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Increased Lymphangiogenesis and Lymphangiogenic Growth Factor Expression in Perivascular Adipose Tissue of Patients with Coronary Artery Disease.

Experimental and human autopsy studies have associated adventitial lymphangiogenesis with atherosclerosis. An analysis of perivascular lymphangiogenesis in patients with coronary artery disease is lacking. Here, we examined lymphangiogenesis and its potential regulators in perivascular adipose tissue (PVAT) surrounding the heart (C-PVAT) and compared it with PVAT of the internal mammary artery (IMA-PVAT). Forty-six patients undergoing coronary artery bypass graft surgery were included. Perioperatively collected C-PVAT and IMA-PVAT were analyzed using histology, immunohistochemistry, real time PCR, and PVAT-conditioned medium using cytokine arrays. C-PVAT exhibited increased PECAM-1 (platelet endothelial cell adhesion molecule 1)-positive vessel density. The number of lymphatic vessels expressing lymphatic vessel endothelial hyaluronan receptor-1 or podoplanin was also elevated in C-PVAT and associated with higher inflammatory cell numbers, increased intercellular adhesion molecule 1 (ICAM1) expression, and fibrosis. Significantly higher expression of regulators of lymphangiogenesis such as vascular endothelial growth factor (VEGF)-C, VEGF-D, and VEGF receptor-3 was observed in C-PVAT compared to IMA-PVAT. Cytokine arrays identified angiopoietin-2 as more highly expressed in C-PVAT vs. IMA-PVAT. Findings were confirmed histologically and at the mRNA level. Stimulation of human lymphatic endothelial cells with recombinant angiopoietin-2 in combination with VEGF-C enhanced sprout formation. Our study shows that PVAT surrounding atherosclerotic arteries exhibits more extensive lymphangiogenesis, inflammation, and fibrosis compared to PVAT surrounding a non-diseased vessel, possibly due to local angiopoietin-2, VEGF-C, and VEGF-D overexpression.

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Knockdown of angiopoietin-like 2 induces clearance of vascular endothelial senescent cells by apoptosis, promotes endothelial repair and slows atherogenesis in mice.

Elimination of senescent cells (SnC) is anti-atherogenic, but the specific contribution of senescent vascular endothelial cells (EC) is unknown. We inactivated angiopoietin like-2 (angptl2), a marker of SnEC and a pro-atherogenic cytokine in LDLr, hApoB atherosclerotic (ATX) mice. Three months after a single vascular delivery of a small hairpin (sh)Angptl2 in 3-month old ATX mice using an adeno-associated virus serotype 1 (AAV1), aortic atheroma plaque progression was slowed by 58% (p<0.0001). In the native aortic endothelium, expression was decreased by 80%, in association with a reduced expression of , a cyclin-dependent kinase inhibitor overexpressed in growth-arrested SnC. Endothelial activation was reduced (lower and expression), decreasing monocyte expression in the endothelium. One week post-injection, the ratio increased in the endothelium only, suggesting that SnEC were eliminated by apoptosis. Four weeks post-injection, the endothelial progenitor marker increased, suggesting endothelial repair. In arteries of atherosclerotic patients, we observed a strong correlation between and (r=0.727, p=0.0002) confirming the clinical significance of -associated senescence. Our data suggest that therapeutic down-regulation of vascular leads to the clearance of SnEC by apoptosis, stimulates endothelial repair and reduces atherosclerosis.

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Engineering small-caliber vascular grafts from collagen filaments and nanofibers with comparable mechanical properties to native vessels.

At the present time, there is no successful synthetic, off-the-shelf small-caliber vascular graft (<6 mm) for the repair or bypass of the coronary or carotid arteries. This stimulates on-going investigations to fabricate an artificial vascular graft that has both sufficient mechanical properties as well as superior biological performance. Collagen has long been considered as a viable material to encourage cell recruitment, tissue regeneration, and revascularization, but its use has been limited by its inferior mechanical properties. In this study, novel electrochemically aligned collagen filaments were used to engineer a bilayer small-caliber vascular graft, by circular knitting the collagen filaments and electrospinning collagen nanofibers. The collagen prototype grafts showed significantly greater bursting strength under dry and hydrated conditions to that of autografts such as the human internal mammary artery and the saphenous vein (SV). The suture retention strength was sufficient under dry condition, but that under hydrated condition needs to be further improved. The radial dynamic compliance of the collagen grafts was similar to that of the human SV. During in vitro cell culture assays with human umbilical vein endothelial cells, the prototype collagen grafts also encouraged cell adhesion and promoted cell proliferation compared to the synthetic poly(lactic acid) grafts. In conclusion, this study demonstrated the feasibility of the use of novel collagen filaments for fabricating small caliber tissue-engineered vascular grafts that provide both sufficient mechanical properties and superior biological performance.

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Spatial Arrangements of Connexin43 in Cancer Related Cells and Re-Arrangements under Treatment Conditions: Investigations on the Nano-Scale by Super-Resolution Localization Light Microscopy.

Cancer studies suggest that the spatial localization of connexin43 (Cx43) could play an important role during tumor genesis and the formation of metastasis. Cx43 has been shown to be upregulated in cancer cells; thereby a shift from Cx43 normal localization in gap junctions in the cell membrane towards a primarily cytoplasmic localization was observed in many studies. So far neither the spatial arrangements of Cx43 in breast cancer cells nor the effects of treatment outcome (ionizing radiation and antibody therapy) on the spatial arrangements of Cx43, have been microscopically studied on the nanoscale. This has brought up the idea to study the micro- and nanoscaled spatial Cx43 arrangements in a model of breast cancer-related cell types, i.e., SkBr3 breast cancer cells, BJ fibroblasts, and primary human internal mammary artery endothelial cells (HIMAECs). The cells were treated with neuregulin1 (NRG1), trastuzumab (Herceptin), or 6MeV-photon irradiation at a dose of 4 Gy. NRG1 stimulates further NRG1 release in the tumor endothelium that may lead to an enhanced tumor protective effect whereas Herceptin, used in antibody treatment, works in an antagonistic fashion to NRG1. After fluorescent labelling with specific antibodies, the molecular positions of Cx43 in the perinuclear cytosol and in the cell periphery at the membrane were determined for the three treatment related applications (NRG1, trastuzumab, 4 Gy irradiation) using confocal laser scanning microscopy (CLSM) and single molecule localization microscopy (SMLM). These techniques enable investigations of Cx43 enrichment and topological arrangements of Cx43 molecules from the micro-scale of a whole cell to the nano-scale of single molecules. In SkBr3 cells with and without radiation treatment high density accumulations were detected which seem to be diluted after NRG1 and trastuzumab treatment although the SMLM distance frequency distributions did not significantly vary. In BJ fibroblasts and HIMAECs differences between periphery and perinuclear cytosol were observed after the different treatment processes. HIMAECs showed significant Cx43 accumulation after NRG1, trastuzumab, and radiation treatment in the perinuclear region whereas in the periphery radiation has less influence as compared to the control. BJ cells were reacting to the treatments by Cx43 accumulations in the perinuclear region but also in the periphery. In conclusion, it was shown that by using CLSM and super-resolution SMLM, treatment effects on the spatial and thus functional arrangements of Cx43 became detectable for investigations of tumor response mechanisms.

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Erythrocytes From Patients With Type 2 Diabetes Induce Endothelial Dysfunction Via Arginase I.

Cardiovascular complications are major clinical problems in type 2 diabetes mellitus (T2DM). The authors previously demonstrated a crucial role of red blood cells (RBCs) in control of cardiac function through arginase-dependent regulation of nitric oxide export from RBCs. There is alteration of RBC function, as well as an increase in arginase activity, in T2DM.

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Effects Of Endothelin-1 On Intracellular Tetrahydrobiopterin Levels In Vascular Tissue.

Tetrahydrobiopterin (BH4) is the essential cofactor of endothelial nitric oxide synthase (eNOS) and intracellular levels of BH4 is regulated by oxidative stress. The aim of this paper was to describe the influence of exogenous endothelin-1 on intracellular BH4 and its oxidation products dihydrobiopterin (BH2) and biopterin (B) in a wide range of vascular tissue.

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Diabetes Mellitus Induces Hyperreactivity of 5-Hydroxytryptamine (5-HT)-Induced Constriction in Human Internal Thoracic Artery and Is Associated with Increase in the Membrane Protein Level of 5-HT Receptor.

Studies indicate that 5-hydroxytryptamine (5-HT) released from activated platelets in coronary artery bypass grafting (CABG) induces 5-HT receptor-mediated graft spasm. We previously reported that 5-HT-induced constriction of human endothelium-denuded saphenous vein (SV) was significantly augmented in patients with diabetes mellitus (DM) than in patients without DM (non-DM), without changes in the levels of the membrane-bound 5-HT receptor of their smooth muscle cells. Although the internal thoracic artery (ITA) is the key graft conduit for CABG, the effect of DM on the ITA graft spasm is still unclear. Therefore, in this study, we investigated the effect of DM on 5-HT-induced vasoconstriction and the level of membrane-bound 5-HT receptor in ITA grafts. 5-HT-induced constriction of the isolated human endothelial-denuded ITA was significantly higher in patients with DM than in patients without DM. In addition, the level of the 5-HT receptor in the membrane fraction of human ITA smooth muscle cells was significantly higher in patients with DM than in those without DM. These results demonstrate that DM is a risk factor for CABG in both venous and arterial conduits, and that it differentially affects the level of the membrane-bound 5-HT receptor in the venous and arterial smooth muscle cells.

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Decreased FENDRR and LincRNA-p21 expression in atherosclerotic plaque.

Cardiovascular diseases are the most important cause of mortality worldwide, particularly atherosclerosis. Recently, lncRNAs affecting atherosclerotic progression have been reported in vascular smooth muscle cells, endothelial cells, and monocytes, suggesting that lncRNAs play an important role in atherosclerosis.

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Endothelium-dependent vasorelaxant effect of procyanidin B2 on human internal mammary artery.

The aim of the present study was to investigate and characterize vasorelaxant effect of procyanidin B2 on human internal mammary artery (HIMA) as one of the mechanisms of its protective effect against vascular risk. Procyanidin B2 induced strong concentration-dependent relaxation of HIMA rings pre-contracted by phenylephrine. Pretreatment with L-NAME, a NO synthase inhibitor, hydroxocobalamin, a NO scavenger, and ODQ, an inhibitor of soluble guanylate cyclase, significantly inhibited procyanidin B2-induced relaxation of HIMA, while indomethacin, a cyclooxygenase inhibitor, considerably reduced effects of low concentrations. Among K channel blockers, iberiotoxin, a selective blocker of large conductance Ca-activated K channels (BK), abolished procyanidin B2-induced relaxation, glibenclamide, a selective ATP-sensitive K(K) channels blocker, induced partial inhibition, while 4-aminopyridine, a blocker of voltage-gated K(K) channels, and TRAM-34, an inhibitor of intermediate-conductance Ca-activated K(IK) channels, slightly reduced maximal relaxation of HIMA. Further, procyanidin B2 relaxed contraction induced by phenylephrine in Ca-free Krebs solution, but had no effect on contraction induced by caffeine. Finally, thapsigargin, a sarcoplasmic reticulum Ca-ATPase inhibitor, significantly reduced relaxation of HIMA produced by procyanidin B2. These results demonstrate that procyanidin B2 produces endothelium-dependent relaxation of HIMA pre-contracted by phenylephrine. This effect is primarily the result of an increased NO synthesis and secretion by endothelial cells and partially of prostacyclin, although it involves activation of BK and K, as well as K and IK channels in high concentrations of procyanidin B2.

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CD14CD16 "nonclassical" monocytes are associated with endothelial dysfunction in patients with coronary artery disease.

Endothelial dysfunction and inflammation are key mechanisms of vascular disease. We hypothesised that heterogeneity of monocyte subpopulations may be related to the development of vascular dysfunction in coronary artery disease (CAD). Therefore, we examined the relationships between monocyte subsets (CD14CD16 "classical - Mon1", CD14CD16 "intermediate - Mon2" and CD14CD16 "nonclassical - Mon3"), endothelial function and risk factor profiles in 130 patients with CAD undergoing coronary artery bypass grafting. This allowed for direct nitric oxide (NO) bioavailability assessment using isometric tension studies ex vivo (acetylcholine; ACh- and sodium-nitropruside; SNP-dependent) in segments of internal mammary arteries. The expression of CD14 and CD16 antigens and activation markers were determined in peripheral blood mononuclear cells using flow cytometry. Patients with high CD14CD16 "nonclassical" and low CD14CD16 "classical" monocytes presented impaired endothelial function. High frequency of CD14CD16 "nonclassical" monocytes was associated with increased vascular superoxide production. Furthermore, endothelial dysfunction was associated with higher expression of activation marker CD11c selectively on CD14CD16 monocytes. Nonclassical and classical monocyte frequencies remained independent predictors of endothelial dysfunction when major risk factors for atherosclerosis were taken into account (β=0.18 p=0.04 and β=-0.19 p=0.03, respectively). In summary, our data indicate that CD14CD16 "nonclassical" monocytes are associated with more advanced vascular dysfunction measured as NO- bioavailability and vascular reactive oxygen species production.

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