Vascular Glycobiology of Oxidative Stress and Endothelial Dysfunction, a Potential Therapeutic Target

The integrity of endothelial glycocalyx is crucial for normal endothelial function and endothelial response to shear stress. Endothelial glycocalyx also tethers and concentrates the extracellular superoxide dismutase isoform 3 (SOD3) which protects the endothelium against the oxidative damage of reactive oxygen species (ROS). Degradation of endothelial glycocalyx results in endothelial dysfunction and vascular remodeling. The proteolytic enzymes Matrix Metalloproteinases (MMPs) and Disintegrin Metalloproteinases (ADAMs) are capable of disrupting endothelial cell surface proteins such as syndecans resulting in derangement of endothelial glycocalyx. We experimentally induced oxidative stress in human adipose microvascular endothelial cells (HAMECs) in the presence or absence of exogenous hydrogen peroxide (H2O2) and endogenous oxidative stress inducer buthionine sulfoximine (BSO) and MMP inhibitors. We found that either H2O2 or BSO, increased MMPs (MMP1, MMP2, and MMP9) and ADAMs (ADAM10 and ADAM17). Consequently, there was a reduction in protein levels of syndecan-1 and SOD3 in the total cell lysate and increases in levels of syndecan-1 ectodomain and SOD3 in cell culture media detected by immunoprecipitation and Western blotting. The upregulation of MMPs was accompanied by a decline in the mRNA and protein levels of their inhibitors, TIMPs. Interestingly, we found that the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) normalized MMP expression and restored TIMP 1 and TIMP 3 after H2O2 or BSO. Pharmacological inhibition of MMPs using marimastat and lisinopril mitigated the effect of oxidative stress on the glycocalyx. Using immunofluorescent labeled wheat germ agglutinin, we found that lisinopril and marimastat effectively restored the cell surface density of heparan sulfate glycosaminoglycans (HS-GAGs), syndecans and SOD3 on the endothelial cell surface. Lastly, we found that lisinopril decreased HDAC mRNA expression induced by H2O2 suggesting an epigenetic mechanism of action for lisinopril on endothelial responses to oxidative stress. In conclusion, our findings suggest that oxidative stress induced loss of the endothelial glycocalyx involves epigenetic regulation of MMPs that results in the shedding of SOD3 and syndecan-1. These data shed light on a therapeutically targetable epigenetic regulatory mechanism by which oxidative stress may induce vascular remodeling and endothelial dysfunction

Syndecan-1 downregulation reduces HSV-1 spread.

<p>Monolayers of HCE cells were exposed to (A) HSV-1 (KOS) or (B) HSV-1 (KOS) K26GFP strains at MOI of 5 at 37°C to allow virus entry. 2 h post-infection, cells were washed, incubated for 1 min with 0.1 M citrate buffer (PH 3.0), then washed with PBS and overlaid with 0.5% methylcellulose medium. After 4 h, cells were washed, dissociated, and approximately 600 cells were plated onto 80% confluent monolayers of uninfected HCE cells that have been transfected with either scrambled siRNA or syndecan-1 siRNA for 72 h in 0.5% methylcellulose medium. (A). The spread of HSV-1 (KOS) from infected cells to the siRNA transfected HCE cells was evaluated by staining and counting the number of plaques formed at the 10× objective (Zeiss Axiovert 200). Results are presented as relative number of plaques (mean ± 1SD) of four independent experiments performed in duplicates. (B) HSV-1 (KOS) K26GFP spread was qualitatively assessed at the 10× objective (Zeiss Axiovert 200). Representative images from one experiment performed in triplicate are shown. <i>SDC1</i>, syndecan-1.</p

Comparison of syncytia number and nuclei count after syndecan-1 overexpression in each CHO cell type.

<p>Average number of syncytial cells, as well as, the average number of nuclei per syncytia was counted in CHO-K1 and CHO-745 cells after overexpressing syndecan-1 on target cells or effector cells. Positive controls are target and effector cells expressing normal levels of syndecan-1. Syncytial cells were counted 72 h post mixing. Syncytia were classified as any red cell having two or more nuclei. Number of syncytia was normalized to the number of syncytia detected in the negative control wells where the effector cell population lacks gB. The average is based on results from two independent experiment performed in duplicate (mean ± 1SD).</p

Downregulation of Syndecan-1 results in reduced production of infectious virus.

<p>HCE cells were transfected with either control scrambled siRNA or syndecan-1 siRNA. 72 h post-transfection cells were infected with HSV-1 (KOS) at an MOI of 0.1. At 0, 5, 24, 48, and 72 h post-infection, infectious virus was quantified by a standard plaque assay on HCE cell monolayers. The titers shown are the mean ± 1 SD of a representative experiment of two independent experiments performed in duplicates. <i>SDC1</i>, syndecan-1.</p

Syndecan-1 knockdown or overexpression do not affect cell viability.

<p>(A). CHO-K1, CHO-745, and HCE cells were transfected with scrambled (scr) siRNA or syndecan-1 (SDC1) siRNA. 72–96 h after transfection, immunoblots of cell lysates were prepared and probed with anti-SDC1 polyclonal Ab. β-actin protein level was measured as loading control. Representative blots are shown. Protein bands were quantified using ImageQuant TL image analysis software (version: 7). SDC1 protein expression (mean ± 1SD), normalized to that of β-actin, of at least three independent experiments was quantified by calculating the relative intensity of each syndecan-1 band relative to the control scrambled siRNA treated bands, and presented as bar graph. (B) Cells were grown in 6-well plates, mock treated or transfected with human SDC1 plasmid for 48 h. Cell surface level of SDC1 was evaluated by flowcytomety. FITC stained cells were used as background control. Results are representative of two independent experiments (C, D). Cells were grown in 96-well plates, transfected with scrambled siRNA or SDC1 siRNA for 48 h (C), or transfected with control GFP plasmid or human SDC1 plasmid for 24 h (D). Triplicate wells were evaluated for cell viability using MTS assay. Results are expressed as 100% wild type (wt) viability where they represent the percent corrected absorbance after subtracting the background absorbance, relative to scrambled siRNA transfected cells (C), or relative to GFP transfected cells (D), and are mean ± 1SD of at least 2 independent experiments.</p

Syncytial cell formation in CHO-K1 and CHO-745 after syndecan-1 overexpression.

<p>(A). An illustration of syncytia assay that was exploited to understand the contribution of syndecan-1 during HSV-1 induced cell-to-cell fusion. Effector cell population that expresses HSV-1 fusion glycoproteins, T7 polymerase as well as a RFP-NES construct that restricts the expression of RFP to the cytoplasm is mixed with the target cell population that expresses nectin-1 as a gD receptor, the luciferase reporter gene under the control of T7 promoter as well as a CFP-NLS construct that restricts the level of CFP to the nucleus. (B). Syncytia formation was observed in cells 72 h after the mix of target cells with effector cells. <i>Top panels</i> show representative syncytia formed in CHO-K1 cells after overexpressing syndecan-1 in target, or effector cell. <i>Bottom panels</i> show representative syncytia formed in CHO-745 cells after overexpressing syndecan-1 in target, or effector cells. Positive controls are target cells mixed with effector cells where both populations have the wild-type level of syndecan-1 on the surface. Negative controls are target cells mixed with effector cells missing gB. (Scale bar = 37.5 µm). <i>SDC1</i>, syndecan-1.</p

Syndecan-1 overexpression on target cells enhances cell fusion, while its overexpression on effector cells inhibits cell fusion.

<p>(A). An illustration of cell fusion assay that was exploited to understand the contribution of syndecan-1 during HSV-1 induced cell-to-cell fusion. Effector cell population that expresses HSV-1 fusion glycoproteins plus T7 polymerase is mixed with the target cell population that expresses nectin-1 as a gD receptor and the luciferase reporter gene under the control of T7 promoter. Luciferase reporter gene activity is determined to quantify cell-to-cell fusion. (B). Target cells for CHO-K1 and CHO-745 cells were either transfected with GFP control plasmid (wild type cells) or transfected with syndecan-1 plasmid and mixed with effector cells 24 h post-transfection. As a negative control, target cells were mixed with effector cells that lack HSV-1 gB (C). Target cells for CHO-K1 and CHO-745 cells were either mock treated (wild type cells) or transfected with syndecan-1 specific siRNA and mixed with effecor cells 48 h post-transfection. As a negative control, target cells were mixed with effector cells that lack HSV-1 gB. (D). Effector cells for CHO-K1 and CHO-745 cells were transfected with either GFP control plasmid (wild type cells) or human syndecan-1 plasmid and mixed with target cells 24 h post-transfection. As a negative control, target cells were mixed with effector cells that lack HSV-1 gB. (B, C, D). Fusion was measured 16 h post mixing. Results are presented as mean ± 1 SD of at least 3 independent experiments. <i>SDC1</i>, syndecan-1.</p

Syndecan-1 knockdown reduces plaque formation in HCE cells.

<p>(A). Monolayers of HCE cells were transfected with either control plasmid GFP, or with human syndecan-1. 24 h post-transfection cells were infected with serial dilution of HSV-1(KOS) stocks. (B). 50% confluent HCE cells were transfected with either control scrambled siRNA or syndecan-1 specific siRNA. 72 h post-transfection, cells were infected with serial dilution of HSV-1(KOS) stocks. (A, B). 72 h post-infection cells were fixed and stained with crystal violet stain. Infectivity was measured by the number of plaque forming units (PFUs). Number of PFUs was counted at the 10× objective (Zeiss Axiovert 200). Plaques consist of 15 or more nuclei were counted. Results are means ± 1 SD of three independent experiments conducted in duplicate. <i>SDC1</i>, syndecan-1.</p

syndecan-1 ectodomain and cytoplasmic domains are important for inhibiting cell fusion when overexpressed on effector cells.

<p>(A). Syndecan-1 truncation and mutants used in the study are illustrated including the full-length wild type (<i>wt</i>) core protein syndecan-1 (SDC1) that includes an extracellular domain, transmembrane domain (TM), and COOH-terminal cytoplasmic domain. Also illustrated are the construct FcR^ectohS1 that is a chimera comprised of the ectodomain of human IgG Fcγ receptor Ia/CD64 fused to the transmembrane and cytoplasmic domains of human syndecan-1, the construct hS1^pLeu™ that has the transmembrane domain replaced with leucine residues, and a truncation mutant hS1^Δcyto that lacks the 33 C-terminal amino acids. (B) Cells were grown in 96-well plates, transfected with control GFP plasmid, full-length <i>wt</i> human SDC1 plasmid, the construct FcR^ectohS1, the construct hS1^pLeu™, or the construct hS1^Δcyto for 24 h. Triplicate wells were evaluated for cell viability using MTS assay. Results are expressed as 100% wild type (wt) viability where they represent the percent corrected absorbance after subtracting the background absorbance, relative to control GFP plasmid transfected cells, and are mean ± 1SD of at least 3 independent experiments. (C). Effector cells for CHO-K1 and CHO-745 cells were transfected with either control GFP plasmid, full-length <i>wt</i> syndecan-1, the construct FcR^ectohS1, the construct hS1^pLeu™, or the construct hS1^Δcyto and mixed with the target cells 24 h post-transfection. Fusion was measured 16 h post mixing. Results are presented as mean ± 1 SD of at least 3 independent experiments. As a negative control, target cells were mixed with effector cells lacking HSV-1 gB.</p

Glycemia-induced miRNA changes: a review.

Diabetes is a rapidly increasing global health concern that significantly strains the health system due to its downstream complications. Dysregulation in glycemia represents one of the fundamental obstacles to achieving glycemic control in diabetic patients. Frequent hyperglycemia and/or hypoglycemia events contribute to pathologies that disrupt cellular and metabolic processes, which may contribute to the development of macrovascular and microvascular complications, worsening the disease burden and mortality. miRNAs are small single-stranded non-coding RNAs that regulate cellular protein expression and have been linked to various diseases, including diabetes mellitus. miRNAs have proven useful in the diagnosis, treatment, and prognosis of diabetes and its complications. There is a vast body of literature examining the role of miRNA biomarkers in diabetes, aiming for earlier diagnoses and improved treatment for diabetic patients. This article reviews the most recent literature discussing the role of specific miRNAs in glycemic control, platelet activity, and macrovascular and microvascular complications. Our review examines the different miRNAs involved in the pathological processes leading to the development of type 2 diabetes mellitus, such as endothelial dysfunction, pancreatic beta-cell dysfunction, and insulin resistance. Furthermore, we discuss the potential applications of miRNAs as next-generation biomarkers in diabetes with the aim of preventing, treating, and reversing diabetes