Primary Murine Endothelial Cells Research Articles

MO056: Alteration of Glycocalyx on Endothelium of Peritubular Capillaries in CKD

Abstract BACKGROUND AND AIMS The glycocalyx is a carbohydrate-rich gel-like mesh, which covers the luminal surface of cells, including the endothelium. The glycocalyx is involved in many regulatory functions of the endothelium, including vascular permeability. In chronic kidney disease (CKD), peritubular capillaries undergo anatomical, structural and functional alterations such as rarefaction, increased tortuosity and increased permeability. Here, we hypothesized that the glycocalyx of peritubular capillaries might be affected in CKD and investigated morphological and ultrastructural pathological alterations of the glycocalyx in different murine CKD models and human kidney tissue. METHOD We stained the glycocalyx of peritubular capillaries in kidney tissue of murine and human CKD specimen using different fluorescently labeled plant-derived lectins. Next, we established the Lanthanum Dysprosium Glycosamino Glycan adhesion (LaDy GAGa) staining technique to visualize the ultrastructure of the glycocalyx using transmission electron microscopy and to perform quantitative analyses. Finally, we analyzed the expression and regulation of glycocalyx components in primary murine endothelial cells. RESULTS Fluorescence stainings using different lectins with high affinity to components of the renal glycocalyx revealed a reduced binding to the endothelium in CKD. We found a similar change in a human kidney tissue. The LaDy GAGa staining technique visualized the ultrastructure of the glycocalyx and enabled quantitive analyses. We found a significant reduction of the glycocalyx thickness and density in two different models of renal fibrosis and CKD, i.e. unilateral ureteral obstruction and ischemia–reperfusion injury. Additionally, mRNA expression of proteins involved in glycocalyx biology, synthesis and turnover, i.e. syndecan 1 and glypican 1, which are main components of the glycocalyx, and exostosin 2, involved in the synthesis of the glycocalyx, were significantly upregulated in endothelial cells isolated from murine CKD models. CONCLUSION Visualization of glycocalyx using specific ultrastructural analyses allows qualitative and quantitative analyses, revealing significant pathological alterations in the glycocalyx of peritubular capillaries in CKD.

Micrornas Mir-147-3p And Mir-298-5p Are Nod-Driven Regulators Of Endothelial Cytokine Expression

Background and Aims: NOD1 and 2 are intracellular pattern recognition receptors mediating vascular inflammatory processes. Since endothelial cells are crucially involved in vascular inflammation, we here investigated the microRNA (miRNA) expression profile in primary murine endothelial cells upon NOD stimulation and their role in endothelial cytokine expression.

The Adaptor CRADD/RAIDD Controls Activation of Endothelial Cells by Proinflammatory Stimuli

A hallmark of inflammation, increased vascular permeability, is induced in endothelial cells by multiple agonists through stimulus-coupled assembly of the CARMA3 signalosome, which contains the adaptor protein BCL10. Previously, we reported that BCL10 in immune cells is targeted by the "death" adaptor CRADD/RAIDD (CRADD), which negatively regulates nuclear factor κB (NFκB)-dependent cytokine and chemokine expression in T cells (Lin, Q., Liu, Y., Moore, D. J., Elizer, S. K., Veach, R. A., Hawiger, J., and Ruley, H. E. (2012) J. Immunol. 188, 2493-2497). This novel anti-inflammatory CRADD-BCL10 axis prompted us to analyze CRADD expression and its potential anti-inflammatory action in non-immune cells. We focused our study on microvascular endothelial cells because they play a key role in inflammation. We found that CRADD-deficient murine endothelial cells display heightened BCL10-mediated expression of the pleotropic proinflammatory cytokine IL-6 and chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) in response to LPS and thrombin. Moreover, these agonists also induce significantly increased permeability in cradd(-/-), as compared with cradd(+/+), primary murine endothelial cells. CRADD-deficient cells displayed more F-actin polymerization with concomitant disruption of adherens junctions. In turn, increasing intracellular CRADD by delivery of a novel recombinant cell-penetrating CRADD protein (CP-CRADD) restored endothelial barrier function and suppressed the induction of IL-6 and MCP-1 evoked by LPS and thrombin. Likewise, CP-CRADD enhanced barrier function in CRADD-sufficient endothelial cells. These results indicate that depletion of endogenous CRADD compromises endothelial barrier function in response to inflammatory signals. Thus, we define a novel function for CRADD in endothelial cells as an inducible suppressor of BCL10, a key mediator of responses to proinflammatory agonists.

Vascular-specific quantification in an in vivo Matrigel chamber angiogenesis assay

The study of angiogenesis as a therapeutic target requires reliable in vivo assays that can provide physiologically relevant data. A murine in vivo Matrigel-based angiogenesis assay is presented here which includes the quantitative assessment of vascular-specific indicators of neovascularization. Matrigel containing 175 ng/ml bFGF is encapsulated in synthetic chambers which are implanted subcutaneously in C57/B16J mice. Ex vivo implants can be imaged to qualitatively view perfused vasculature within the chambers, or histologically processed to confirm the presence of vascular-specific tissue within the Matrigel. Viable cells are recovered from the excised chambers and quantified cytometrically using endothelial cell-specific markers CD34 and CD144, and for a marker of nucleated cells, Hoechst 33342. Thalidomide, 200 mg/kg/day, was tested using the assay and was found to inhibit angiogenesis by 46%. Angiogenesis inhibitors secreted by LL/M27 tumors were also characterized, where tumor-bearing mice showed a 73% inhibition of angiogenesis compared to tumor-free controls. Analysis of the number of nucleated cells in these samples failed to show a strong correlation with the number of endothelial cells, indicating that quantification of nonvascular-specific tissue in in vivo angiogenesis assays may not be sufficient. This new assay provides an objective, comprehensive determination of the vasculature-specific response of both endogenous and exogenous angiogenesis inhibitors in vivo, and also creates new opportunities for obtaining primary murine endothelial cells.

Isolation of murine aortic endothelial cells in culture and the effects of sex steroids on their growth.

The lack of commercially available primary murine endothelial cells prompted us to isolate and cultivate this cell type. We report here the effect of sex steroids on the in vitro growth of murine aortic endothelial cells. Murine aortic endothelial cells were isolated by a combination of explant outgrowth from aortic rings and enzymatic digestion. The endothelial nature of the cells was verified by uptake of acylated low-density lipoprotein and positive staining for CD-31. Murine aortic endothelial cell growth is stimulated by physiological concentrations of estrogen. Progesterone, when given simultaneously with estrogen, inhibited the stimulatory growth effect of estrogen. Murine aortic endothelial cells grown in vitro continue to express messenger ribonucleic acid for proteins related to endothelial growth. These include vascular endothelial growth factor, its receptors Flt-1 and Flk-1, and the angiogenesis-associated transcription factor, Ets-1.