Bioactive coating for tissue-engineered smalldiameter vascular grafts

Objective: to develop a method for modifying composite small-diameter porous tubular biopolymer scaffolds based on bacterial copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and gelatin modified with a double-layered bioactive coating based on heparin (Hp) and platelet lysate (PL) that promote adhesion and proliferation of cell cultures.Materials and methods. Composite porous tubular biopolymer scaffolds with 4 mm internal diameter were made by electrospinning from a 1 : 2 (by volume) mixture of a 10% solution of poly(3-hydroxybutyrateco- 3-hydroxyvalerate) copolymer, commonly known as PHBV, and a 10% solution of gelatin, respectively, in hexafluoro-2-propanol. The structure of the scaffolds was stabilized with glutaraldehyde vapor. The scaffolds were modified with a bioactive Hp + PL-based coating. The surface morphology of the samples was analyzed using scanning electron microscopy. Biological safety of the modified scaffolds in vitro (hemolysis, cytotoxicity) was evaluated based on the GOST ISO 10993 standard. Interaction with cultures of human endothelial cell line (EA. hy926) and human adipose-derived mesenchymal stem cells (hADMSCs) was studied using vital dyes.Results. We developed a method for modifying small-diameter composite porous tubular biopolymer scaffolds obtained by electrospinning from a mixture of PHBV and gelatin modified with double-layered bioactive coating based on covalently immobilized Hp and human PL. The modified scaffold was shown to have no cytotoxicity and hemolytic activity in vitro. It was also demonstrated that the developed coating promotes hADMSC adhesion and proliferation on the external surface and EA.hy926 on the internal surface of the composite porous tubular biopolymer scaffolds in vitro.Conclusion. The developed coating can be used for the formation of in vivo tissueengineered small-diameter vascular grafts.

Inhibitory Effects of Syzygium jambos Extract on Biomarkers of Endothelial Cell Activation

Background: Disordered endothelial cell activation plays an important role in the pathophysiology of atherosclerosis, cancer, sepsis, viral infections, and inflammatory responses. There is interest in developing novel therapeutics to regulate endothelial cell function in atherothrombotic, metabolic, vascular, and hematological diseases. Extracts from leaves of the Syzygium jambos (L.) Alston (S. jambos) trees have been proposed to treat cardiovascular diseases and diabetes through unclear mechanisms. We investigated the effects of the S. jambos extract on biomarkers of endothelial dysfunction and immune responses in the human endothelial cell line, EA.hy926. Methods: Leaves of S. jambos were collected, concocted and lyophilized. To study the effects of S. jambos on endothelial cell activation, we used the human endothelial cell line. IL-6 levels were measured using qPCR and ELISA. PDI activity was measured using Insulin Turbidity and Di-E-GSSG assays. CM-H2DCFDA was used to study ROS levels. Migration assay was used to study S. jambos effect on ex vivo human polymorphonuclear and human mononuclear cells.Results: Our results show that incubation of EA.hy926 cells with ET-1 led to a 6.5 ± 1.6 fold increase in IL-6 expression by qPCR, an event that was blocked by S. jambos. Also, we observed that ET-1 increased extracellular protein disulfide isomerase (PDI) activity that was likewise dose-dependently blocked by S. jambos (IC50=14µg/mL). Consistent with these observations, ET-1 stimulated ex vivo human polymorphonuclear and mononuclear cell migration that also was dose-dependently blocked by S. jambos. In addition, ET-1 stimulation led to significant increases in ROS production that were sensitive to S. jambos. Conclusion: Our results suggest that the S. jambos extract represents a novel cardiovascular protective pharmacological approach to regulate endothelial cell activation, IL-6 expression, and immune-cell responses.

Genetic depletion studies inform receptor usage by virulent hantaviruses in human endothelial cells

Hantaviruses are RNA viruses with known epidemic threat and potential for emergence. Several rodent-borne hantaviruses cause zoonoses accompanied by severe illness and death. However, assessments of zoonotic risk and the development of countermeasures are challenged by our limited knowledge of the molecular mechanisms of hantavirus infection, including the identities of cell entry receptors and their roles in influencing viral host range and virulence. Despite the long-standing presumption that β3/β1-containing integrins are the major hantavirus entry receptors, rigorous genetic loss-of-function evidence supporting their requirement, and that of decay-accelerating factor (DAF), is lacking. Here, we used CRISPR/Cas9 engineering to knockout candidate hantavirus receptors, singly and in combination, in a human endothelial cell line that recapitulates the properties of primary microvascular endothelial cells, the major targets of viral infection in humans. The loss of β3 integrin, β1 integrin, and/or DAF had little or no effect on entry by a large panel of hantaviruses. By contrast, loss of protocadherin-1, a recently identified entry receptor for some hantaviruses, substantially reduced hantavirus entry and infection. We conclude that major host molecules necessary for endothelial cell entry by PCDH1-independent hantaviruses remain to be discovered.

Genetic depletion studies define receptor usage by virulent hantaviruses in human endothelial cells

Hantaviruses are a large group of RNA viruses that include known epidemic threats and other agents poised for emergence. Several rodent-borne hantaviruses cause zoonoses accompanied by severe illness and death. However, assessments of zoonotic risk and the development of countermeasures alike are challenged by our limited knowledge of the molecular mechanisms of hantavirus infection, including the identities of cell entry receptors and their roles in influencing viral host range and virulence. Previous work has implicated several cell-surface molecules, most notably β3- and β1-containing integrin heterodimers, decay-accelerating factor (DAF), and the cadherin superfamily protein protocadherin-1 (PCDH1), in hantavirus entry in endothelial cells, the major targets of viral infection in humans. Despite the fact that β3/β1 integrins have been presumed to be the major hantavirus entry receptors for over two decades, rigorous genetic evidence supporting their requirement, and that of DAF as an entry cofactor, is lacking. Here, we used CRISPR/Cas9 engineering to knock out four candidate hantaviral receptors, singly and in combination, in a human endothelial cell line that recapitulates the properties of primary microvascular endothelial cells. PCDH1 loss substantially reduced entry and infection by a subset of hantaviruses endemic to the Americas. In contrast, the loss of β3 integrin, β1 integrin, and/or DAF had little or no effect on entry by any of a large panel of hantaviruses tested. We conclude that the major host molecules necessary for endothelial cell entry by PCDH1-independent hantaviruses remain to be discovered.

Evaluation of histone deacetylase inhibitor substituted zinc and indium phthalocyanines for chemo- and photodynamic therapy

3-Hydroxypyridin-2-thione bearing zinc and indium phthalocyanine derivatives, as photosensitizer agents have been synthesized and evaluated for their anti-cancer efficacy on two breast cancer cell lines, MDA-MB-231 and MCF-7 as well as a human endothelial cell line, HUVEC.