Different DOACs Control Inflammation in Cardiac Ischemia-Reperfusion Differently

See the Data Supplement and Major Resources Table for additional information. The data that support the findings of this study are available from the corresponding authors upon reasonable request.

RNA sequencing (RNAseq) data have been deposited in the Gene Expression Omnibus database (accession number GSE161325).

Wild-type mice (C57BL/6; age, 8–9 weeks) were obtained from Janvier Lab (S.A.S., Saint-Berthevin Cedex, France). Only male mice were used throughout this study in accordance with the approved procedures. All animal experiments were conducted according to standards and procedures approved by the local Animal Care and Use Committee (Landesverwaltungsamt Halle and Landesverwaltungsamt, Leipzig, Germany).

Mice (age, 9–10 weeks) were treated with anticoagulants for 7 days before the interventions. In some experiments (tail-bleeding time; FeCl₃ in vivo thrombosis model, see below), increasing doses of a direct thrombin inhibitor (fIIai; dabigatran: range, 5–30 mg/kg, mixed in chow diet) or direct fXai (rivaroxaban: range, 1–20 mg/kg, mixed in chow diet) were used. In the myocardial IRI model (see below), mice received fXai (3 mg/kg) or fIIai (10 mg/kg) for 7 days before surgery, and treatment was reinitiated 2 hours post-surgery and continued for 24 hours (short-term model) or 28 days (long-term model). A subgroup of fXai-treated mice was concomitantly injected with the monoclonal antibody MAPC1591 (10 mg/kg, IP, every second day), which blocks aPC anticoagulant activity, or with the monoclonal antibody MPC1609 (10 mg/kg, IP, every second day), which blocks the anticoagulant and signaling activity of aPC.^32,33 A subgroup of fIIai-treated mice was concomitantly injected with wild-type aPC (1 mg/kg, IP) or a recombinant aPC variant specifically lacking its anticoagulant function (3K3A-aPC, 1 mg/kg, IP) 30 minutes before myocardial IRI.^27,34,35

The data are summarized as the mean±SEM or as box plots. Statistical analyses are mentioned in the Data Supplement for analyses of RNAseq data and in the corresponding figure legends. Post hoc comparisons of ANOVA were corrected with the Tukey method. Statistical significance was accepted at values of P<0.05. The Kolmogorov-Smirnov test or the D’Agostino-Pearson normality test was used to determine whether the data were consistent with a gaussian distribution. Statistics XL (www.statistixl.com) and Prism 5 (www.graphpad.com) software were used for statistical analyses.

We conducted dose-escalation studies in mice to identify dosing regimens that resulted in comparable anticoagulant effects of direct fIIai and fXai. First, tail-bleeding time was determined in mice that received increasing doses of a direct thrombin inhibitor (fIIai; dabigatran: 5–30 mg/kg, mixed in chow diet) or a direct fXai (rivaroxaban: 1–20 mg/kg, mixed in chow diet). Tail-bleeding time was determined 1 week after starting anticoagulant treatment. The average tail-bleeding time in untreated control mice (Cont) was 4±1 minutes. As expected, both fIIai and fXai dose dependently prolonged the tail-bleeding time. The D₅₀ (dose at which the tail-bleeding time was 50% of the maximal observed tail-bleeding time) in mice, which is defined as the dose at which the tail-bleeding time was 50% of the maximal observed tail-bleeding time, was ≈10 mg/kg for fIIai and ≈3 mg/kg for fXai (Figure 1A and 1B).

Next, we used an in vivo thrombosis assay. We induced thrombosis in mesenteric arterioles by local application of FeCl₃. Mice were pretreated for 1 week with either the determined D₅₀ (10 mg/kg for fIIai and 3 mg/kg for fXai) or the lowest dose that resulted in the maximal prolongation of bleeding time (D₁₀₀ [lowest dose at which maximum tail bleeding time was observed]; 20 mg/kg for fIIai and 10 mg/kg for fXai). To avoid excessive hemorrhage during the surgical procedure, anticoagulants were withheld 12 hours before the intervention. At the lower dose (D₅₀), the time until the appearance of the first thrombi was not different in treated mice compared with control mice, while the time to occlusion was comparably prolonged for both anticoagulants (Figure 1C through 1E). At the higher dose (D₁₀₀), both anticoagulants comparably prolonged the time until the appearance of the first thrombi, and both anticoagulants had a comparable effect on the time to occlusion (Figure 1F through 1H).

Using the D₅₀ dosing scheme, we observed plasma levels of anti-fIIa and anti-fXai in the range of plasma concentrations observed in patients (dabigatran: ≈22 ng/mL after 16 hours; rivaroxaban: ≈46 ng/mL after 4 hours; Figure IA and IB in the Data Supplement).^36,37 Additionally, using the D₅₀ dosing scheme, comparable effects on prothrombin time and plasma levels of TAT (thrombin-antithrombin) complex and PF4 (platelet factor 4) were observed (Figure IC through IE in the Data Supplement), corroborating that the chosen dosing resulted in similar anticoagulation effects. We observed no spontaneous bleeding in any of the treated mice. Taken together, we identified dosing regimens for fXai and fIIai with comparable anticoagulant effects both in vivo (tail-bleeding time, in vivo thrombus formation) and ex vivo.

We next determined the impact of fIIai and fXai in a myocardial IRI (ischemia repurfusion injury) model using dosing regimens with comparable anticoagulant effects (D₅₀; 10 mg/kg for fIIai and 3 mg/kg for fXai; left anterior descending ligation for 90 minutes followed by 24 hours of reperfusion; Figure 2A). Mice were pretreated for 1 week with either fIIai or fXai. To avoid excessive bleeding during surgically induced myocardial IRI, both anticoagulants were withheld 12 hours before IRI. Infarct size was determined after 24 hours and was markedly and comparably reduced by both anticoagulants (Figure 2B and 2C). The comparable reduction in infarct size in mice treated with either direct anticoagulant was in agreement with the comparable anticoagulant effects observed using the D₅₀ in the tail-bleeding time analysis and the mesenteric thrombosis model.

Since directly targeting coagulation proteases with DOACs may differentially affect coagulation protease-dependent signaling despite a similar anticoagulant effect, we next conducted unbiased expression analyses (RNAseq). In infarcted heart tissue of mice undergoing myocardial IRI without anticoagulant treatment (control group), the expression of 689 genes was induced and that of 239 genes was reduced compared with those of the hearts of sham-operated mice (Figure 2D and 2E). Of the 689 induced genes and the 239 repressed genes in IRI, fXai treatment resulted in at least 1.5-fold reversal of 519 genes (75.3%) and 182 genes (76.15%), respectively (Figure 2D and 2E). Gene expression in the hearts of fIIai-treated IRI mice was strikingly different from that of fXai-treated IRI mice and more closely followed the changes observed in control IRI hearts (Figure 2D and 2E). Of the 689 induced genes and 239 repressed genes in IRI, fIIai treatment resulted in at least 1.5-fold reversal of 259 genes (37.5%) and 79 genes (33.05%), respectively (Figure 2D and 2E). While genes regulated by fXai and fIIai overlapped significantly (229 upregulated and 69 repressed; Figure 2E), the number of genes specifically reversed by fXai were larger than the number of genes specifically reversed by fIIai (fXai: 290 upregulated and 113 repressed; fIIai: 30 upregulated and 10 repressed, respectively; Figure 2E). Hence, fXai treatment prevented changes in gene expression upon myocardial IRI to a larger extent than fIIai treatment.

We performed functional annotation to study the pathways that contributed to these differential effects. Genes that were induced due to myocardial IRI were mainly involved in cytokine-cytokine receptor interaction, TNF (tumor necrosis factor) signaling, NOD (nucleotide-binding oligomerization domain)-like receptor signaling, Jak-STAT (janus kinase-signal transducer and activator of transcription) signaling, but also chemokine-signaling, phosphatidylinositol signaling, or NF-κB (nuclear factor kappa B) signaling (Figure II in the Data Supplement). Of these IRI-induced and pathway-related genes, fXai treatment prevented the induction of genes related to the cytokine-cytokine receptor interaction, TNF signaling, NOD-like receptor signaling, and NF-κB signaling (Figure III in the Data Supplement). Genes that were suppressed by fIIai treatment were related to pathways involved in aldosterone synthesis and secretion, TGF-β (transforming growth factor beta) signaling, calcium signaling, and phosphatidylinositol signaling (Figure IV in the Data Supplement).

We further performed gene ontology analysis on genes that were induced by myocardial IRI and repressed upon fIIai or fXai treatment. Gene ontology analysis of differentially expressed genes revealed that among genes upregulated due to IRI, the most relevant biological process was involved in inflammatory response, including 49 differential genes assessed (Figure 2F). While genes that were regulated by fXai treatment were predominately involved in biological processes related to inflammatory responses (Figure 2F and 2G), those that were regulated by fIIai treatment were not. Taken together, although fIIai and fXai resulted in comparable infarct sizes, unbiased gene expression analyses revealed marked differences, particularly in pathways related to inflammation and NOD-like receptor signaling (inflammasome) associated genes (Figure 2F and 2G).

To validate the data obtained from the unbiased analyses, we determined the expression levels of sterile inflammation and inflammasome regulators in injured tissue by qRT-PCR (quantitative real time polymerase chain reaction). mRNA expression of IL (interleukin)-6, IL-1β, TNF-α, MIF (macrophage migration inhibitory factor), and MCP-1 (monocyte chemoattractant protein-1) was increased in injured tissue in comparison to sham-operated mice (Figure 3A). The expression of IL-6, IL-1β, TNF-α, and MCP-1 was increased to the same or an even greater extent in fIIai-treated mice, while the expression of mRNA for these cytokines was reduced in fXai-treated mice compared with that of control mice (Figure 3A). Thus, fXai specifically reduced mRNA expression of inflammation-associated genes. Alternative gene expression patterns as identified by RNAseq were likewise confirmed by qRT-PCR, including genes not reduced by either anticoagulant (Pik3r5 [phosphoinositide-3-kinase regulatory subunit 5] and Tlr2 [toll like receptor 2]), regulated by both anticoagulants (Prkcg [protein kinase C gamma]), or specifically regulated by fIIai (Ucp3 [mitochondrial uncoupling protein 3]; Figure V in the Data Supplement). Expression of the proinflammatory cytokines IL-1β, IL-6, TNF-α, and IL-6 is, in part, controlled by NF-κB.³⁸ Congruently, phosphorylation of IκBα (NF-κB inhibitor alpha) and expression of p65 NF-κB were increased in mice with myocardial IRI compared with those of sham-operated mice (Figure 3B and 3C). In fXai-treated but not fIIai-treated mice, IκBα phosphorylation and p65 NF-κB levels were markedly reduced (Figure 3B and 3C). Concurrently, monocyte/macrophage frequency was increased to the same extent in control and fIIai-treated mice, while monocyte/macrophage frequency was markedly lower in fXai-treated mice (Figure VI in the Data Supplement). The blunted inflammatory response in fXai-treated mice was associated with improved VE-cadherin staining (linear staining pattern and higher staining intensity) and less signs of intramural edema upon electron microscopical analyses as compared with fIIai-treated or control mice (Figure VII in the Data Supplement), reflecting improved vascular barrier function post-myocardial IRI in fXai-treated mice. These results establish that fIIai less potently inhibits the inflammatory response associated with myocardial IRI than fXai, despite a comparable anticoagulant effect and a comparable reduction in infarct size.

Activation of the NF-κB pathway drives NLRP3 (NLR family pyrin domain containing 3) inflammasome activation, thus promoting myocardial IRI-associated sterile inflammation.^39,40 Accordingly, both NF-κB and the NLRP3 inflammasome are linked with the severity of myocardial IRI.^27,41 Hence, we next determined whether fIIai and fXai differentially modulate myocardial IRI-associated NLRP3 inflammasome activation. The expression of NLRP3 and cleaved caspase-1 and IL-1β was increased following myocardial IRI compared with that of sham-operated mice (Figure 3D and 3E). fXai but not fIIai markedly reduced NLRP3 expression and cleavage of procaspase-1 and pro-IL-1β (Figure 3D and 3E). Concurrently, plasma levels of the inflammasome-associated cytokines IL-1β and IL-18 were reduced in fXai-treated but not fIIai-treated mice (Figure 3F and 3G; Figure VIII in the Data Supplement). Thus, fXai but not fIIai efficiently restricts inflammasome activation associated with myocardial IRI.

The pronounced difference in regard to gene expression and inflammasome activation upon myocardial IRI in mice pretreated with fIIai or fXai raises the question as to whether anticoagulant treatment preconditions the heart and affects the NLRP3 inflammasome or gene expression already before myocardial IRI and whether the observed changes may be linked with altered epigenetic regulation. Analyses of mice after 1 week of treatment with fIIai or fXai did not result in different expression of selected cytokines and in particular had no impact on the NLRP3 inflammasome (Figure 4A and 4B). Apparently IRI-dependent inflammasome activation is required to uncover differences in inflammasome regulation. However, unbiased expression analyses (RNAseq) revealed altered gene expression (Figure 4C and 4D). Thus, compared with controls, 69 genes were induced by both fIIai and fXai (significant overlap), and a comparable number of genes were specifically induced by fIIai or fXai (23 and 20, respectively; Figure 4D). Additionally, 32 genes were suppressed by both fIIai and fXai (significant overlap), while a low but significantly different number of genes were specifically suppressed by fIIai or fXai (5 and 20 genes, respectively; Figure 4D). Thus, the total number of genes regulated by both anticoagulants or specifically by either anticoagulant was lower in the resting state compared with changes observed post-myocardial IRI. Accordingly, the total number of pathways identified was less as compared with the observations made post-myocardial IRI (Figures IX and X in the Data Supplement). Neither anticoagulant targeted pathways related to cytokine-cytokine receptor interaction or NOD-like receptor signaling, which is in agreement with the above results obtained by qRT-PCR and immunoblotting (Figure 4A and 4B). In addition, we observed differential effects of fXai and fIIai on H3K9me3 (tri-methyl-histone H3 lysin 9), an epigenetic histone H3 modification reflecting heterochromatin, and epigenetic regulators (DNMT1 [DNA methyltransferase 1] and SIRT1; Figure 4E and 4F). Taken together, these results demonstrate that anticoagulation with fXai or fIIai primes or preconditions the myocardium but that additional factors are required for the differential inflammasome regulation during myocardial IRI.

Considering potential mechanisms underlying the observed differences in sterile inflammation and the NLRP3 inflammasome upon treatment with fIIai and fXai, we evaluated the differential regulation of the serine protease aPC.^30,42 As activation of the zymogen protein C depends on the thrombin-TM complex, direct fIIai and fXai may interfere with aPC generation to variable extents. Differential regulation of aPC appeared to be an attractive explanation, as the anti-inflammatory effects of aPC are well established and depend at least, in part, particularly in the context of myocardial IRI, on NLRP3 inflammasome inhibition.²⁷ To determine whether direct fIIai and fXai differentially interfere with protein C activation, we used an established in vivo protein C activation assay.^43,44 Myocardial IRI was induced in mice after 7 days of fIIai or fXai treatment, following the same dosing regimens as above (D₅₀; 10 mg/kg for fIIai and 3 mg/kg for fXai), and in vivo aPC generation was determined after 24 hours. In vivo aPC generation was markedly reduced in mice following myocardial IRI (Figure 5A). In vivo aPC generation was slightly but significantly further reduced in fIIai-treated animals (Figure 5A; P=0.028). Conversely, in vivo aPC generation was increased in fXai-treated mice compared with that of control IRI mice (Figure 5A). These data indicate that direct fIIai interferes with aPC generation, while fXai does not impair and even appears to promote aPC generation in comparison to that of control myocardial IRI mice.

aPC has been shown to induce a gene expression profile that is associated with cytoprotection.^45–47 The increased aPC generation in fXai- and reduced aPC generation in fIIai-treated mice suggests that the observed differences in gene expression reflect aPC-dependent gene regulation. To directly determine whether the observed changes in gene expression reflect, at least in part, aPC-dependent effects, we compared gene expression in fXai- or fIIai-treated mice to that of aPC-treated mice.²⁷ In support of the proposed aPC-dependent gene regulation in fXai-treated mice, changes in gene expression in aPC-treated mice were similar to those observed in fXai-treated mice but were strikingly different from those in fIIai-treated mice (Figure 5B). Thus, of the 689 induced genes and 239 repressed genes upon myocardial IRI, aPC treatment resulted in an at least 1.5-fold reversal of 575 of the induced genes (83.4%) and of 161 of the repressed genes (67.3%). There was a large overlap of genes that were regulated by aPC or fXai treatment: 469 (245+224, 68.06%) of the 689 IRI-induced genes and 131 (72+59, 54.8%) of the 239 IRI-repressed genes were similarly (1.5-fold) reversed by either intervention (Figure 5C). Similar to fXai treatment, aPC treatment in the myocardial IRI mouse model repressed genes that are involved in cytokine-cytokine receptor interactions, NOD-like receptor signaling, pathways related to TNF signaling, NF-κB signaling, and biological processes related to inflammatory responses (Figures III and XI in the Data Supplement).

In contrast, the overlap of genes regulated by fIIai with genes regulated by aPC was less pronounced: of 689 induced genes and 239 suppressed genes in the control group, 246 (224+22, 35.7%) of the IRI-induced genes and 66 (59+7, 27.6%) of the IRI-repressed genes were similarly (1.5-fold) reversed by both aPC and fIIai (Figure 5C). The overlapping pathways regulated by aPC and fIIai treatment were related to HIF-1 signaling and phosphatidylinositol signaling (Figures IV and XI in the Data Supplement). The number of IRI-induced genes normalized by aPC and fXai was larger than the number of genes regulated by aPC and fIIai (P_DI [fXai-fIIai]; Figure 5C). Thus, in the context of myocardial IRI, fXai promotes aPC generation and alters gene expression profiles that mimics the effects of aPC and results in suppression of inflammatory pathways.

We next investigated whether restoring aPC signaling in mice treated with fIIai is sufficient to mimic the cytoprotective effect observed in response to fXai. To this end, mice that were treated only with the fIIai (following the same protocol as above) were compared with fIIai-treated mice that received a signaling-selective aPC mutant (3K3A-aPC: 1 mg/kg, IP, 30 minutes before I/R; controls: PBS, equal volume, IP). 3K3A-aPC is an aPC variant that is largely devoid of its anticoagulant function but retains the cytoprotective properties of aPC.^27,34,35 3K3A-aPC on top of fIIai markedly reduced infarct size (Figure 5D and 5E). The proinflammatory effect of fIIai on cytokine expression was lost upon concomitant 3K3A-aPC treatment (Figure XIIA and XIIB in the Data Supplement). Furthermore, 3K3A-aPC treatment in addition to fIIai restricted NLRP3 expression, cleavage of procaspase-1 and pro-IL-1β (Figure 5H; Figure XIIA and XIIC in the Data Supplement), and plasma levels of NLRP3 inflammasome–associated cytokines (IL-1β and IL-18) following myocardial IRI (Figure 5F and 5G). Hence, the unfavorable proinflammatory profile following myocardial IRI and pretreatment with fIIai can be compensated for by restoring aPC signaling.

To determine whether the beneficial anti-inflammatory effects of fXai depend on increased generation of endogenous aPC, we used inhibitor antibodies either to completely block all aPC activities using the mAb MPC1609 or selectively block the anticoagulant activity of aPC while retaining its cytoprotective activities using the mAb MAPC1591.³² Mice were treated for 1 week with fXai (3 mg/kg) and randomly assigned to injections with either MPC1609, MAPC1591 (each 10 mg/kg, IP, every second day), or PBS (control: equal volume, IP). Myocardial IRI was induced, and the mice were analyzed after 24 hours as described above (Figure 6A). Again, fXai markedly reduced infarct size (Figure 6B and 6C) and mRNA expression of IL-6, TNF-α, MIF, and MCP-1 in comparison to those of control IRI mice (Figure 6D through 6G). The protective effect of fXai on infarct size and cytokine expression was lost upon concomitant MPC1609 treatment, whereas MAPC1591 treatment did not impede the protective function of fXai (Figure 6B through 6G). Furthermore, fXai-mediated inhibition of the NLRP3 inflammasome following myocardial IRI was lost upon MPC1609 but not MAPC1591 treatment, indicating that fXai-mediated inflammasome inhibition is independent of aPC-mediated coagulation inhibition but depends on aPC signaling (Figure 7). Taken together, these data demonstrate that fXai does not interfere with aPC generation or with cytoprotective signaling by aPC following myocardial IRI, while fIIa inhibition by fIIai abolishes aPC-dependent protective effects.

Failure to resolve inflammation following myocardial IRI facilitates tissue destruction, ultimately leading to replacement of the myocardium with extracellular matrix.⁴⁸ Considering the differential effect of fIIai and fXai on myocardial IRI-associated inflammation, we hypothesized that these anticoagulant strategies differentially affect cardiac fibrosis after myocardial infarction. We evaluated cardiac fibrosis 28 days post-myocardial IRI in control mice and mice that were treated with fIIai or fXai (Figure 8A; Figure XIII in the Data Supplement). Masson trichrome staining revealed marked cardiac fibrosis post-myocardial IRI in control IRI mice compared with sham-operated mice (Figure 8B and 8C; Figure XIV in the Data Supplement). Concomitantly, mRNA expression of fibrosis-related genes (collagen 1α1, collagen 3α1, α-SMA [alpha smooth muscle actin], and CTGF [connective tissue growth factor]) was markedly increased 28 days post-myocardial IRI in control mice (Figure 8D through 8G). Thus, despite comparable infarct sizes 24 hours after myocardial IRI (Figure 2), the extent of fibrosis 28 days after myocardial IRI, as reflected by Masson trichrome staining and mRNA expression of fibrosis-related genes, was markedly reduced in fXai-treated but not fIIai-treated mice (Figure 8B through 8G).