissues in inflammation-associated lymphangiogenesis. Induction of VEGF-C by IFN-γ was detected also in other myeloid cells, such as blood-purified CD1c+ DCs, CD14+ monocytes and in monocyte-derived macrophages. In all these cell types, VEGF-C was found associated with the cell membrane by low affinity, heparan sulphate-mediated, interactions. Therefore, human DCs should be considered as new players in inflammation-associated lymphangiogenesis.

Monocyte recruitment to polymer implants regulates the in situ generation of macrophages and vascular remodeling

Monocyte recruitment to polymer implants regulates the in situ generation of macrophages and vascular remodeling

β4 integrin is not essential for localization of hemidesmosome proteins plectin and CD151 in cerebral vessels.

Objective:In the central nervous system (CNS), β4 integrin is predominantly expressed by endothelial cells lining arterioles. As β4 integrin plays an essential role in epithelial tissues, organizing structural proteins into specialized adhesive structures called hemidesmosomes (HD), the aim of this study was to determine whether it plays a similar role in CNS endothelium.Methods:Dual-immunofluorescence was used to examine the relationship between β4 integrin expression and co-expression of the HD proteins plectin and CD151 in frozen sections of mouse brain, both under normoxic (control) conditions and following chronic mild hypoxia. The requirement of β4 integrin for the localization of HD proteins was examined in transgenic mice lacking β4 integrin expression specifically in endothelial cells (β4-EC-KO mice).Results:Immunofluorescence revealed that in the normal adult CNS, plectin and CD151 strongly co-localized with β4 integrin in arterioles. However, in the chronic mild hypoxia model, in which extensive cerebrovascular remodeling is observed, plectin and CD151 were strongly upregulated on all cerebral vessels, but surprisingly, in capillaries, this occurred in a β4 integrin-independent manner. Unexpectedly, absence of endothelial β4 integrin (in β4-EC-KO mice) had no impact on the expression level or distribution pattern of plectin and CD151 within stable or remodeling cerebral vessels.Conclusions:These results demonstrate that the HD proteins plectin and CD151 are closely associated with β4 integrin on arterioles in normal brain, and are strongly upregulated on remodeling blood vessels. However, unlike its described role in the epidermis, β4 integrin is not essential for localization or regulation of expression of plectin and CD151 in cerebral vessels.

Distinct RNA transcriptome patterns are potentially associated with angiogenesis in Tie2-expressing monocytes

Tie2-expressing Monocytes (TEMs) were previously identified as a novel subset of monocytes and were believed to have prominent pro-angiogenesis activities in human tumors. While the molecular mechanism of the angiogenesis promoting capacity of TEMs remains unclear. RNA transcriptome pattern, including non-coding RNAs as microRNA (miRNA) and long non-coding RNA (lncRNA), plays important role in cell differentiation and functions. However, little is known about the transcriptome patterns of TEMs, including those non-coding RNAs. We explore the transcriptome of TEMs and the matched monocytes that do not express Tie2 (Tie2−monocytes) isolated from peripheral blood of healthy adults employing the Agilent Human miRNA(8*60K,Design ID: 046064)microarray and the Agilent lncRNA Gene Expression(4*180K, Design ID: 042818)microarray. A total of 141 mRNAs, 142 lncRNAs and 75 miRNAs were found dysregulated in TEMs compared to Tie2−monocytes. TEMs have the distinct RNA transcriptome patterns according to the Hierarchical clustering and then the gene expression patterns were confirmed by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Functional annotation by Gene Ontology (GO) analyses showed that the up-regulated mRNAs in TEMs were associated to blood vessel remodeling and positive regulation of epithelial cell proliferation, and the up-regulated insulin like growth factor 1(IGF1) mRNA was involved in both pathways. For functional analysis of those dysregulated non-coding RNAs, target genes of the miRNAs were predicted and cis/trans-regulation analysis of the lncRNAs were performed.

Nose-only cigarette smoke exposure plus airway lipopolysaccharide inhalation induced chronic obstructive pulmonary disease and associated pulmonary hypertension in mice

To establish a mouse model of chronic obstructive pulmonary disease (COPD) and associated pulmonary hypertension (COPD-PH) induced by nose-only cigarette smoking exposure plus airway lipopolysaccharide (LPS) inhalation. There were 24 male C57B6 mice divided into a control group and a model group at random. The model group was given LPS by intranasal inhalation on day 1 and day 14 and exposed to the cigarette smoke in a nose-only exposure system, while the control group was given physiological saline and exposed to normal air. The model establishment was evaluated according the following parameters: the lung function and the right heart pressure, the total and differential cell numbers in bronchial alveolar lavage fluid (BALF), and the pathological changes of lung tissues. The functional residual capacity data of the model group and the control group were (0.402 ± 0.057) and (0.243 ± 0.064) ml respectively (P<0.05). The inspiratory resistance data of the model group and the control group were (1.056 ± 0.121) and (0.789 ± 0.063) cmH(2)O · ml(-1) · s(-1) (1 cmH(2)O=0.098 kPa) respectively (P<0.05). The static lung compliance data of the model group and the control group were (0.084 ± 0 .007) and (0.056 ± 0.004) cmH(2)O/ml respectively (P<0.05). The right ventricular mean pressure of the model group and the control group were (11.3 ± 1.3) and (7.9 ± 1.1) mmHg (1 mmHg=0.133 kPa) respectively (P<0.05), while the right ventricular hypertrophy index of the model group and the control group were (0.267 ± 0.019) and (0.195 ± 0.023) respectively (P<0.05). Moreover, the histological staining showed that goblet cell hyperplasia, lung inflammation and thickening of smooth muscle layers of bronchial and pulmonary small vessels occurred in the model group, which indicated ongoing airway and blood vessel remodeling. A COPD-PH mouse model was established by nose-only cigarette smoking exposure plus airway LPS inhalation in a short period of time, and this method was more similar to the smoking behavior of human.

Synergistic Actions of Blocking Angiopoietin-2 and Tumor Necrosis Factor-α in Suppressing Remodeling of Blood Vessels and Lymphatics in Airway Inflammation

Remodeling of blood vessels and lymphatics are prominent features of sustained inflammation. Angiopoietin-2 (Ang2)/Tie2 receptor signaling and tumor necrosis factor-α (TNF)/TNF receptor signaling are known to contribute to these changes in airway inflammation after Mycoplasma pulmonis infection in mice. We determined whether Ang2 and TNF are both essential for the remodeling on blood vessels and lymphatics, and thereby influence the actions of one another. Their respective contributions to the initial stage of vascular remodeling and sprouting lymphangiogenesis were examined by comparing the effects of function-blocking antibodies to Ang2 or TNF, given individually or together during the first week after infection. As indices of efficacy, vascular enlargement, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic vessel sprouting were assessed. Inhibition of Ang2 or TNF alone reduced the remodeling of blood vessels and lymphatics, but inhibition of both together completely prevented these changes. Genome-wide analysis of changes in gene expression revealed synergistic actions of the antibody combination over a broad range of genes and signaling pathways involved in inflammatory responses. These findings demonstrate that Ang2 and TNF are essential and synergistic drivers of remodeling of blood vessels and lymphatics during the initial stage of inflammation after infection. Inhibition of Ang2 and TNF together results in widespread suppression of the inflammatory response.

Role of Slit2/Robo1 in trophoblast invasion and vascular remodeling during ectopic tubal pregnancy

IntroductionFor ectopic tubal pregnancy to be viable, it requires a supporting vascular network and functioning trophoblast. Slit2/Robo1 signaling plays an important role in placental angiogenesis during normal pregnancy. Hence, we here investigated whether or not Slit2/Robo1 signaling also had an impact in ectopic tubal pregnancy. MethodsThe Slit2 and Robo1 expression pattern relevant to trophoblast invasive behavior and vascular remodeling was studied in human tubal placenta obtained from patients with ectopic pregnancy (5–8weeks gestation), The trophoblast development, vascular architecture and Robo1 expression pattern were observed in Slit2 overexpression (Slit2-Tg) and C57BL mice placenta (E13.5 and E15.5). ResultsMarked with CK-7 and Vimentin, the vessel profiles of fallopian tube were classified into four stages. In the presence of extravillous trophoblast (EVT), stellate-shaped and polygonal-shaped EVTs were observed, and the stellate-shaped EVT showed the higher Slit2 expression (P < 0.01) but lower Robo1 expression (P < 0.05) than polygonal-shaped cells. By contrast, a temporary Slit2 up-regulation in remodeling vessel and Slit2 down-regulation in remodeled vessel of polygonal-shape extravillous trophoblast cells occurred in tubal pregnancies. In Slit2-Tg mice E13.5 and E15.5 placenta, Slit2 overexpression promoted vascular remodeling by increasing in the diameter of the maternal blood sinusoids and fetal capillaries, but enhanced the thickness of trophoblast and vasculature at E15.5 Slit2-Tg mice. ConclusionsThe varying Slit2 and Robo1 expression in EVTs was associated with trophoblast invasion and probably plays an important role in the events of blood vessel remodeling of the fallopian tube tissues.

Abstract 4169: Tumor endothelium regulates microenvironment-mediated migration via the proteolysis of extracellular TFPI-2 by trypsinogen 4

Abstract Proteases secreted from endothelial cells (EC) play an important part in angiogenesis, tumor metastasis and blood coagulation processes. In particular, perivascular proteases influence tumor vascularization in many different ways: regulating migration and invasion of endothelial and mural cells, activating chemokines, growth factors, growth factor receptors, break down extracellular matrix and modifying cellular adhesion molecules. Understanding the complexity of protease activities and their regulation by endothelium in response to different microenvironment stimuli might help identify new targets for the inhibition of vascularization in cancer and angiogenesis related diseases. The expression of serine protease 3 (PRSS3) a member of the trypsinogen family, in vivo is associated to the tumors vasculature and in vitro characterizes EC isolated from cancer specimens [Ghilardi, BMC Genomics 2008]. Herein, we used EC isolated from cancer specimens (tumor-derived EC) to elucidate a mechanism linking micro-environment cues to endothelium migration. We show that trypsinogen 4 isoform of PRSS3 is the trypsin family member expressed by human endothelium, and its expression is up-regulated by a tumor pro-angiogenic milieu (i.e. the combined action of VEGF, FGF-2 and EGF; particularly rich in the cancer environment). Trypsinogen 4 depletion profoundly impaired the migratory capability of tumor-derived EC initiated and driven by the tumor pro-angiogenic milieu. We identified Tissue Factor Pathway Inhibitor-2 (TFPI-2) as the functional proteolitic target. TFPI-2 negatively regulated the migration of tumor-derived EC; trypsinogen 4 was capable to cleave TFPI-2 and also to displace it from the extracellular matrix (ECM) deposited by tumor-derived EC. Accordingly, we observed an increased accumulation of TFPI-2 in the extracellular matrix of tumor-derived EC silenced for trypsinogen IV. Altogether, the results indicate that the tumor microenvironment triggers and sustains the expression of trypsinogen 4 to impair TFPI-2 function and permits the migration of endothelium; essential for angiogenesis and blood vessel remodeling. Our findings contribute to advance the understanding of tumor vascular biology, and the comprehension of an active role for trypsinogen 4 and TFPI-2 in microenvironment-mediated tumor neo-vascularization. The identification of the specific protease and its functional target may be of aid for the design of inhibitor(s) exploitable as anti-cancer therapeutics. Supported by the Italian Association for Cancer Research (AIRC) and the CARIPLO foundation. Citation Format: Carmen Ghilardi, Figini Sara, Monica Lupi, Alessia Anastasia, Raffaella Giavazzi, Mariarosa Bani. Tumor endothelium regulates microenvironment-mediated migration via the proteolysis of extracellular TFPI-2 by trypsinogen 4. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4169. doi:10.1158/1538-7445.AM2015-4169

Lymphatic vessel development: fluid flow and valve-forming cells.

Hemodynamic forces regulate many aspects of blood vessel disease and development, including susceptibility to atherosclerosis and remodeling of primary blood vessels into a mature vascular network. Vessels of the lymphatic circulatory system are also subjected to fluid flow-associated forces, but the molecular and cellular mechanisms by which these forces regulate the formation and maintenance of lymphatic vessels remain largely uncharacterized. This issue of the JCI includes two articles that begin to address how fluid flow influences lymphatic vessel development and function. Sweet et al. demonstrate that lymph flow is essential for the remodeling of primary lymphatic vessels, for ensuring the proper distribution of smooth muscle cells (SMCs), and for the development and maturation of lymphatic valves. Kazenwadel et al. show that flow-induced lymphatic valve development is initiated by the upregulation of GATA2, which has been linked to lymphedema in patients with Emberger syndrome. Together, these observations and future studies inspired by these results have potential to lead to the development of strategies for the treatment of lymphatic disorders.

Trypsinogen 4 boosts tumor endothelial cells migration through proteolysis of tissue factor pathway inhibitor-2.

Proteases contribute to cancer in many ways, including tumor vascularization and metastasis, and their pharmacological inhibition is a potential anticancer strategy. We report that human endothelial cells (EC) express the trypsinogen 4 isoform of the serine protease 3 (PRSS3), and lack both PRSS2 and PRSS1. Trypsinogen 4 expression was upregulated by the combined action of VEGF-A, FGF-2 and EGF, angiogenic factors representative of the tumor microenvironment. Suppression of trypsinogen 4 expression by siRNA inhibited the angiogenic milieu-induced migration of EC from cancer specimens (tumor-EC), but did not affect EC from normal tissues. We identified tissue factor pathway inhibitor-2 (TFPI-2), a matrix associated inhibitor of cell motility, as the functional target of trypsinogen 4, which cleaved TFPI-2 and removed it from the matrix put down by tumor-EC. Silencing tumor-EC for trypsinogen 4 accumulated TFPI2 in the matrix. Showing that angiogenic factors stimulate trypsinogen 4 expression, which hydrolyses TFPI-2 favoring a pro-migratory situation, our study suggests a new pathway linking tumor microenvironment signals to endothelial cell migration, which is essential for angiogenesis and blood vessel remodeling. Abolishing trypsinogen 4 functions might be an exploitable strategy as anticancer, particularly anti-vascular, therapy.

Abstract 304: Varicose Vein Development in Mice Is Blocked by Ator- and Rosuvastatin

Venous diseases such as varicose veins and chronic venous insufficiency constitute a substantial source of morbidity. Despite their high prevalence, neither molecular mechanisms initiating these diseases nor pharmaceutic therapies have been explored so far. We recently reported that chronically increased filling pressure or biomechanical venous wall stress is sufficient to cause development of varicose-like veins in mice through a mechanism preceded by activation of the transcription factor activator protein 1 (AP-1). Considering these findings, we hypothesized that pharmacologic inhibition of AP-1 activity may attenuate venous remodeling. To prove this therapeutic concept, human venous smooth muscle cells (SMCs) were subjected to biomechanical stretch (13%, 0.5Hz) and treated with statins - HMG-CoA reductase inhibitors that are supposed to interfere with the activity of AP-1. EMSA and immunofluorescence studies revealed that ator- and rosu- but not simvastatin blocked stretch-induced AP-1 activity. Furthermore, ator- and rosuvastatin inhibited expression of the AP-1 target monocyte chemotactic protein 1 (MCP-1) and proliferation of these cells. In mouse veins exposed to an increased level of intraluminal pressure, atorvastatin diminished the abundance of MCP-1 and MMP-2 by ~90 % and inhibited proliferation of endothelial cells and SMCs (n=5, p&lt;0.05). In line with this observation, growth of varicose-like veins in mice was suppressed by almost ~80 % upon treatment with rosu- and atorvastatin but not simvastatin (n=5, p&lt;0.05) which was accompanied by a decrease in cellular proliferation as well as MMP-2 abundance in remodeling blood vessels. Consequent analyses of human varicose vein samples from patients chronically treated with statins indicated a decrease in SMC proliferation, MCP-1 and MMP-2 abundance (50-60 %, n=10, p&lt;0.05). Collectively, our current findings imply that both ator- and rosuvastatin effectively inhibit the development of varicose veins presumably by interfering with the wall stress-mediated activity of AP-1 in VSMCs. For the first time, this study revealed a possible pharmaceutic treatment that may be suitable to prevent growth of varicose veins and to limit formation of recurrences after varicose vein surgery.

587. MiRNA-92a Is Involved in the Regulation of Adipose-Derived Stromal Cell (ADSC) Angiogenic Properties

Reparative and regenerative processes after tissue damage are strongly dependent on successful restoration of vascular network. Mesenchymal stem/stromal cells (MSC) including ADSC located within the vessel wall and closely interacted with endothelial cells appear to be key regulators of angiogenesis and regeneration in adult tissues. Mechanisms of MSC ability to stimulate blood vessel growth and remodeling are extensively studying during last decades. microRNAs (miRs), small noncoding RNAs that regulate gene expression by binding to target mRNAs reducing their stability and/or inhibiting translation, play an important role in MSC regulation. It was demonstrated that miR-92a expressed in endothelial cells was induced in ischemia and involved in the regulation of angiogenesis and vessel patterning. We studied miR-92a impact in ADSC angiogenic properties regulation.ADSC were isolated from subcutaneous fat tissue of healthy young donors (n=4) and cultured up to 2-3 passages. ADSC phenotype characterized by flow cytometry was CD90+/CD73+/CD105+/CD45-/CD31- for all samples and these cells were capable of adipogenic and osteogenic differentiation. We analyzed miR profile in ADSC using Illumina microarrays and found that miR-92a was one of the highly expressed miRs in all ADSC samples. ADSC transfection by pre-miR-92a or anti-miR-92a led to the coordinated changes of miR-92a well-known target genes expression (integrin alpha 5, ITGA5, and mitogen activated protein kinase, MEK4). ADSC with over-expression of miR-92a completely lost the ability to stimulate capillary-like tube formation by endothelial cells (HUVEC) in vitro compared to ADSC transfected by the control gene. However, transfection of ADSC by anti-miR-92a did not significantly increase their stimulating effect in tube formation assay. As paracrine mechanisms are considered to be the most important for realizing of MSC angiogenic potential, we analyzed gene expression (qRT-PCR) and secretion (Bio-Plex assay and ELISA) of some key angiogenic factors in ADSC. We found that mRNA level of vascular endothelial growth factor (VEGF), angiogenin and leptin was slightly increased in ADSC transfected by anti-miR-92a and this tendency was also observed for VEGF secretion. When miR-92a was over-expressed in ADSC secretion of hepatocyte growth factor (HGF) and angiopoetin-1 by these cells significantly decreased compared to the control cells.We conclude that miR-92a might play an important role in the regulation of ADSC ability to stimulate angiogenesis and key angiogenic factors such as VEGF, HGF and angiopoetin-1 are involved in this process. The underlying mechanisms of miR-92a-mediated direct or indirect effects in ADSC need to be further investigated. Anyway, miR-92a could be considered as a promising target for the modulation of ADSC angiogenic potential.

Matrix metalloproteinase-9 mediates post-hypoxic vascular pruning of cerebral blood vessels by degrading laminin and claudin-5.

Vascular remodeling involves a highly coordinated break-down and build-up of the vascular basal lamina and inter-endothelial tight junction proteins. In light of the important role of matrix metalloproteinases (MMPs) in tissue remodeling, the goal of this study was to examine the role of MMP-9 in remodeling of cerebral blood vessels, both in hypoxia-induced angiogenesis and in the vascular pruning that accompanies the switch from hypoxia back to normoxia. In a chronic mild hypoxia model of cerebrovascular remodeling, gel zymography revealed that MMP-9 levels were increased, both during hypoxic-induced angiogenesis and in the post-hypoxic pruning response. Interestingly, compared to wild-type mice, MMP-9 KO mice showed no alteration in hypoxic-induced angiogenesis, but did show marked delay in post-hypoxic vascular pruning. In wild-type mice, vascular pruning was associated with fragmentation of vascular laminin and the tight junction protein claudin-5, while this process was markedly attenuated in MMP-9 KO mice. In vitro experiments showed that hypoxia stimulated MMP-9 expression in brain endothelial cells but not pericytes. These results show that while MMP-9 is not essential for hypoxic-induced cerebral angiogenesis, it plays an important role in post-hypoxic vascular pruning by degrading laminin and claudin-5.

Molecular modeling as a new approach to the development of urokinase inhibitors.

Proteolytic activity of urokinase plays an important role in negative remodeling of blood vessels, restenosis, tumor angiogenesis, and metastasizing, which necessitates the development of selective urokinase inhibitors. Using methods of computer modeling (docking, post processing, and direct docking) and quantum chemistry, we selected substances from the large compound database, analyzed their structures, and experimentally verified their inhibitor activity. New urokinase inhibitor candidates were proposed based on the theoretical predictions and experimental verification of compound activities. The process of modifying urokinase inhibitors based on (benzothiazol-3-yl)guanidine was developed. A new urokinase inhibitor (5-brom-benzothiazol-3-yl)guanidine, that can be effective for regulation of vascular remodeling and tumor angiogenesis, was created.

Progress in histopathologic and pathogenetic research in a retinitis pigmentosa model.

Retinitis pigmentosa is a major cause of visual impairment and blindness, affecting millions of people worldwide. The mechanisms of and effective treatments for the disease, however, remain to be further investigated. The Royal College of Surgeons rat is one of the most widely used animal models for the study of retinal degeneration diseases. The mutation in the mer tyrosine kinase proto-oncogene of this model leads to deficient phagocytosis in the retinal pigment epithelium cells and the accumulation of photoreceptor out segments in the subretinal space, ultimately resulting in retinal degeneration. The retina begins to change as early as 17 days after birth and becomes gradually thinner with the death and remodeling of cells and blood vessels. Retinal cell apoptosis plays a dominant role in this degeneration, with some cells being activated by the secondary alterations of the retinal neurotransmitter and other related factors.