Vascular miR-181b controls tissue factor-dependent thrombogenicity and inflammation in type 2 diabetes

Background Diabetes mellitus is characterized by chronic vascular inflammation leading to pathological expression of the thrombogenic full length (fl) tissue factor (TF) and its isoform alternatively-spliced (as) TF. Blood-borne TF promotes factor (F) Xa generation resulting in a pro-thrombotic state and cardiovascular complications. MicroRNA (miR)s impact gene expression on the post-transcriptional level and contribute to vascular homeostasis. Their distinct role in the control of the diabetes-related procoagulant state remains poorly understood. Methods In a cohort of patients with poorly controlled type 2 diabetes (n = 46) plasma levels of miR-181b were correlated with TF pathway activity and markers for vascular inflammation. In vitro, human microvascular endothelial cells (HMEC)-1 and human monocytes (THP-1) were transfected with miR-181b or anti-miR-181b and exposed to tumor necrosis factor (TNF) α or lipopolysaccharides (LPS). Expression of TF isoforms, vascular adhesion molecule (VCAM) 1 and nuclear factor (NF) κB nuclear translocation was assessed. Moreover, aortas, spleen, plasma, and bone marrow-derived macrophage (BMDM)s of mice carrying a deletion of the first miR-181b locus were analyzed with respect to TF expression and activity. Results In patients with type 2 diabetes, plasma miR-181b negatively correlated with the procoagulant state as evidenced by TF protein, TF activity, d-dimer levels as well as markers for vascular inflammation. In HMEC-1, miR-181b abrogated TNFα-induced expression of flTF, asTF, and VCAM1. These results were validated using the anti-miR-181b. Mechanistically, we confirmed a miR-181b-mediated inhibition of importin-α3 (KPNA4) leading to reduced nuclear translocation of the TF transcription factor NFκB. In THP-1, miR-181b reduced both TF isoforms and FXa generation in response to LPS due to targeting phosphatase and tensin homolog (PTEN), a principal inducer for TF in monocytes. Moreover, in miR-181−/− animals, we found that reduced levels of miR-181b were accompanied by increased TF, VCAM1, and KPNA4 expression in aortic tissue as well as increased TF and PTEN expression in spleen. Finally, BMDMs of miR-181−/− mice showed increased TF expression and FXa generation upon stimulation with LPS. Conclusions miR-181b epigenetically controls the procoagulant state in diabetes. Reduced miR-181b levels contribute to increased thrombogenicity and may help to identify individuals at particular risk for thrombosis.


Background
Under diabetic conditions, chronic inflammatory signaling in the vasculature sustains endothelial dysfunction, leukocyte infiltration, and a pro-thrombotic environment [1,2].
Tissue factor (TF), the membrane-bound receptor for coagulation factor (F) VIIa, is constitutively expressed in the perivascular space but under physiological conditions not present in the vasculature or blood [3]. However, hyperglycemia and pro-inflammatory molecules, such as tumor necrosis factor (TNF)α or lipopolysaccharides (LPS), induce the highly thrombogenic full length (fl) TF and its less thrombogenic isoform alternativelyspliced (as) TF [4][5][6]. Vessel wall-derived flTF mainly drives inflammatory signaling via protease-activated receptor (PAR)s [7][8][9], can lead to vascular dysfunction under oxidative stress [10], and may contribute to atherothrombosis through release in TF-positive procoagulant microvesicles (MVs) [11,12]. In the blood, hematopoietic cells, including monocytes and macrophages, and their released procoagulant MVs represent the main source of TF [13,14]. When in contact with blood, flTF triggers the FVIIa-dependent generation of FXa prompting thrombin accumulation and clotting [15]. Particularly, patients with poorly controlled diabetes show pathological TF activity in the blood accounting for vascular complications [16].
Recently, a critical role of microRNA (miR)s in the pathogenesis of cardiovascular diseases (CVD) and diabetes-related complications has been demonstrated [17][18][19]. Binding of specific target transcripts by miRs leads to mRNA degradation and translational repression. We have recently shown that miR-126 and miR-19a target the TF transcript thereby contributing to the epigenetic control of vascular inflammation and coagulation [20,21]. Thus, a procoagulant state in diabetes can also be considered a result of a defective miR-mediated control of coagulation factors.
Among endothelial miRs, miR-181b emerged as a powerful regulator of vascular homeostasis. Several lines of evidence point to implication of miR-181b into TF-related thrombogenicity: In endothelial cells (ECs) in vitro, miR-181b targets importin-α3 (KPNA4) and thereby reduces nuclear translocation of nuclear factor (NF) κB, one of the main transcription factors for vascular TF expression [22]. Moreover, Henao Mejia et al. found that miR-181b targets phosphatase and tensin homolog (PTEN), a principal inducer for TF expression in hematopoietic cells [23]. In monocytes, PTEN drives LPS-induced TF expression by negatively regulating the phosphoinositide 3-kinase (PI3K)/AKT pathway. Accordingly, PTEN−/− mice exhibit reduced TF activity [24]. In vivo, Lin et al. reported that miR-181b administration reduced arterial thrombosis in a photochemical injury-model [25], a feature that is also observed in mice with reduced vascular TF expression [26,27]. Finally, miR-181b expression is lowered via hyperglycemia and pro-inflammatory cytokines that are all known to induce TF expression in the vasculature [28].
In the present study, we sought to assess the role of miR-181b in the hemostatic balance in advanced diabetes.
In a cohort of patients with poorly controlled type 2 diabetes, we provide novel evidence that miR-181b expression strongly correlates with reduced activation of the TF-pathway assessed by TF protein, TF activity, and d-dimer levels as well as reduced vascular inflammation. In vitro, TF expression and FXa generation was reduced by miR-181b in human ECs and monocytes. Using animals with a deletion of the first miR-181b locus we provide in vivo evidence that miR-181b contributes to the control of vascular TF activity derived from the endothelium and hematopoietic cells. Our data suggests that the epigenetic control by circulating miR-181b is essential to ensure the hemostatic balance by reducing inflammation-driven vascular TF expression.

Patient study
The study protocol was approved by the local ethics committee and was performed in accordance to the ethics principles in the Declaration of Helsinki. Prior to participation in the study, each patient gave a written informed consent. 46 patients with known diabetes mellitus type 2 admitted for poor glycemic control at the Diabetes Center NRW Bad Oeynhausen, Germany, were included in the study. Table 1 shows the patient characteristics.
free conditions in the central animal facility at the Institute for Molecular Medicine in Frankfurt, Germany.

ELISA experiments and glucose measurements
The protein plasma concentrations were assessed by using specific ELISA systems (Table 2) according to the manufacturer's instructions. Fasting blood glucose levels in patients were measured by a standardized laboratory test, while further glucose levels during the day were obtained by a finger-prick test from capillary blood.

TF activity
The measurement of TF activity was performed as described before [2]. The recombinant FVIIa was kindly provided by Novo Nordisk.

Transfection and stimulation experiments
HMEC-1 and THP-1 were transfected with 200 nM of the oligonucleotides listed in Table 2

Real time PCR
For real-time PCR, total mRNA was isolated from cultured cells and murine tissue with peqGOLD Trifast (Peqlab) and for patient blood plasma using the mirVana Paris Kit (Life Technologies). Gene expression was determined using GoTaq ® Probe qPCR Master Mix (Promega) with FAM-tagged TaqMan ® gene expression assays (life Technologies) or via SYBR Green assays using SYBR ® Select Master Mix (applied Biosystems) with specific primers ( Table 2). Relative gene expression was determined via the comparative C(t) (ΔΔCt) method with GAPDH or 18S ribosomal RNA as endogenous control for mRNA and U6 snRNA for miR.

Western blot
Western blots were performed as described before [3]. Nuclear and cellular lysates were isolated using the NE-PER ™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) following the manufacturer's instructions. Antibodies used are listed in Table 2. The western blots have been performed ≥ 3 times.

Immunofluorescence
For protein staining aortas and spleen were embedded in Tissue-Tek and immunofluorescence was performed with specific antibodies and probes (Table 2).

Whole blood microvesicle-associated FXa generation
TF activity of whole blood mouse MVs was assessed as previously described [4]. Briefly, whole blood was drawn

Culture of bone marrow-derived macrophages (BMDMs)
BMDMs were in vitro-differentiated as described before [5]. Briefly, isolated bone marrow cells were cultivated in DMEM supplemented with 20% L cell medium, 10% FCS, 1 mM l-glutamine, penicillin, and streptomycin. On day 7 macrophages were seeded at a density of 1 × 10 6 per well. On the next day, macrophages were stimulated with 1 µg/mL LPS (Enzo Life Sciences) for 4 h. The cells were then lysed for RNA analysis, western blot or measurement of FXa generation as described above.

Statistical analysis
The statistical analyses were performed using the software GraphPad Prism 7. Correlation of patient characteristics were performed via Pearson correlation for normally distributed values or Spearman correlation in case of not normal distribution. For binary variables, a point-biserial correlation was performed. Differences between 2 groups were examined using a Mann-Whitney test or student's t test (2-tailed

Results
We hypothesized an association of miR-181b with vascular TF expression in the setting of diabetes. . No association of miR-181b with blood glucose levels was observed. However, circulating miR-181b showed a strong negative correlation with TF protein (Fig. 1a, r = − 0.48, p < 0.001) and TF activity (Fig. 1b, r = − 0.82, p < 0.0001). Moreover, miR-181b correlated with reduced levels of d-dimers of the TF pathway (r = − 0.34, p < 0.05, Table 1). Furthermore, miR-181b was related to a lower grade of inflammation as evidenced by a negative correlation with fibrinogen and leukocyte count (Fig. 1c, d). In line with this, miR-181b was positively associated with metformin treatment that is known to reduce vascular inflammation (r = 0.462, p < 0.001, Table 1). Plasma miR-181b also correlated with the antithrombotic miR-126 and miR-19a (r = 0.79, p < 0.0001 and r = 0.73, p < 0.0001, respectively, Fig. 1e, f ).
In human microvascular endothelial cells (HMEC)-1, hyperglycemia and the pro-inflammatory cytokine TNFα reduced miR-181b levels, whereas it induced both TF isoforms and the NFκB-dependent vascular cell adhesion molecule (VCAM) 1 (Additional file 2: Figure S1A-H). We analyzed the effect of miR-181b on the inflammationinduced TF expression in ECs. HMEC-1 were therefore transfected with miR-181b or anti-miR-181b as well as the appropriate controls and stimulated with 10 ng/ mL TNFα. In miR-181b transfected cells, we observed a reduction of both TF isoforms on the mRNA and protein level in cells stimulated with TNFα for 2 h and 6 h, respectively, whereas anti-miR-181b increased asTF and flTF expression under inflammatory conditions (Fig. 2ae). We confirmed that miR-181b reduces and anti-miR-181b induces VCAM1 in HMEC-1 (Fig. 2f-h). As the mechanism for inhibition of TNFα-induced TF expression we found a downregulation of KPNA4 in HMEC-1 mRNA and protein (Additional file 3: Figure S2A, B) consistent with a decrease in NFκB nuclear translocation exhibited by miR-181b (Fig. 2i).
To assess whether miR-181b also reduces TF expression in human monocytes, the main source of TF activity in the blood, THP-1 cells were transfected with miR-181b and then stimulated with LPS to induce TF expression. Due to the low endogenous expression level of miR-181b in monocytes, a gain of function approach was used. We found that miR-181b reduced mRNA levels of both TF isoforms (Fig. 3a, b) and FXa generation (Fig. 3c) following LPS treatment of THP-1. In contrast to ECs, LPS-induced NFκB was not restricted to the cytoplasmatic compartment in THP-1 (Fig. 3d, upper panel) nor were KPNA4 mRNA levels reduced by miR-181b (Fig. 3e). However, miR-181b led to a marked reduction in NFκB phosphorylation and subsequent nuclear translocation (Fig. 3d, both panels) suggesting an upstream effect on NFκB activation. We suspected that control of PTEN expression may contribute to the miR-181b-mediated reduction of TF activity in monocytes. Indeed, PTEN expression was reduced in THP-1 cells transfected with miR-181b (Fig. 3f, g). siRNAmediated knock down of PTEN reduced LPS-induced expression of flTF and asTF in THP-1 and phenocopied the miR-181b effect (Fig. 3h-j).
To assess the significance of our experimental findings in vivo, animals carrying a deletion of the first miR-181b locus (as well as miR-181a-1; B6.Mirc14 tm1.1Ankr , here referred to as miR-181−/− mice) with reduced vascular miR-181b expression were analyzed (Fig. 4a). While no change for asTF mRNA was observed, we found an increase in flTF mRNA and protein expression in miR-181−/− aortas as compared to wt mice, (Fig. 4b-d). Immunofluorescence using a murine TF-specific antibody revealed that the rise in TF protein in miR-181−/− aortas originated from the endothelial layer (Fig. 4e,  upper panel). Moreover, expression of KPNA4 (Fig. 4f ) as well as VCAM1 mRNA and protein (Fig. 4g, h, e lower panel) were increased in aortas of miR-181−/− animals. To assess TF expression in hematopoietic cells, the spleen of miR-181−/− mice was analyzed. Here, miR-181b was reduced (Additional file 5: Figure S4a) while both TF isoforms and PTEN mRNA were increased as compared to wt animals ( Fig. 4i-m).
To investigate the blood procoagulant activity of miR-181b−/− animals, whole blood was stimulated with LPS and the FXa generation of MVs assessed. We did not observe a difference in MV-associated FXa generation between blood of wt and ko animals (Additional file 5: Figure S4B). In line, no differences in plasma thrombin anti-thrombin complexes was seen between wt and ko animals (Additional file 5: Figure S4C). However, isolated bone marrow-derived macrophages (BMDM), that have been shown to contribute to blood thrombogenicity, showed a strong reduction in miR-181b expression in the miR-181−/− animals (Fig. 5a) and exhibited higher levels of asTF and flTF mRNA (Fig. 5b, c) as well as more TF protein and FXa generation (Fig. 4d, e) in response to LPS than wt cells.

Discussion
Here, we report that miR-181b controls thrombogenicity in the vasculature and correlates with reduced circulating TF and vascular inflammation in patients with diabetes. Using human ECs and monocytic cells, we demonstrate that miR-181b reduces the inflammation-induced vascular TF expression and FXa generation. In a murine miR-181 ko system, we provide in vivo evidence that loss of miR-181b promotes endothelial and hematopoietic TF expression. Our work demonstrates that miR-181b is a Moreover, miR-181b expression was related to lower levels of the pro-inflammatory fibrinogen (c) and lower leukocyte counts (d). We also observed a correlation of miR-181b with the TF-specific miR-126 (e) and miR-19a (f). n = 46. Upon normality testing a Spearman or Pearson correlation was performed, r-values and p-values are indicated pivotal regulator of thrombogenicity and contributes to vascular homeostasis in the presence of poorly controlled diabetes.

Low miR-181b: association with a pro-thrombotic and pro-inflammatory state
In the healthy vasculature, circulating miRs epigenetically control pro-inflammatory transcription factors and their gene products to preserve vascular homeostasis [3,[30][31][32]. However, in a diabetic environment this protective miR transcriptome is disrupted [33]. In particular, diabetes-related loss of TF-specific miRs, such as miR-126 or miR-223 contributes to vascular inflammation and a pro-thrombotic state [33,34].
Here we show that miR-181b, a key regulator for endothelial function, beta cell function, peripheral insulin sensitivity, and NFκB signaling [22,28,35], participates in the control of vascular TF activity in diabetes. In poorly controlled diabetics, miR-181b was associated with reduced activation of the TF pathway, lower leukocyte count, and fibrinogen, which both constitute independent risk factors for cardiovascular mortality among patients with diabetes [36].
In contrast to markers of inflammation, miR-181b levels did not correlate with blood glucose or HbA1c in our cohort. Although we and Sun et al. [28] found that miR-181b expression in HMEC-1 and human umbilical vein endothelial cells (HUVEC) is reduced by high glucose concentrations, Yang et al. [37] reported activation of the miR-181a/b-specific transcription factor signal transducer and activator of transcription (STAT) 3 in HUVEC treated with sera from diabetics pointing to a more complex regulation of miR-181b expression in humans. This may explain why the miR-181b expression in our healthy controls did not differ from those in poorly controlled diabetes (Additional file 6: Figure S5A). In contrast, miR-126 that has been shown to be reduced in diabetes in the population-based Bruneck cohort, was significantly lower in our diabetes cohort compared to healthy controls confirming the quality of our measurements (Additional file 6: Figure S5B). We assume that diabetic conditions beyond glycemia impact miR-181b expression resulting in differential expression levels among diabetics. Although we cannot rule out an impact of co morbidities and anti-diabetic drugs on miR-181b expression in our cohort, it seems likely that chronic inflammation in an advanced state of the disease is a more relevant factor than blood glucose for miR-181b suppression. In accordance, we found co expression of miR-181b with miR-126 and miR-19a that are both reduced by inflammatory cytokines [20,21].

TF in the endothelium is reduced by miR-181b under inflammatory conditions
Our data indicates that miR-181b restrains NFκBdepending TF expression in human and murine ECs via targeting of KPNA4. The direct contribution of vessel wall-derived TF to thrombosis has been a matter of debate [4]. Yet, through allosteric activation of FVIIa and/ or FXa generation, TF promotes pro-inflammatory PAR signaling leading to sustained vascular dysfunction. Consequently, an EC-specific deletion of TF reduced circulating inflammatory cytokines [38]. Recently, clinical trials provided strong evidence that the TF/VIIa/Xa pathway contributes to vascular complications and pharmacological blockage of FXa at low doses prevented ischemic CVD endpoints [39]. However, inhibition of vascular TF and PARs are challenged by bleeding issues due to their role in hemostasis and platelet function, respectively [40].
Our study identifies miR-181b as a potent regulator for pathological TF-induced vascular inflammation. Further clinical research needs to assess the therapeutic potential of miR-181b in the setting of diabetes and CVD.  Figure S3). KPNA4 mRNA (e), PTEN mRNA (f) and protein (g) in THP-1 transfected with a control miR or miR-181b. THP-1 were transfected with a control siRNA or siRNA against PTEN. PTEN knock down was confirmed via quantification of PTEN mRNA (h) and led to reduced mRNA levels of flTF (i) and asTF (j). n ≥ 3, groups were compared by 2-way ANOVA test with global p-values for treatment with LPS and Sidak's multiple comparison post hoc test (a, b) or student's t-test

Endothelial miR-181b governs hematopoietic TF activity via PTEN
In the vasculature, hematopoietic cells represent the main pool of circulating TF and contribute to thrombosis [13,26]. Here, we show that miR-181b reduces TF activity in human monocytes through control of PTEN. The strong negative correlation of circulating miR-181b with plasma TF activity in our patient cohort suggests that the mostly endothelial-derived miR-181b impacts monocytic TF production in humans. Recent studies reported that ECs can modulate the phenotype of monocytes via release of endothelial MVs. Njock et al. [41] showed that miR-181b along with other anti-inflammatory miRs is enriched in EC-derived MVs. Treatment of recipient THP-1 with EC-MVs or with miR-181b mimics repressed LPS-induced inflammatory gene expression in monocytes. These data along with our study highlight that endothelial miR-181b exerts anti-thrombotic properties beyond the vasculature by also modulating the thrombotic phenotype of monocytic cells and support the hypothesis that the endothelium at least indirectly participates in the regulation of plasma TF activity.
In contrast to ECs, miR-181b did not alter NFκB nuclear translocation in human monocytes. Different host cell factors that preclude down regulation of KPNA4 may explain this observation. However, we are the first to show that miR-181b controls upstream NFκB activation in monocytes via targeting PTEN. The PI3K/AKT pathway was previously shown to negatively regulate LPSinduced TF transcription in human monocytes through downstream control of NFκB along with the TF-specific transcription factors activator protein (AP)-1 and early growth response (EGR)-1 [42]. Consequently, targeting of the PI3K/AKT negative regulator PTEN by miR-181b reduces LPS-induced TF expression and possibly other inflammatory factors.

Differences of miR-181b's effect in the murine system
In the murine system, miR-181b deficiency failed to alter monocyte-derived MV-TF activity but increased TF expression in spleen and BMDM.
In line with previous reports showing that miR-181b does not alter NFκB reporter activity of mouse peripheral blood mononuclear cell (PBMC)s [32], we did not observe changes in MV-associated monocytic TF activity or TAT levels. As suggested by Sun et al., differential expression of importin isoforms in mouse monocytes may be the underlying cause. However, our study shows that miR-181b reduces hematopoietic TF production in murine BMDM. Macrophages not only produce TF-positive thromboinflammatory MVs [14] but also account for TF accumulation in atherosclerotic plaques [43]. Reduced TF expression in macrophages may contribute to the observation that miR-181b overexpression in Apo E−/− mice protects from atherosclerosis [32].
Limitations miR expression in patient plasma samples was normalized to U6 which can be altered in some diseases. However, this has not been reported in patients with diabetes and our expression profiles in both cohorts are in accordance with previously published data [34]. Moreover, the miR-181−/− mouse model analyzed here carries a deletion of miR-181b-1 and the adjacent miR-181a-1 as well. Due to transcriptional co-regulation and high sequence similarities both miRs share largely the same functions. Accordingly, some of our findings might also be explained by loss of miR-181a.

Conclusions
Our work shows that circulating miR-181b contributes to the control of diabetes-related thrombogenicity and vascular inflammation (Fig. 6). Low miR-181b expression may identify patients at particular risk for thromboembolic complications. Vice versa, posttranscriptional control of vascular TF expression by miR-181b represents a potential avenue to improve endothelial dysfunction and thrombosis risk without facing bleeding issues as observed by direct inhibition of inflammatory coagulation factors. Further clinical trials are warranted to