Hierarchical cluster analysis of the tested genes and microRNAs of the monocytes of Ecuadorian type 2 diabetic patients and controls in the validation cohort.

<p>On the left, the fold change values between the T2D group and the non-diabetic controls were determined from normalized Ct values (Ct gene/Ct reference gene ABL) by the ΔΔCt method (2−ΔΔCt, User Bulletin 2; Applied Biosystems, Foster City, CA). Data were standardized to the non-diabetic control subjects. The fold change of each gene in the non-diabetic control subjects is therefore 1. Differences between groups were tested using t tests for independent samples. This table shows that 2 microRNAs (MiR-34c-5p and miR-576-3p) were significantly higher expressed in the monocytes of the T2D patients compared to non-diabetic controls. Also, 4 genes (of the 24 tested) were significantly different expressed (PTGS2 lower, and CD9, DHRS3 and PTPN7 significantly higher). The heatmap and dendrogram present the result of the hierarchical clustering of the genes. Three major clusters were found: Cluster A contains inflammatory compounds and includes miR-410 and miR-576-3p. Cluster B contains inflammatory compounds and factors involved with migration/differentiation/metabolism; Cluster C only consists of migration/metabolic factors. MiR-138, miR-574-3p, miR-146a and miR-34c-5p formed a sub-cluster within cluster C and strongly clustered together.</p

Dendrogram and heatmap of hierarchical clustering of T2D patients and non-diabetic controls of the validation cohort using microRNA and mRNA expression as determined by qPCR.

<p>This figure shows that two main subject clusters (X and Y) could be identified. Cluster X contained 5 diabetics and 7 non-diabetic subjects, and cluster Y comprised 17 diabetics and 12 non-diabetics. This approach did not distinguish between T2D patients and non-diabetic controls.</p

TLR Accessory Molecule RP105 (CD180) Is Involved in Post-Interventional Vascular Remodeling and Soluble RP105 Modulates Neointima Formation

<div><p>Background</p><p>RP105 (CD180) is TLR4 homologue lacking the intracellular TLR4 signaling domain and acts a TLR accessory molecule and physiological inhibitor of TLR4-signaling. The role of RP105 in vascular remodeling, in particular post-interventional remodeling is unknown.</p><p>Methods and Results</p><p>TLR4 and RP105 are expressed on vascular smooth muscle cells (VSMC) as well as in the media of murine femoral artery segments as detected by qPCR and immunohistochemistry. Furthermore, the response to the TLR4 ligand LPS was stronger in VSMC from RP105^−/− mice resulting in a higher proliferation rate. In RP105^−/− mice femoral artery cuff placement resulted in an increase in neointima formation as compared to WT mice (4982±974 µm² vs.1947±278 µm²,p = 0.0014). Local LPS application augmented neointima formation in both groups, but in RP105^−/− mice this effect was more pronounced (10316±1243 µm² vs.4208±555 µm²,p = 0.0002), suggesting a functional role for RP105. For additional functional studies, the extracellular domain of murine RP105 was expressed with or without its adaptor protein MD1 and purified. SEC-MALSanalysis showed a functional 2∶2 homodimer formation of the RP105-MD1 complex. This protein complex was able to block the TLR4 response in whole blood ex-vivo. In vivo gene transfer of plasmid vectors encoding the extracellular part of RP105 and its adaptor protein MD1 were performed to initiate a stable endogenous soluble protein production. Expression of soluble RP105-MD1 resulted in a significant reduction in neointima formation in hypercholesterolemic mice (2500±573 vs.6581±1894 µm²,p<0.05), whereas expression of the single factors RP105 or MD1 had no effect.</p><p>Conclusion</p><p>RP105 is a potent inhibitor of post-interventional neointima formation.</p></div

Neointima formation in WT and RP105^−/− mice.

<p>Neointima formation after femoral artery cuff placement in RP105^−/− and wild type mice. Areas of femoral arterial sections were quantified by using 6 sequential sections per segment and are expressed in micrometers squared (mean±SEM). Increased neointima formation in RP105^−/− mice compared to WT (wild type) controls (A). Increased intima/media ratio in RP105^−/− mice compared to WT controls (B). Representative pictures of Elastin von Giesson (C) HPS (D) and α-smooth muscle cell actin (F) of RP105^−/− and WT controls. * = P<0.05 Arrows indicate the Internal Elastic Lamina (IEL) and the External Elastic Lamina (EEL).</p

Soluble RP105 protein – characterization.

<p>Purification and analysis of RP105, MD1 and RP105-MD1 complex. RP105 (lane 1) and RP105-MD1 complex (lane 2) purified by nickel- and streptactin- tandem affinity purification and analysed by SDS-PAGE (10%) and subsequent silverstaining. Lane 3 shows MD1 purified by nickel-affinity purification and analysed by SDS-PAGE (12%) and subsequent Coomassie staining (A). SDS-PAGE analysis of PNGase deglycosylation of SDS denatured MD1 (B). Size exclusion chromatography (Superdex200 PC3.2/30) of the purified RP105 (dashed line) and RP105-MD1 complex (solid line). Molecular weight determination of the RP105-MD1 complex using SEC-MALS (inset). The dashed horizontal lines indicate a molecular weight of 180 kD and 220 kD, respectively (C).</p

RT-PCR of TLR4 (A) or RP105 (B) on wild type VSMC that were incubated with PBS, 1 ng/ml LPS or 10 ng/ml LPS.

<p>Immunostaining of TLR4 (green) or RP105 (red) or both (overlay) on wild type VSMC that were incubated with PBS (C) or 1 ng/ml LPS. (D) Colocalized TLR4 and RP105 staining on VSMC (E). RP105 staining in media area of a murine femoral artery (F). Proliferation of VSMC of WT and RP105^−/− (G) Liquid-scintillation counting after 16 hours of WT and RP105^−/− VSMC. VSMC of show a RP105^−/−an increased proliferative response to LPS. VSMC were cultured in medium containing 10%FCS or 10%FCS with1 ng/ml LPS. Starved WT and RP105^−/− VSMC were used as controls. White bars represent WT VSMC, black bars represent RP105^−/− VSMC.</p

LPS induced neointima formation.

<p>Neointima formation after femoral artery cuff placement with LPS in RP105^−/− and wild type mice. Areas of femoral arterial sections were quantified by using 6 sequential sections per segment and are expressed in micrometers squared (mean±SEM). Increased neointima formation in RP105^−/− mice compared to WT controls after local LPS application (A). Increased intima/media ratio in RP105^−/− mice compared to WT controls after local LPS application (B). Increased outward remodeling in RP105^−/− mice compared to WT controls after local LPS application (C) Representative pictures of Elastin von Giesson (D), HPS (E) and α-smooth muscle cell actin (F) of RP105^−/− and WT controls. * = P<0.05 Arrows indicate the Internal Elastic Lamina (IEL) and the External Elastic Lamina (EEL).</p

Soluble RP105 protein – functionality.

<p>Whole blood stimulation. Blood stimulated with LPS and PBS or combined with purified solRP105 protein or purified solRP105-MD1 protein. Supernatant from triplicates was used for TNFα measurement. * = P<0.05.</p

SolRP105-MD1 reduced neointima formation.

<p>Neointima formation after femoral artery cuff placement with LPS in hypercholesterolemic APOE3Leiden mice. Areas of femoral arterial sections were quantified by using 6 sequential sections per segment and are expressed in micrometers squared (mean±SEM). Decreased neointima formation in mice that had overexpression of solRP105-MD1 (A). Intima Media ratio (B) Pictures of Elastin von Giesson (C) HPS (D) α-smooth muscle cell actin (E) and CD45 (F) of Luc(1) solRP105 (2) MD1 (3) solRP105-MD1 (4) * = P<0.05.</p