The effect of high glucose on the inhibitory action of C21, a selective AT2R agonist, of LPS-stimulated tissue factor expression in human mononuclear cells

Human PBMCs were obtained from unpooled buffy coats left over from blood bank draws taken from healthy donors with the approval of the local ethics committee. According to local procedures, individuals with a history of diabetes and hypertension, either on antihypertensive drugs or not, are excluded from blood donation. As detailed elsewhere [22], leukocytes were isolated from fresh buffy coats diluted 1:1 with sodium citrate 0.38 % in saline solution, mixed gently with 0.5 volume of 2 % Dextran T500 and left for 40 min for erythrocyte sedimentation. The leukocyte-rich supernatant was recovered and centrifuged for 10 min at 200xg. The pellet was resuspended in 30 ml of sodium citrate solution, layered over 15 ml of Ficoll-Hypaque and centrifuged for 30 min at 350xg at 20 °C. The PBMC-rich ring was recovered, washed twice in sodium citrate 0.38 % and resuspended in no glucose RPMI 1640 medium (Sigma Chemical, St Louis, Missouri, USA) supplemented with 100 U/ml penicillin-streptomycin. The final PBMC preparations typically contain 25–35 % monocytes, 65–75 % lymphocytes and less than 5 % neutrophils [22].

After isolation, cells resuspended in polypropylene tubes (3×10⁶ cells/ml) were exposed to experimental drugs or their appropriate vehicle in presence of two different D-glucose concentrations (5.5 mM, hereafter referred to as Normal Glucose, NG, or 50 mM heretofore referred to as High Glucose, HG) 30 min prior to LPS (Escherichia coli 026:B6 LPS; Sigma Chemical), 100 ng/ml, and left to incubate for 18 hs at 37 °C in a 5 % CO₂ atmosphere until assay. Previous experiments had shown the D-glucose concentration-dependent increase in TF procoagulant activity (see below) as well as the neutral effect of L-Glucose used as a control for osmolarity changes [13] and, therefore, were not repeated. Untreated PBMCs were also included in each experimental series to obtain baseline values. All reagents and solutions used for cell isolation and culture were prepared with endotoxin-free water and glassware was rendered endotoxin-free by exposure to high temperature. Drugs were kept in stock solution and diluted in serum-free RPMI at the appropriate concentrations immediately before use. Cell viability as assessed by dimethyl thiazolyl diphenyl tetrazolium (MTT) was verified (85 % or more of viable cells) throughout all experimental phases.

Statistical differences were assessed by non parametric tests (Wilkoxon’s and Mann-Whitney for paired and unpaired comparisons respectively) on either absolute data or percent changes from control conditions, these latter taken as a measure of drug effect. Data were reported as means ± SD unless otherwise reported. A two-tailed p-level <0.05 was the threshold for statistical significance.

Resting and LPS-stimulated TF PCA, Ag and mRNA were greater in HG than NG (Table 1). In both experimental conditions, BAY 11-7082 (10⁻⁵M) abolished LPS-stimulated PCA (Fig. 1).

Both C21 and OLM downregulated LPS-induced TF PCA (Fig. 2). However, the extent of inhibition exerted by C21 was by and large not influenced by D-glucose concentrations (Fig. 2, left panel) in contrast with OLM whose effect was potentiated in HG (Fig. 2, right panel).

A similar behaviour was found when analyzing the effect of the two drugs on TFAg and TFmRNA (Fig. 3, left and right panel respectively).

PD123,319 (10⁻⁶M) abolished the effect of C21 in both NG and HG (Fig. 4, left and right panel respectively).

The interpretation of the results of this study are facilitated by some further discussion of the involvement of ATII in innate immunity, a biological system by which germline-encoded receptor proteins recognize specific patterns presented by groups of pathogens [20]. Among them, LPS, the proinflammatory stimulus used in our experimental series, constitutes a major marker for the recognition of intruding Gram-negative bacteria that, by binding to Toll-like receptor(TLR)4 s and CD14 and recruiting adaptor proteins to the cytoplasmic Toll/interleukin-1 receptor, initiates the pathogen-induced inflammatory response of which activation of coagulation is a prominent component [1, 20]. ATII contributes to the innate immune response through complex and interrelated transcriptional and posttranscriptional mechanisms including upregulation of the TLR4 expression leading to a more intense NF-kB activation e.g. [27‐29]. In turn, LPS or its intracellular proxies such as TNF-alpha [20] stimulate ATII generation [30] by activating renin, the earlier step of its biosynthetic chain [31], and increasing the number of ATII receptors available for stimulation by the peptide [32]. Thus, immunomodulation appears as an integral component of RAS functions far away from the conventional view of a hormonal system mainly involved in systemic blood pressure and body fluid volume control.

Although primarily aimed at the acute defence against infection and tissue damage, a growing body of evidence implicates innate immunity in several chronic conditions including type 2 diabetes [33] in which raised glucose concentrations, the hallmark of that disease, induce overexpression of Toll-like receptor(TLR) [34, 35] and CD14 [36], promote de-novo local synthesis of RAS components [37, 38] and potentiate a wide array of ATII-mediated biological actions e.g. [39‐41]. By stimulating NADPH oxidase and mitochondrial metabolism, HG also accelerates reactive oxygen species (ROS) generation activating NFkB [16‐18], thus initiating TF gene transcription along with a host of other proinflammatory cytokines. That concept is fully in line with our results showing upregulation by HG of both quiescent and LPS-induced TF PCA, mRNA and Ag expression and abolition of activated PCA by BAY11-7082, a NF-kB inhibitor [25], demonstrates the complete dependency upon the NFkB signalling pathway of the procoagulant effect of endotoxin in human PBMCs.

Within the frame of reference summarized in the previous paragraph, our data confirm the inhibitory action of AT2R agonism by C21 on LPS-stimulated TF expression in human PBMCs [14] and the complete antagonism exerted by PD123,319 reassures about the specificity of the response. The parallel decrease of TFAg and mRNA is consonant with transcriptional modulation of TF protein likely by NFkB downregulation [41], an interpretation fully supported by recent reports showing attenuated cytokine production by C21 in LPS-stimulated monocytic cells [42]. In addition to those relevant albeit confirmatory findings, however, the main and original pathophysiological implications of this study arise from the contrast between the neutral effect of HG on C21-induced TF inhibition and the potentiated effect of AT1R blockade by OLM in PBMCs exposed to high D-glucose. That divergent behaviour, in fact, indicates that HG impacts differently on AT1R- and AT2R-mediated signal transduction pathways although our data do not allow any obvious explanation for that divergence. On a speculative basis, increased AT1Rs available for binding as a consequence of higher D-glucose concentrations [32] might potentiate the effect of OLM although the same should theoretically apply to AT2R stimulation given the sensitivity of that receptor subset to glucose concentrations [43]. The greater inhibitory effect of OLM might also relate to increased ANGII production in PBMCs grown in HG media [37, 38] although AT2R antagonism by PD 123, 329 per se was totally neutral on the procoagulant potential of PBMCs in our previous experience [14]. Thus, TF inhibition by C21 may merely represent the result of a pharmacological manipulation of physiologically silent binding sites activated by an agonist attaining concentrations at the receptor site far exceeding those achieved by ATII, the endogenous ligand [44]. Other mechanisms peculiar to AT1R blockers may also be at play including modulation of TLR4 expression and activity possibly independent of AT1R blockade [45] but this as well as all the above outlined possibilities are speculative and our data cannot provide any solid evidence in favour or against them.