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Toll-like receptor 2-deficiency on bone marrow-derived cells augments vascular healing of murine arterial lesions
Life Sciences 242 (2020) 117189
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Toll-like receptor 2-deficiency on bone marrow-derived cells augments vascular healing of murine arterial lesions☆ W. Liua, J.-C. Eczkob, M. Ottoa, R. Bajoratb, B. Vollmarc, J.-P. Roesnerb, N.-M. Wagnera,
T
⁎
a
Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Münster, Münster, Germany Department of Anesthesia and Intensive Care, University Medical Center Rostock, Rostock, Germany c Institute for Experimental Surgery, University Medical Center Rostock, Rostock, Germany b
A R T I C LE I N FO
A B S T R A C T
Keywords: Toll-like receptor 2 TLR2 Arterial injury Restenosis Neointima Smooth muscle cells
Aims: Neointimal hyperplasia contributes to arterial restenosis after percutaneous transluminal coronary angioplasty or vascular surgery. Neointimal thickening after arterial injury is determined by inflammatory processes. We investigated the role of the innate immune receptor toll-like receptor 2 (TLR2) in neointima formation after arterial injury in mice. Materials and methods: Carotid artery injury was induced by 10% ferric chloride in C57Bl/6J wild type (WT), TLR2 deficient (B6.129-Tlr2tm1Kir/J, TLR2−/−) and WT mice treated with a TLR2 blocking antibody. 21 days after injury, carotid arteries were assessed histomorphometrically and for smooth muscle cell (SMC) content. To identify the contribution of circulating cells in mediating the effects of TLR2-deficiency, arterial injury was induced in WT/TLR2−/−-chimeric mice and the paracrine modulation of bone marrow-derived cells from WT and TLR2−/− on SMC migration compared in vitro. Key findings: TLR2−/− mice and WT mice treated with TLR2 blocking antibodies exhibited reduced neointimal thickening (23.7 ± 4.2 and 6.5 ± 3.0 vs. 43.1 ± 5.9 μm, P < 0.05 and P < 0.01), neointimal area (5491 ± 1152 and 315 ± 76.7 vs. 13,756 ± 2627 μm2, P < 0.05 and P < 0.01) and less luminal stenosis compared to WT mice (8.5 ± 1.6 and 5.0 ± 1.3 vs. 22.4 ± 2.2%, both P < 0.001n = 4–8 mice/group). The phenotypes of TLR2−/− vs. WT mice were completely reverted in WT/TLR2−/− bone marrow chimeric mice (5.9 ± 1.5 μm neointimal thickness, 874.2 ± 290.2 μm2 neointima area and 2.7 ± 0.6% luminal stenoses in WT mice transplanted with TLR2−/− bone marrow vs. 23.6 ± 5.1 μm, 3555 ± 511 μm2 and 12.0 ± 1.3% in WT mice receiving WT bone marrow, all P < 0.05, n = 6/group). Neointimal lesions of WT and WT mice transplanted with TLR2−/− bone marrow chimeric mice showed increased numbers of SMC (10.8 ± 1.4 and 12.6 ± 1.4 vs. 3.8 ± 0.9 in TLR2−/− and 3.5 ± 1.1 cells in WT mice transplanted with TLR2−/− bone marrow, all P < 0.05, n = 6). WT bone marrow cells stimulated SMC migration more than TLR2-deficient bone marrow cells (1.7 ± 0.05 vs. 1.3 ± 0.06-fold, P < 0.05, n = 7) and this effect was aggravated by TLR2 stimulation and diminished by TLR2 blockade (1.1 ± 0.03-fold after stimulation with TLR2 agonists and 0.8 ± 0.02-fold after TLR2 blockade vs. control treated cells defined as 1.0, P < 0.05, n = 7). Significance: TLR2-deficiency on hematopoietic but not vessel wall resident cells augments vascular healing after arterial injury. Pharmacological blockade of TLR2 may thus be a promising therapeutic option to improve vessel patency after iatrogenic arterial injury.
1. Introduction
endarterectomy or angioplasty. Maladaptive regenerative processes in arterial lesions after injury can endanger outcome of patients after interventional or surgical vascular procedures and can result in restenosis or in-stent stenosis that reduce vessel patency due to vessel and graft
Iatrogenic arterial injury frequently occurs during percutaneous transluminal coronary angioplasty (PTCA), coronary bypass surgery,
☆ Statement of authorship: All authors take responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation. ⁎ Corresponding author at: Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Münster, Albert-Schweitzer-Campus 1, 48161 Münster, Germany. E-mail address: [email protected] (N.-M. Wagner).
https://doi.org/10.1016/j.lfs.2019.117189 Received 12 November 2019; Received in revised form 12 December 2019; Accepted 16 December 2019 Available online 28 December 2019 0024-3205/ © 2019 Elsevier Inc. All rights reserved.
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The background of the mice was confirmed by genotyping, and age matched mouse groups were used for the experiments.
occlusion. Thus, a detailed understanding of the pathophysiology of impaired healing of arterial lesions is of particular importance and therapeutic options to optimize outcome after vascular surgery are urgently needed. Neointimal hyperplasia is an integral part of maladaptive arterial regeneration and is characterized by the invasion of smooth muscle cells (SMCs) into the intimal layer of the vessel wall. SMCs make up for the majority of the neointima constituting cells and the degree of SMC mobilization determines scar formation and thus the degree of luminal stenosis after injury [1]. Although it has been controversially discussed whether intimal SMCs arise from differentiating circulating progenitor cells [2] or from resident medial SMCs or adventitial fibroblasts [3,4], the paradigm that the initial SMC mobilization into neointimal lesions after injury is driven by inflammatory processes has been widely accepted [5]. Toll-like receptors (TLRs) are innate immune receptors that respond to and integrate a variety of inflammatory processes. In addition to exogenous pathogen associated molecular patterns (PAMPs), TLRs recognize endogenous mediators termed “danger associated molecular patterns” (DAMPs) that are liberated upon tissue injury [6]. TLR2 is expressed on cells of the vessel wall including smooth muscle cells [7] and endothelial cells [8] and shows ubiquitous expression in the vascular tree [9]. In addition, TLR2 is found on circulating bone marrowderived cells (BMCs) of myeloid origin, making TLR2 a potential modulator of both vessel wall inherent remodeling processes as well as a potential mediator of BMC allocation to sites of arterial injury [10]. In the development of murine and human atherosclerosis, loss of TLR2 expression in the vessel wall is associated with protective effects against disease progression [11–13]. In turn, activation of TLR2 by synthetic ligands can increase disease burden in atherosclerosis susceptible mice and this is mediated by TLR2 expressed on BMCs only [12]. TLR2 agonists are naturally liberated upon tissue injury, suggesting that TLR2 activation on BMCs can modulate the inflammation-driven responses to arterial injury. Indeed, it has previously been suggested that TLR2 genetic deficiency confers protection against injury-induced arterial occlusion [14]. However, the role of TLR2 and the cellular localization of its expression in arterial remodeling after arterial injury remains unknown and a potential therapeutic importance of antagonizing TLR2 for improving arterial lesion healing unevaluated. In this investigation, we aimed to identify the contribution of TLR2 expressed on local cells of the vessel wall vs. TLR2 on BMCs to functional regeneration of arterial lesions after chemical injury. We further dissected the role of TLR2 presence on circulating BMCs for SMC migratory capacity and for the first time tested the application of a TLR2blocking antibody to augment arterial regeneration after injury in mice.
2.2. Ferric-chloride-induced arterial injury Mice were anesthetized by intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine and placed on a heating pat in the supine position. After removal of hair and careful disinfection, the left carotid artery was dissected free and a silicon peace slid under the vessel. Arterial injury was induced by subjecting the vessel to 10% ferric chloride (Sigma Aldrich, Germany) soaked in a 2 × 2 mm filter paper piece for 3 min. Ferric chloride was removed and the skin was sutured using 0.7 Ethicon sutures. Three weeks after injury, mice were sacrificed and arterial segments were harvested in 4% formalin and subjected to histological analysis. 2.3. Histologic analysis Immunohistochemistry was performed on paraffin-embedded carotid artery sections. For quantitative morphometric analysis, carotid arteries were stained with hematoxylin/eosin. The neointima and media area, the intima/media ratio, and the degree of luminal stenosis were determined using ImagePro Plus software (Media Cybernetics, United Kingdom). Five sections equally spaced throughout the injured arterial segment (at 200-um intervals) were evaluated, and the results were averaged for each animal. The presence of smooth muscle cells was assessed using a rabbit polyclonal anti-mouse α-actin antibody (Dako, Denmark). 2.4. Irradiation and bone marrow transplantation
2. Methods
For bone marrow transplantation experiments prior to arterial injury, mice were irradiated twice daily (BiD) using a dose of 5 Gy applied each at an 4 h interval from a linear accelerator (VARIAN, Palo Alto, CA, USA). 24 h later, mice were transplanted with unfractionated whole bone marrow using 1 × 108 freshly isolated bone marrow-derived cells in a volume of 150 μL sterile PBS applied via tail vein injection. Wild type mice received either bone marrow from WT mice (WT ➔ WT, serving as controls) or TLR2−/− mice (TLR2−/− ➔ WT) while TLR2−/− mice received either bone marrow from TLR2−/− mice (TLR2−/− ➔ TLR2−/−, serving as controls) or WT mice (WT ➔ TLR2−/−). Mice were housed in isolation containers with filtered air for the following 4 weeks and inspected and weighed daily. After 4 weeks of recovery, mice were subjected to arterial injury. Genotype of circulating cells was confirmed by qPCR on the day of sacrifice (3 weeks after lesion induction, Suppl. Fig. 1).
2.1. Experimental animals
2.5. Isolation of CD117 (c-Kit)-positive cells from bone marrow
TLR2 knockout (B6.129-Tlr2tm1Kir/J, TLR2−/−, n = 30) mice were purchased from Jackson Laboratories. Together with C57Bl/6J mice (n = 35) serving as controls, mice were housed in a pathogen-free facility with a 12/12 light and dark cycle and were given free access to water and standard rodent chow. Murine genotypes were verified by qPCR (Suppl. Fig. 1). A subset of WT mice was treated with 2.5 mg/kg bodyweight of a monoclonal antibody against murine TLR2 (clone 2.5, HycultBiotech, Wayne, USA). The antibody was dissolved in 200 μL of sterile saline and applied via left ventricular injection on POD 2 and 4 after arterial lesion induction. All animal procedures were in accordance with institutional, national and European guidelines for the care and use of laboratory animals (European Communities Council Directive of November 24th, 1986 [86/609/EEC]). The protocol was approved by the Ethical Committee of the Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei, MecklenburgVorpommern, Germany (permit number LALLF M-V/TSD/7221.3-1.1072/12). All efforts were made to minimize suffering of the animals.
c-Kit positive cells were isolated form 8–10 week old wild type or TLR2-deficient mice as described previously [15]. Murine bone marrow was isolated from femur and tibial bone and cell suspensions were incubated with magnetic microbeads coated with anti-c-Kit monoclonal antibody (Miltenyi Biotec, Bergisch Gladbach, Germany). MS columns and the MiniMacs cell separator system were employed to obtain c-Kitpositive cell fractions. 2.6. Smooth muscle cell migration assay Human aortic smooth muscle cells were purchased from PromoCell (Heidelberg, Germany) and cultured at 37 °C in DMEM supplemented with 10% FBS, 2 mmol/L L-glutamine, 100 U mL−1 penicillin, and 100 μg/mL streptomycin, in a 5% CO2 incubator. To assess their migratory activity, 1 × 104 smooth muscle cells were seeded in 24-well plates and incubated for 24 h. From confluent cells, the culture medium was removed, 2 mL fresh culture medium were 2
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Transplantation of WT bone marrow into WT mice or bone marrow of TLR2−/− mice into TLR2−/− mice served as control. The genotype of circulating cells was verified by Northern blot by the time of carotid artery harvest (Suppl. Fig. 1). All mice quickly recovered following lethal irradiation and transplantation and exhibited no more than ~10% initial reduction in body weight followed by a rapid increase in body weight. Reconstitution of bone marrow with WT bone marrow cells in TLR2−/− mice and vice versa completely reversed previous findings: transplantation of TLR2-deficient bone marrow into WT mice rescued the impaired arterial regeneration in WT mice by resulting in reduced neointima thickness (5.9 ± 1.5 μm in WT mice transplanted with TLR2−/− bone marrow vs. 23.7 ± 5.2 μm after transplantation of WT bone marrow into WT mice, P < 0.05, n = 6 animals/group), while transplantation of WT bone marrow into TLR2−/− mice resulted in the phenotype of WT mice exhibiting increased intimal hyperplasia (30.9 ± 6.4 μm in TLR2−/− mice transplanted with WT bone marrow vs. 13.9 ± 4.1 μm after transplantation of TLR2−/− bone marrow into TLR2−/− mice, P < 0.05, n = 6 mice/group, Fig. 2A, B). Chimeric WT mice with TLR2-deficient bone marrow also exhibited reduced neointima area (874.2 ± 290 vs. 3555 ± 511 μm2 in WT mice transplanted with WT bone marrow, P < 0.05) and a lesser degree of luminal stenosis following arterial injury (2.7 ± 0.6 vs. 12 ± 1.4% in WT mice transplanted with WT bone marrow, P < 0.05, n = 6), while transplantation of WT bone marrow into TLR2-deficient mice resulted in increased neointimal area (8318 ± 290 vs. 3040 ± 954 μm2 in TLR2−/− mice transplanted with TLR2−/− bone marrow, P < 0.05, n = 6) and increased luminal stenosis (12.7 ± 2.6 vs. 4.2 ± 2.1% in TLR2−/− mice transplanted withTLR2−/− bone marrow, P < 0.05, n = 6, Fig. 2C, D). Reduced neointima formation in WT mice with a TLR2−/− genotype of hematopoietic cells was moreover associated with reduced accumulation of SMA-positive smooth muscle cells (3.5 ± 1.1 smooth muscle cells in WT mice transplanted with TLR2−/− bone marrow vs. 8.8 ± 1.9 in WT mice transplanted with WT bone marrow, P < 0.05). In contrast, transplantation of WT bone marrow into TLR2−/− mice resulted in increased numbers of smooth muscle cells in neointimal lesions (12.7 ± 1.4 vs. 3.1 ± 0.5 in TLR2−/− mice transplanted with TLR2−/− bone marrow, P < 0.001, n = 6 mice/group, Fig. 2E).
added, and the cells were cultured for additional 2 h. Intact smooth muscle cell layers were then wounded by scratching twice with a pipette tip in a 90° angle. Cells were washed with DMEM to remove debris and the remaining cells were incubated with regular growth medium. 1 × 105 freshly isolated bone marrow cells from either TLR2−/− or WT mice were then co-incubated and scratch wound sizes were assessed after 12 h using an inverted microscope. For TLR2 blockade, 1 mg/mL of TLR2-blocking antibody (clone 2.5, HycultBiotech, Wayne, USA) was used. For TLR2 stimulation, 1 μg/mL of the synthetic TLR2/TLR1 ligand Pam3CSK4 (Invivogen, USA) or 100 μg/mL of the natural TLR2 specific agonist lipoteichoic acid from Staphylococcus aureus (Sigma Aldrich, Germany) was used and incubated simultaneously with bone marrow derived cells for 6 h. 2.7. Statistical analysis Data was analyzed using One-way ANOVA followed by Bonferroni correction for multiple comparisons. Data is presented as mean ± standard error of mean (SEM) and differences were considered significant if P values were below 0.05. 3. Results 3.1. TLR2-deficient mice are protected against neointimal lesion development after arterial injury Histological analysis of mouse carotid arteries 21 days after arterial injury by ferric chloride revealed reduced neointima formation in TLR2−/− compared to WT mice (neointima thickness, 23.7 ± 4.3 vs. 43.1 ± 5.9 μm, P < 0.05; neointima area, 5491 ± 1152 vs. 13,756 ± 2627 μm2, P < 0.05, n = 8 mice/group, Fig. 1A, B). Reduced neointima formation in TLR2−/− mice was associated with reduced luminal stenosis in TLR2−/− mice compared to WT mice (8.6 ± 1.7 vs. 22.4 ± 2.2%, P < 0.001, n = 8 mice/group). Further characterization of the increased neointima detected in WT mice revealed smooth muscle cells are the primary cellular constituent of the neointima in WT mice. In contrast, less smooth muscle cells were found in TLR2−/− mice (10.9 ± 1.4 in WT vs. 3.9 ± 0.9 smooth muscle actin (SMA)-positive cells in TLR2−/− mice, P < 0.01, n = 8 mice/ group, Fig. 1C, D). Of note, no difference existed between the number of SMA-positive cells in the intima and media of WT compared to TLR2−/− mice (Suppl. Fig. 2).
3.4. TLR2−/− CD117-positive bone marrow cells promote smooth muscle cell migration to a lesser extent than WT bone marrow cells in vitro Employment of an in vitro scratch wound assay of confluent smooth muscle cells incubated in the presence of either WT or TLR2-deficient CD117-positive bone marrow cells revealed that both types of hematopoietic cells promote smooth muscle cell migration (Fig. 3A, B). However, TLR2-deficient bone marrow cells stimulated migration of smooth muscle cells to a lesser extent than WT bone marrow cells (1.38 ± 0.06-fold vs. 1.78 ± 0.05-fold compared to smooth muscle cell migration in the presence of medium alone defined as 1.0 [ctrl], P < 0.05, n = 7 independent experiments). The potency of WT bone marrow cells to induce SMC migration was diminished in the presence of a TLR2 blocking antibody (0.88 ± 0.02-fold, P < 0.05, n = 5) and increased in the presence of the TLR2 specific agonist lipoteichoic acid (LTA) (P < 0.05 vs. ctrl, n = 5) (Fig. 3C).
3.2. The beneficial effects of TLR2 deficiency can be mimicked by TLR2blocking antibody administration into WT mice To test a therapeutic implication of the findings above, we subjected WT mice to intracardiac treatment with 2.5 mg/kg bodyweight TLR2blocking antibody T2.5 on POD 2 and 4 following arterial injury [16]. Compared to WT mice subjected to treatment with saline serving as vehicle only, antibody-treated mice exhibited reduced neointima thickness (6.6 ± 1.5 μm in antibody treated vs. 43.1 ± 5.9 μm in untreated mice, P < 0.01, n = 4 mice/group, Fig. 1A, B), neointima area (315 ± 76.7 vs. 13,756 ± 2627 μm2, P < 0.01) and luminal stenosis (5.0 ± 1.4 vs. 22.4 ± 2.2%, P < 0.001). Antibody-treated mice also exhibited reduced numbers of SMA-positive SMCs in neointimal lesions when compared to untreated mice (3.5 ± 1.0 vs. 10.9 ± 1.4 cells, P < 0.01, n = 4 mice/group), Fig. 1C, D.
4. Discussion In the present study, we provide evidence for protective effects of genetic TLR2 deficiency against maladaptive arterial lesion regeneration after arterial injury. TLR2 deficiency was associated with reduced intimal hyperplasia and subsequently reduced luminal stenosis in murine carotid arteries three weeks after injury. Strikingly, the phenotype of increased neointimal formation in wild type mice could be completely rescued by the transplantation of TLR2-deficient bone marrow. In contrast, protection against neointimal thickening after
3.3. Neointima formation in WT mice can be reduced to the level of TLR2−/− mice by transplantation of TLR2-deficient bone marrow In order to test whether reduced neointima formation in TLR2−/− mice is mediated by TLR2-deficiency of cells in the vessel wall or TLR2deficiency on circulating cells, we generated chimeric mice by transplanting bone marrow of WT mice into TLR2−/− mice and vice versa. 3
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Fig. 1. Genetic TLR2 deficiency or application of TLR2 blocking antibodies reduces neointimal hyperplasia after carotid artery injury. A Representative hematoxylin/ eosin-stained carotid artery sections and magnifications showing increased neointimal hyperplasia in wild type (WT) but not TLR2-deficient (TLR2−/−) or TLR2blocking antibody (AB)-treated WT mice. Neointima thickness is indicated by arrows. Bar indicates 100 μm. B Quantitative summary of the histomorphological assessment of n = 8 mice/group and n = 4 mice in the antibody-treated groups. Mean and SEM, One-Way ANOVA/Bonferroni, *P < 0.05, **P < 0.01 and ***P < 0.001 vs. WT. C Representative histological findings after staining of paraffin-embedded sections for alpha-smoot muscle actin (SMA). Bar indicates 10 μm. D Quantitative summary of SMA-positive cell assessment. Mean and SEM, One-Way ANOVA/Bonferroni, **P < 0.01 vs. WT, n = 4–8 mice/group.
arterial injury in TLR2−/− mice was completely reverted by transplantation of bone marrow from wild type mice into TLR2−/− mice. In 2010, Daniel et al. pioneered in the dissection of the role of circulating bone marrow-derived cells into neointimal lesions. The authors showed that homing of circulating cells and thus the contribution of these cells to the cellular compartment of the neointima is limited to the first 1–3 weeks after arterial injury [3]. These cells were mostly of myeloid origin and had differentiated into cells exhibiting characteristics typical for monocytes and macrophages, identical to the bone marrow-derived cellular subsets identified to express TLR2 [10]. Upon tissue injury, injured cells release intracellular molecules termed danger associated
molecular patterns (DAMPs) that serve as agonists on innate immune receptors such as TLR2 [17]. Activation of TLR2 on monocytes can induce a distinct inflammatory response that results in increased expression and release of pro-inflammatory cytokines that can trigger the creation of a pro-inflammatory milieu at sites of arterial injury [18]. The accumulation of pro-inflammatory mediators is considered a key event in triggering smooth muscle cell migration from the tunica media into neointimal lesions [1]. The results of the present study support the current notion that bone marrow-derived cells are key mediators of the initial vascular remodeling process after injury since both phenotypes of WT and TLR2−/− mice were reverted in chimeric WT/TLR2−/− 4
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Fig. 1. (continued)
that was topically applied to carotid arteries to induce arterial injury in the present study, penetrates the vessel wall and traverses the endothelium by an endocytic-exocytic pathway into the arterial lumen where it causes complete endothelial denudation [20]. Involvement of TLR2 was described for most of the processes induced by arterial injury mentioned above. For example, TLR2 signaling increases platelet reactivity, promotes thrombus formation [21] and the absence of TLR2 on circulating progenitor cells augments endothelial cell function [22], suggesting alternative mechanisms of bone marrow-derived cells or platelets to contribute to improved arterial lesion regeneration in TLR2−/− vs. wild type mice. We have previously characterized CD117positive myeloid stem and progenitor cells from TLR2-deficient compared to TLR2 competent wild type mice and showed that cells from TLR2−/− mice exhibit an increased potency to facilitate vascular regeneration by the secretion of paracrine factors [23]. Particularly on decellularized or denuded surfaces, stem and progenitor cells contribute to neointima formation [24,25] and we have thus in the present study tested the hypothesis that progenitor cells of myeloid origin can modulate the migratory activity of smooth muscle cells that were the primary cellular constituent of intimal lesions. In vitro experiments revealed that CD117-positive progenitors from wild type mice more potently triggered the migratory activity of smooth muscle cells compared to those isolated from TLR2−/− mice. Although these observations suggest that the paracrine characteristics of myeloid progenitors from wild type mice are of pro-inflammatory nature compared to those of cells isolated from TLR2-deficient mice, we can only speculate on the relevance of CD117-positive cells for homing to sites of arterial injury and the subsequent effects on arterial remodeling thereafter. In addition, it is of limitation to the present study that the paracrine mediators released from TLR2-deficient compared to TLR2 competent cells from wild type mice remain unspecified. However, our results mechanistically support the hypothesis that bone marrow-derived cells such as progenitors of myeloid origin transiently reside at vascular lesions, gain
mice after bone marrow transplantation. It can thus be speculated that the presence of TLR2 on circulating bone marrow-derived cells in wild type mice 1) resulted in an increased homing of circulating cells to sites of arterial injury, 2) lead to the homing of cells with increased proinflammatory characteristics or 3) extended the duration of circulating cells in neointimal lesions that in summary determined the maladaptive vascular regeneration process in wild type compared to TLR2−/− mice exhibited 3 weeks after injury. In this regard, it is of limitation to the present study that the degree of homing of bone marrow-derived cells to sites of arterial injury in carotid arteries was not assessed during the early phase post injury (i.e. between 1 and 3 weeks after the insult), while 3 weeks after injury the identification of bone marrow-derived cells in lesions was unlikely [3]. We can thus only speculate that the increased numbers of intimal smooth muscle cells in wild type mice 3 weeks after injury were the result of a differential initial homing or pro-inflammatory phenotype of bone marrow-derived cells in wild type vs. TLR2−/− mice. Histomorphological analysis of neointimal lesions in the present study revealed that smooth muscle cells made up for almost half of the cellular components in intimal lesions in wild type mice while lesions of TLR2−/− mice exhibited only scarce distribution of α-smooth muscle actin-positive cells in the intima. These results are in line with studies in porcine models that evaluated the cellular components of neointimal lesion developing after self-expanding stent implantations and percutaneous transluminal coronary angioplasty (PTCA) [19]. Smooth muscle cells normally reside in the medial layer of the vessel wall and exhibit a mostly quiescent and contractile phenotype. Early responses to luminal injury of the vessel wall such as platelet activation and thrombus formation, endothelial activation, endothelial dysfunction or even denudation and leukocyte recruitment can induce the local expression of matrix metalloproteinases. These matrix metalloproteinases facilitate breakdown of the laminal elastica interna and thus trigger smooth muscle cell migration into the intimal layer [1]. Ferric chloride, 5
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Fig. 2. Generation of WT/TLR2−/− chimeric mice reverted the phenotype of vascular lesion development. A Representative graphics depicting the transplantation modalities mice had undergone prior to arterial injury and a schematic depiction of the morphologic changes detected in the carotid arteries (upper chart). In the lower chart, representative hematoxylin/eosin-stained carotid artery sections after generation of chimeric mice or controls are shown. BM indicates bone marrow, SMC smooth muscle cells. White arrows in histological sections indicate neointimal thickness. Bar indicates 10 μm. B, C, D, E Quantitative summary of the histomorphological assessment of n = 6 mice/group. Mean and SEM, One-way ANOVA/Bonferroni, *P < 0.05 and **P < 0.01 and ***P < 0.001 as indicated, n = 6 mice/group.
Despite various efforts to understand the pathophysiology underlying neointima formation, therapeutic options for the prevention or treatment of in-stent stenosis or restenosis following arterial injury are not available to date. TLR2 antagonists have been suggested for the treatment of cardiovascular disease [26] and we have previously shown that they can mimic the beneficial effects of genetic TLR2 ablation on vascular regeneration and function [16,27]. In this study, we tested the
monocytic characteristics and determine the fate of vascular remodeling in a paracrine manner [3]. Based on this hypothesis and in line with our previous investigations, TLR2 deficiency on these cell types or pharmacological TLR2 blockade would thus support vascular remodeling by resulting in a less inflammatory, more regeneration-facilitating panel of mediators that limits scar formation and thus luminal stenosis after vascular injury. 6
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Fig. 2. (continued)
5. Conclusion
hypothesis that pharmacological blockade of TLR2 initially after chemically induced arterial injury can reduce neointimal hyperplasia and luminal stenosis. Using an established and previously tested TLR2-targeting antibody and an intracardiac route of application on POD 2 and 4 after arterial injury, vascular remodeling 3 weeks after injury revealed reduced lesion formation and luminal stenosis in wild type mice that were comparable to those exhibited in TLR2-deficient mice. Future studies have to evaluate whether blockade of innate immune receptors in the early phase after vascular insults may display a promising therapeutic tool to augment vascular regeneration and patient outcome. Chronic exposure to TLR2 antagonists in contrast appears to less likely become an option for the treatment of cardiovascular disease, since functional TLR2 has also been reported to be an important cornerstone of collateral arterial remodeling and vascular regeneration [28–30].
In summary, this study provides evidence that TLR2 genetic deficiency or pharmacological blockade beneficially modulates neointimal hyperplasia in response to arterial injury and that these effects are primarily mediated by bone marrow-derived circulating cells. Lack of TLR2 signaling in these cells may facilitate arterial regeneration in a paracrine manner. Future studies will have to dissect whether blockade of innate immune receptor signaling can reduce maladaptive vascular regenerative processes and scar formation in the vessel wall by limiting the effects of danger associated molecular patterns released upon tissue injury. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lfs.2019.117189.
Fig. 3. Effects of WT and TLR2-deficient CD117-positive bone marrow cells on smooth muscle cell migration. A Representative images of co-culture of CD117-positive bone marrow cells with smooth muscle cells. Depicted are smooth muscle cell layer scratch wounds 12 h after scratching in the presence of either bone marrow-derived cells from wild type animals (WT) or TLR2-deficient (TLR2−/−) mice or media only serving as vehicle control (ctrl). Bar indicates 100 μm. B Quantitative summary of n = 7 independent experiments. Mean and SEM, One-way ANOVA/Bonferroni, *P < 0.05 and **P < 0.01 and ***P < 0.001 vs. ctrl or as indicated. C Effects of blockade or stimulation of TLR2 on bone marrow derived cells from wild type mice on SMC migration. AB antibody, LTA lipoteichoic acid. Mean and SEM, One-way ANOVA/Bonferroni, *P < 0.05 and ***P < 0.001 vs. ctrl or as indicated.
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Funding
Res. Commun. 345 (2006) 1446–1453. [15] D. Furlani, P. Donndorf, I. Westien, et al., HMGB-1 induces c-kit+ cell microvascular rolling and adhesion via both toll-like receptor-2 and toll-like receptor-4 of endothelial cells, J. Cell. Mol. Med. 16 (2012) 1094–1105, https://doi.org/10. 1111/j.1582-4934.2011.01381.x. [16] N.M. Wagner, L. Bierhansl, G. Noldge-Schomburg, et al., Toll-like receptor 2blocking antibodies promote angiogenesis and induce ERK1/2 and AKT signaling via CXCR4 in endothelial cells, Arterioscler. Thromb. Vasc. Biol. 33 (2013) 1943–1951, https://doi.org/10.1161/ATVBAHA.113.301783. [17] K.L. Rock, E. Latz, F. Ontiveros, et al., The sterile inflammatory response, Annu. Rev. Immunol. 28 (2009) 321–342. [18] P. Lacerte, A. Brunet, B. Egarnes, et al., Overexpression of TLR2 and TLR9 on monocyte subsets of active rheumatoid arthritis patients contributes to enhance responsiveness to TLR agonists, Arthritis Res. Ther. 18 (2016) 10. [19] T. Christen, V. Verin, M.-L. Bochaton-Piallat, et al., Mechanisms of neointima formation and remodeling in the porcine coronary artery, Circulation. 103 (2001) 882–888. [20] M.T. Tseng, A. Dozier, B. Haribabu, et al., Transendothelial migration of ferric ion in FeCl3 injured murine common carotid artery, Thromb. Res. 118 (2006) 275–280. [21] S. Biswas, A. Zimman, D. Gao, et al., TLR2 plays a key role in platelet hyperreactivity and accelerated thrombosis associated with hyperlipidemia, Circ. Res. 121 (2017) 951–962. [22] N.-M. Wagner, L. Bierhansl, G. Noeldge-Schomburg, et al., Toll-like receptor 2 (TLR2) blocking antibodies promote angiogenesis and induce ERK1/2 and AKT signaling via CXCR4 in endothelial cells, Arteriosclerosis, thrombosis, and vascular biology 33 (8) (2013) 1943–1951, https://doi.org/10.1161/ATVBAHA. [23] N.-M. Wagner, L. Bierhansl, A. Butschkau, et al., TLR2-deficiency of cKit+ bone marrow cells is associated with augmented potency to stimulate angiogenic processes, Int. J. Clin. Exp. Pathol. 6 (2013) 2813. [24] S. Tsai, J. Butler, S. Rafii, et al., The role of progenitor cells in the development of intimal hyperplasia, J. Vasc. Surg. 49 (2009) 502–510, https://doi.org/10.1016/j. jvs.2008.07.060. [25] T.-N. Tsai, J.P. Kirton, P. Campagnolo, et al., Contribution of stem cells to neointimal formation of decellularized vessel grafts in a novel mouse model, Am. J. Pathol. 181 (2012) 362–373. [26] E. Lin, J.E. Freedman, L.M. Beaulieu, Innate immunity and toll-like receptor antagonists: a potential role in the treatment of cardiovascular diseases, Cardiovasc. Ther. 27 (2009) 117–123, https://doi.org/10.1111/j.1755-5922.2009.00077.x. [27] S. Bergt, A. Guter, A. Grub, et al., Impact of Toll-like receptor 2 deficiency on survival and neurological function after cardiac arrest: a murine model of cardiopulmonary resuscitation, PLoS One 8 (2013) e74944, , https://doi.org/10.1371/ journal.pone.0074944. [28] D. de Groot, I.E. Hoefer, S. Grundmann, et al., Arteriogenesis requires toll-like receptor 2 and 4 expression in bone-marrow derived cells, J. Mol. Cell. Cardiol. 50 (2011) 25–32. [29] H. Wu, X.W. Cheng, L. Hu, et al., Cathepsin S activity controls injury-related vascular repair in mice via the TLR2-mediated p38MAPK and PI3K− Akt/p-HDAC6 signaling pathway, Arterioscler. Thromb. Vasc. Biol. 36 (2016) 1549–1557. [30] C. He, P. Lai, J. Wang, et al., TLR2/4 deficiency prevents oxygen-induced vascular degeneration and promotes revascularization by downregulating IL-17 in the retina, Sci. Rep. 6 (2016) 27739.
The present study was funded by departmental funds of the Clinic for Anesthesiology and Intensive Care Medicine of the Rostock University Medical Center. Declaration of competing interest The authors declare no conflicts of interest. References [1] M.A. Zain, W.J. Siddiqui, StatPearls, StatPearls Publishing, 2018. [2] M. Sata, A. Saiura, A. Kunisato, et al., Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis, Nat. Med. 8 (2002) 403. [3] J.-M. Daniel, W. Bielenberg, P. Stieger, et al., Time-course analysis on the differentiation of bone marrow-derived progenitor cells into smooth muscle cells during neointima formation, Arterioscler. Thromb. Vasc. Biol. 30 (2010) 1890–1896. [4] A. Clowes, M. Reidy, M. Clowes, Mechanisms of stenosis after arterial injury, Lab. Investig. 49 (1983) 208–215. [5] P. Libby, Inflammation in atherosclerosis, Arterioscler. Thromb. Vasc. Biol. 32 (2012) 2045–2051. [6] L. de Oliviera Nascimento, P. Massari, L.M. Wetzler, The role of TLR2 in infection and immunity, Front. Immunol. 3 (2012) 79. [7] G.-L. Lee, Y.-W. Chang, J.-Y. Wu, et al., TLR 2 induces vascular smooth muscle cell migration through cAMP response element-binding protein-mediated interleukin-6 production, Arterioscler. Thromb. Vasc. Biol. 32 (2012) 2751–2760. [8] J. Fan, R.S. Frey, A.B. Malik, TLR4 signaling induces TLR2 expression in endothelial cells via neutrophil NADPH oxidase, J. Clin. Invest. 112 (2003) 1234–1243, https:// doi.org/10.1172/JCI18696. [9] O. Pryshchep, W. Ma-Krupa, B.R. Younge, et al., Vessel-specific Toll-like receptor profiles in human medium and large arteries, Circulation. 118 (2008) 1276. [10] T.H. Flo, Ø. Halaas, S. Torp, et al., Differential expression of Toll-like receptor 2 in human cells, J. Leukoc. Biol. 69 (2001) 474–481. [11] A.E. Mullick, K. Soldau, W.B. Kiosses, et al., Increased endothelial expression of Toll-like receptor 2 at sites of disturbed blood flow exacerbates early atherogenic events, J. Exp. Med. 205 (2008) 373–383, https://doi.org/10.1084/jem.20071096. [12] A.E. Mullick, P.S. Tobias, L.K. Curtiss, Modulation of atherosclerosis in mice by Tolllike receptor 2, J. Clin. Invest. 115 (2005) 3149–3156, https://doi.org/10.1172/ JCI25482. [13] A.H. Schoneveld, M.M. Oude Nijhuis, B. van Middelaar, et al., Toll-like receptor 2 stimulation induces intimal hyperplasia and atherosclerotic lesion development, Cardiovasc. Res. 66 (2005) 162–169, https://doi.org/10.1016/j.cardiores.2004.12. 016. [14] T. Shishido, N. Nozaki, H. Takahashi, et al., Central role of endogenous Toll-like receptor-2 activation in regulating inflammation, reactive oxygen species production, and subsequent neointimal formation after vascular injury, Biochem. Biophys.
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Toll-like receptor 2-deficiency on bone marrow-derived cells augments vascular healing of murine arterial lesions☆ W. Liua, J.-C. Eczkob, M. Ottoa, R. Bajoratb, B. Vollmarc, J.-P. Roesnerb, N.-M. Wagnera,
T
⁎
a
Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Münster, Münster, Germany Department of Anesthesia and Intensive Care, University Medical Center Rostock, Rostock, Germany c Institute for Experimental Surgery, University Medical Center Rostock, Rostock, Germany b
A R T I C LE I N FO
A B S T R A C T
Keywords: Toll-like receptor 2 TLR2 Arterial injury Restenosis Neointima Smooth muscle cells
Aims: Neointimal hyperplasia contributes to arterial restenosis after percutaneous transluminal coronary angioplasty or vascular surgery. Neointimal thickening after arterial injury is determined by inflammatory processes. We investigated the role of the innate immune receptor toll-like receptor 2 (TLR2) in neointima formation after arterial injury in mice. Materials and methods: Carotid artery injury was induced by 10% ferric chloride in C57Bl/6J wild type (WT), TLR2 deficient (B6.129-Tlr2tm1Kir/J, TLR2−/−) and WT mice treated with a TLR2 blocking antibody. 21 days after injury, carotid arteries were assessed histomorphometrically and for smooth muscle cell (SMC) content. To identify the contribution of circulating cells in mediating the effects of TLR2-deficiency, arterial injury was induced in WT/TLR2−/−-chimeric mice and the paracrine modulation of bone marrow-derived cells from WT and TLR2−/− on SMC migration compared in vitro. Key findings: TLR2−/− mice and WT mice treated with TLR2 blocking antibodies exhibited reduced neointimal thickening (23.7 ± 4.2 and 6.5 ± 3.0 vs. 43.1 ± 5.9 μm, P < 0.05 and P < 0.01), neointimal area (5491 ± 1152 and 315 ± 76.7 vs. 13,756 ± 2627 μm2, P < 0.05 and P < 0.01) and less luminal stenosis compared to WT mice (8.5 ± 1.6 and 5.0 ± 1.3 vs. 22.4 ± 2.2%, both P < 0.001n = 4–8 mice/group). The phenotypes of TLR2−/− vs. WT mice were completely reverted in WT/TLR2−/− bone marrow chimeric mice (5.9 ± 1.5 μm neointimal thickness, 874.2 ± 290.2 μm2 neointima area and 2.7 ± 0.6% luminal stenoses in WT mice transplanted with TLR2−/− bone marrow vs. 23.6 ± 5.1 μm, 3555 ± 511 μm2 and 12.0 ± 1.3% in WT mice receiving WT bone marrow, all P < 0.05, n = 6/group). Neointimal lesions of WT and WT mice transplanted with TLR2−/− bone marrow chimeric mice showed increased numbers of SMC (10.8 ± 1.4 and 12.6 ± 1.4 vs. 3.8 ± 0.9 in TLR2−/− and 3.5 ± 1.1 cells in WT mice transplanted with TLR2−/− bone marrow, all P < 0.05, n = 6). WT bone marrow cells stimulated SMC migration more than TLR2-deficient bone marrow cells (1.7 ± 0.05 vs. 1.3 ± 0.06-fold, P < 0.05, n = 7) and this effect was aggravated by TLR2 stimulation and diminished by TLR2 blockade (1.1 ± 0.03-fold after stimulation with TLR2 agonists and 0.8 ± 0.02-fold after TLR2 blockade vs. control treated cells defined as 1.0, P < 0.05, n = 7). Significance: TLR2-deficiency on hematopoietic but not vessel wall resident cells augments vascular healing after arterial injury. Pharmacological blockade of TLR2 may thus be a promising therapeutic option to improve vessel patency after iatrogenic arterial injury.
1. Introduction
endarterectomy or angioplasty. Maladaptive regenerative processes in arterial lesions after injury can endanger outcome of patients after interventional or surgical vascular procedures and can result in restenosis or in-stent stenosis that reduce vessel patency due to vessel and graft
Iatrogenic arterial injury frequently occurs during percutaneous transluminal coronary angioplasty (PTCA), coronary bypass surgery,
☆ Statement of authorship: All authors take responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation. ⁎ Corresponding author at: Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Münster, Albert-Schweitzer-Campus 1, 48161 Münster, Germany. E-mail address: [email protected] (N.-M. Wagner).
https://doi.org/10.1016/j.lfs.2019.117189 Received 12 November 2019; Received in revised form 12 December 2019; Accepted 16 December 2019 Available online 28 December 2019 0024-3205/ © 2019 Elsevier Inc. All rights reserved.
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The background of the mice was confirmed by genotyping, and age matched mouse groups were used for the experiments.
occlusion. Thus, a detailed understanding of the pathophysiology of impaired healing of arterial lesions is of particular importance and therapeutic options to optimize outcome after vascular surgery are urgently needed. Neointimal hyperplasia is an integral part of maladaptive arterial regeneration and is characterized by the invasion of smooth muscle cells (SMCs) into the intimal layer of the vessel wall. SMCs make up for the majority of the neointima constituting cells and the degree of SMC mobilization determines scar formation and thus the degree of luminal stenosis after injury [1]. Although it has been controversially discussed whether intimal SMCs arise from differentiating circulating progenitor cells [2] or from resident medial SMCs or adventitial fibroblasts [3,4], the paradigm that the initial SMC mobilization into neointimal lesions after injury is driven by inflammatory processes has been widely accepted [5]. Toll-like receptors (TLRs) are innate immune receptors that respond to and integrate a variety of inflammatory processes. In addition to exogenous pathogen associated molecular patterns (PAMPs), TLRs recognize endogenous mediators termed “danger associated molecular patterns” (DAMPs) that are liberated upon tissue injury [6]. TLR2 is expressed on cells of the vessel wall including smooth muscle cells [7] and endothelial cells [8] and shows ubiquitous expression in the vascular tree [9]. In addition, TLR2 is found on circulating bone marrowderived cells (BMCs) of myeloid origin, making TLR2 a potential modulator of both vessel wall inherent remodeling processes as well as a potential mediator of BMC allocation to sites of arterial injury [10]. In the development of murine and human atherosclerosis, loss of TLR2 expression in the vessel wall is associated with protective effects against disease progression [11–13]. In turn, activation of TLR2 by synthetic ligands can increase disease burden in atherosclerosis susceptible mice and this is mediated by TLR2 expressed on BMCs only [12]. TLR2 agonists are naturally liberated upon tissue injury, suggesting that TLR2 activation on BMCs can modulate the inflammation-driven responses to arterial injury. Indeed, it has previously been suggested that TLR2 genetic deficiency confers protection against injury-induced arterial occlusion [14]. However, the role of TLR2 and the cellular localization of its expression in arterial remodeling after arterial injury remains unknown and a potential therapeutic importance of antagonizing TLR2 for improving arterial lesion healing unevaluated. In this investigation, we aimed to identify the contribution of TLR2 expressed on local cells of the vessel wall vs. TLR2 on BMCs to functional regeneration of arterial lesions after chemical injury. We further dissected the role of TLR2 presence on circulating BMCs for SMC migratory capacity and for the first time tested the application of a TLR2blocking antibody to augment arterial regeneration after injury in mice.
2.2. Ferric-chloride-induced arterial injury Mice were anesthetized by intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine and placed on a heating pat in the supine position. After removal of hair and careful disinfection, the left carotid artery was dissected free and a silicon peace slid under the vessel. Arterial injury was induced by subjecting the vessel to 10% ferric chloride (Sigma Aldrich, Germany) soaked in a 2 × 2 mm filter paper piece for 3 min. Ferric chloride was removed and the skin was sutured using 0.7 Ethicon sutures. Three weeks after injury, mice were sacrificed and arterial segments were harvested in 4% formalin and subjected to histological analysis. 2.3. Histologic analysis Immunohistochemistry was performed on paraffin-embedded carotid artery sections. For quantitative morphometric analysis, carotid arteries were stained with hematoxylin/eosin. The neointima and media area, the intima/media ratio, and the degree of luminal stenosis were determined using ImagePro Plus software (Media Cybernetics, United Kingdom). Five sections equally spaced throughout the injured arterial segment (at 200-um intervals) were evaluated, and the results were averaged for each animal. The presence of smooth muscle cells was assessed using a rabbit polyclonal anti-mouse α-actin antibody (Dako, Denmark). 2.4. Irradiation and bone marrow transplantation
2. Methods
For bone marrow transplantation experiments prior to arterial injury, mice were irradiated twice daily (BiD) using a dose of 5 Gy applied each at an 4 h interval from a linear accelerator (VARIAN, Palo Alto, CA, USA). 24 h later, mice were transplanted with unfractionated whole bone marrow using 1 × 108 freshly isolated bone marrow-derived cells in a volume of 150 μL sterile PBS applied via tail vein injection. Wild type mice received either bone marrow from WT mice (WT ➔ WT, serving as controls) or TLR2−/− mice (TLR2−/− ➔ WT) while TLR2−/− mice received either bone marrow from TLR2−/− mice (TLR2−/− ➔ TLR2−/−, serving as controls) or WT mice (WT ➔ TLR2−/−). Mice were housed in isolation containers with filtered air for the following 4 weeks and inspected and weighed daily. After 4 weeks of recovery, mice were subjected to arterial injury. Genotype of circulating cells was confirmed by qPCR on the day of sacrifice (3 weeks after lesion induction, Suppl. Fig. 1).
2.1. Experimental animals
2.5. Isolation of CD117 (c-Kit)-positive cells from bone marrow
TLR2 knockout (B6.129-Tlr2tm1Kir/J, TLR2−/−, n = 30) mice were purchased from Jackson Laboratories. Together with C57Bl/6J mice (n = 35) serving as controls, mice were housed in a pathogen-free facility with a 12/12 light and dark cycle and were given free access to water and standard rodent chow. Murine genotypes were verified by qPCR (Suppl. Fig. 1). A subset of WT mice was treated with 2.5 mg/kg bodyweight of a monoclonal antibody against murine TLR2 (clone 2.5, HycultBiotech, Wayne, USA). The antibody was dissolved in 200 μL of sterile saline and applied via left ventricular injection on POD 2 and 4 after arterial lesion induction. All animal procedures were in accordance with institutional, national and European guidelines for the care and use of laboratory animals (European Communities Council Directive of November 24th, 1986 [86/609/EEC]). The protocol was approved by the Ethical Committee of the Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei, MecklenburgVorpommern, Germany (permit number LALLF M-V/TSD/7221.3-1.1072/12). All efforts were made to minimize suffering of the animals.
c-Kit positive cells were isolated form 8–10 week old wild type or TLR2-deficient mice as described previously [15]. Murine bone marrow was isolated from femur and tibial bone and cell suspensions were incubated with magnetic microbeads coated with anti-c-Kit monoclonal antibody (Miltenyi Biotec, Bergisch Gladbach, Germany). MS columns and the MiniMacs cell separator system were employed to obtain c-Kitpositive cell fractions. 2.6. Smooth muscle cell migration assay Human aortic smooth muscle cells were purchased from PromoCell (Heidelberg, Germany) and cultured at 37 °C in DMEM supplemented with 10% FBS, 2 mmol/L L-glutamine, 100 U mL−1 penicillin, and 100 μg/mL streptomycin, in a 5% CO2 incubator. To assess their migratory activity, 1 × 104 smooth muscle cells were seeded in 24-well plates and incubated for 24 h. From confluent cells, the culture medium was removed, 2 mL fresh culture medium were 2
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Transplantation of WT bone marrow into WT mice or bone marrow of TLR2−/− mice into TLR2−/− mice served as control. The genotype of circulating cells was verified by Northern blot by the time of carotid artery harvest (Suppl. Fig. 1). All mice quickly recovered following lethal irradiation and transplantation and exhibited no more than ~10% initial reduction in body weight followed by a rapid increase in body weight. Reconstitution of bone marrow with WT bone marrow cells in TLR2−/− mice and vice versa completely reversed previous findings: transplantation of TLR2-deficient bone marrow into WT mice rescued the impaired arterial regeneration in WT mice by resulting in reduced neointima thickness (5.9 ± 1.5 μm in WT mice transplanted with TLR2−/− bone marrow vs. 23.7 ± 5.2 μm after transplantation of WT bone marrow into WT mice, P < 0.05, n = 6 animals/group), while transplantation of WT bone marrow into TLR2−/− mice resulted in the phenotype of WT mice exhibiting increased intimal hyperplasia (30.9 ± 6.4 μm in TLR2−/− mice transplanted with WT bone marrow vs. 13.9 ± 4.1 μm after transplantation of TLR2−/− bone marrow into TLR2−/− mice, P < 0.05, n = 6 mice/group, Fig. 2A, B). Chimeric WT mice with TLR2-deficient bone marrow also exhibited reduced neointima area (874.2 ± 290 vs. 3555 ± 511 μm2 in WT mice transplanted with WT bone marrow, P < 0.05) and a lesser degree of luminal stenosis following arterial injury (2.7 ± 0.6 vs. 12 ± 1.4% in WT mice transplanted with WT bone marrow, P < 0.05, n = 6), while transplantation of WT bone marrow into TLR2-deficient mice resulted in increased neointimal area (8318 ± 290 vs. 3040 ± 954 μm2 in TLR2−/− mice transplanted with TLR2−/− bone marrow, P < 0.05, n = 6) and increased luminal stenosis (12.7 ± 2.6 vs. 4.2 ± 2.1% in TLR2−/− mice transplanted withTLR2−/− bone marrow, P < 0.05, n = 6, Fig. 2C, D). Reduced neointima formation in WT mice with a TLR2−/− genotype of hematopoietic cells was moreover associated with reduced accumulation of SMA-positive smooth muscle cells (3.5 ± 1.1 smooth muscle cells in WT mice transplanted with TLR2−/− bone marrow vs. 8.8 ± 1.9 in WT mice transplanted with WT bone marrow, P < 0.05). In contrast, transplantation of WT bone marrow into TLR2−/− mice resulted in increased numbers of smooth muscle cells in neointimal lesions (12.7 ± 1.4 vs. 3.1 ± 0.5 in TLR2−/− mice transplanted with TLR2−/− bone marrow, P < 0.001, n = 6 mice/group, Fig. 2E).
added, and the cells were cultured for additional 2 h. Intact smooth muscle cell layers were then wounded by scratching twice with a pipette tip in a 90° angle. Cells were washed with DMEM to remove debris and the remaining cells were incubated with regular growth medium. 1 × 105 freshly isolated bone marrow cells from either TLR2−/− or WT mice were then co-incubated and scratch wound sizes were assessed after 12 h using an inverted microscope. For TLR2 blockade, 1 mg/mL of TLR2-blocking antibody (clone 2.5, HycultBiotech, Wayne, USA) was used. For TLR2 stimulation, 1 μg/mL of the synthetic TLR2/TLR1 ligand Pam3CSK4 (Invivogen, USA) or 100 μg/mL of the natural TLR2 specific agonist lipoteichoic acid from Staphylococcus aureus (Sigma Aldrich, Germany) was used and incubated simultaneously with bone marrow derived cells for 6 h. 2.7. Statistical analysis Data was analyzed using One-way ANOVA followed by Bonferroni correction for multiple comparisons. Data is presented as mean ± standard error of mean (SEM) and differences were considered significant if P values were below 0.05. 3. Results 3.1. TLR2-deficient mice are protected against neointimal lesion development after arterial injury Histological analysis of mouse carotid arteries 21 days after arterial injury by ferric chloride revealed reduced neointima formation in TLR2−/− compared to WT mice (neointima thickness, 23.7 ± 4.3 vs. 43.1 ± 5.9 μm, P < 0.05; neointima area, 5491 ± 1152 vs. 13,756 ± 2627 μm2, P < 0.05, n = 8 mice/group, Fig. 1A, B). Reduced neointima formation in TLR2−/− mice was associated with reduced luminal stenosis in TLR2−/− mice compared to WT mice (8.6 ± 1.7 vs. 22.4 ± 2.2%, P < 0.001, n = 8 mice/group). Further characterization of the increased neointima detected in WT mice revealed smooth muscle cells are the primary cellular constituent of the neointima in WT mice. In contrast, less smooth muscle cells were found in TLR2−/− mice (10.9 ± 1.4 in WT vs. 3.9 ± 0.9 smooth muscle actin (SMA)-positive cells in TLR2−/− mice, P < 0.01, n = 8 mice/ group, Fig. 1C, D). Of note, no difference existed between the number of SMA-positive cells in the intima and media of WT compared to TLR2−/− mice (Suppl. Fig. 2).
3.4. TLR2−/− CD117-positive bone marrow cells promote smooth muscle cell migration to a lesser extent than WT bone marrow cells in vitro Employment of an in vitro scratch wound assay of confluent smooth muscle cells incubated in the presence of either WT or TLR2-deficient CD117-positive bone marrow cells revealed that both types of hematopoietic cells promote smooth muscle cell migration (Fig. 3A, B). However, TLR2-deficient bone marrow cells stimulated migration of smooth muscle cells to a lesser extent than WT bone marrow cells (1.38 ± 0.06-fold vs. 1.78 ± 0.05-fold compared to smooth muscle cell migration in the presence of medium alone defined as 1.0 [ctrl], P < 0.05, n = 7 independent experiments). The potency of WT bone marrow cells to induce SMC migration was diminished in the presence of a TLR2 blocking antibody (0.88 ± 0.02-fold, P < 0.05, n = 5) and increased in the presence of the TLR2 specific agonist lipoteichoic acid (LTA) (P < 0.05 vs. ctrl, n = 5) (Fig. 3C).
3.2. The beneficial effects of TLR2 deficiency can be mimicked by TLR2blocking antibody administration into WT mice To test a therapeutic implication of the findings above, we subjected WT mice to intracardiac treatment with 2.5 mg/kg bodyweight TLR2blocking antibody T2.5 on POD 2 and 4 following arterial injury [16]. Compared to WT mice subjected to treatment with saline serving as vehicle only, antibody-treated mice exhibited reduced neointima thickness (6.6 ± 1.5 μm in antibody treated vs. 43.1 ± 5.9 μm in untreated mice, P < 0.01, n = 4 mice/group, Fig. 1A, B), neointima area (315 ± 76.7 vs. 13,756 ± 2627 μm2, P < 0.01) and luminal stenosis (5.0 ± 1.4 vs. 22.4 ± 2.2%, P < 0.001). Antibody-treated mice also exhibited reduced numbers of SMA-positive SMCs in neointimal lesions when compared to untreated mice (3.5 ± 1.0 vs. 10.9 ± 1.4 cells, P < 0.01, n = 4 mice/group), Fig. 1C, D.
4. Discussion In the present study, we provide evidence for protective effects of genetic TLR2 deficiency against maladaptive arterial lesion regeneration after arterial injury. TLR2 deficiency was associated with reduced intimal hyperplasia and subsequently reduced luminal stenosis in murine carotid arteries three weeks after injury. Strikingly, the phenotype of increased neointimal formation in wild type mice could be completely rescued by the transplantation of TLR2-deficient bone marrow. In contrast, protection against neointimal thickening after
3.3. Neointima formation in WT mice can be reduced to the level of TLR2−/− mice by transplantation of TLR2-deficient bone marrow In order to test whether reduced neointima formation in TLR2−/− mice is mediated by TLR2-deficiency of cells in the vessel wall or TLR2deficiency on circulating cells, we generated chimeric mice by transplanting bone marrow of WT mice into TLR2−/− mice and vice versa. 3
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Fig. 1. Genetic TLR2 deficiency or application of TLR2 blocking antibodies reduces neointimal hyperplasia after carotid artery injury. A Representative hematoxylin/ eosin-stained carotid artery sections and magnifications showing increased neointimal hyperplasia in wild type (WT) but not TLR2-deficient (TLR2−/−) or TLR2blocking antibody (AB)-treated WT mice. Neointima thickness is indicated by arrows. Bar indicates 100 μm. B Quantitative summary of the histomorphological assessment of n = 8 mice/group and n = 4 mice in the antibody-treated groups. Mean and SEM, One-Way ANOVA/Bonferroni, *P < 0.05, **P < 0.01 and ***P < 0.001 vs. WT. C Representative histological findings after staining of paraffin-embedded sections for alpha-smoot muscle actin (SMA). Bar indicates 10 μm. D Quantitative summary of SMA-positive cell assessment. Mean and SEM, One-Way ANOVA/Bonferroni, **P < 0.01 vs. WT, n = 4–8 mice/group.
arterial injury in TLR2−/− mice was completely reverted by transplantation of bone marrow from wild type mice into TLR2−/− mice. In 2010, Daniel et al. pioneered in the dissection of the role of circulating bone marrow-derived cells into neointimal lesions. The authors showed that homing of circulating cells and thus the contribution of these cells to the cellular compartment of the neointima is limited to the first 1–3 weeks after arterial injury [3]. These cells were mostly of myeloid origin and had differentiated into cells exhibiting characteristics typical for monocytes and macrophages, identical to the bone marrow-derived cellular subsets identified to express TLR2 [10]. Upon tissue injury, injured cells release intracellular molecules termed danger associated
molecular patterns (DAMPs) that serve as agonists on innate immune receptors such as TLR2 [17]. Activation of TLR2 on monocytes can induce a distinct inflammatory response that results in increased expression and release of pro-inflammatory cytokines that can trigger the creation of a pro-inflammatory milieu at sites of arterial injury [18]. The accumulation of pro-inflammatory mediators is considered a key event in triggering smooth muscle cell migration from the tunica media into neointimal lesions [1]. The results of the present study support the current notion that bone marrow-derived cells are key mediators of the initial vascular remodeling process after injury since both phenotypes of WT and TLR2−/− mice were reverted in chimeric WT/TLR2−/− 4
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Fig. 1. (continued)
that was topically applied to carotid arteries to induce arterial injury in the present study, penetrates the vessel wall and traverses the endothelium by an endocytic-exocytic pathway into the arterial lumen where it causes complete endothelial denudation [20]. Involvement of TLR2 was described for most of the processes induced by arterial injury mentioned above. For example, TLR2 signaling increases platelet reactivity, promotes thrombus formation [21] and the absence of TLR2 on circulating progenitor cells augments endothelial cell function [22], suggesting alternative mechanisms of bone marrow-derived cells or platelets to contribute to improved arterial lesion regeneration in TLR2−/− vs. wild type mice. We have previously characterized CD117positive myeloid stem and progenitor cells from TLR2-deficient compared to TLR2 competent wild type mice and showed that cells from TLR2−/− mice exhibit an increased potency to facilitate vascular regeneration by the secretion of paracrine factors [23]. Particularly on decellularized or denuded surfaces, stem and progenitor cells contribute to neointima formation [24,25] and we have thus in the present study tested the hypothesis that progenitor cells of myeloid origin can modulate the migratory activity of smooth muscle cells that were the primary cellular constituent of intimal lesions. In vitro experiments revealed that CD117-positive progenitors from wild type mice more potently triggered the migratory activity of smooth muscle cells compared to those isolated from TLR2−/− mice. Although these observations suggest that the paracrine characteristics of myeloid progenitors from wild type mice are of pro-inflammatory nature compared to those of cells isolated from TLR2-deficient mice, we can only speculate on the relevance of CD117-positive cells for homing to sites of arterial injury and the subsequent effects on arterial remodeling thereafter. In addition, it is of limitation to the present study that the paracrine mediators released from TLR2-deficient compared to TLR2 competent cells from wild type mice remain unspecified. However, our results mechanistically support the hypothesis that bone marrow-derived cells such as progenitors of myeloid origin transiently reside at vascular lesions, gain
mice after bone marrow transplantation. It can thus be speculated that the presence of TLR2 on circulating bone marrow-derived cells in wild type mice 1) resulted in an increased homing of circulating cells to sites of arterial injury, 2) lead to the homing of cells with increased proinflammatory characteristics or 3) extended the duration of circulating cells in neointimal lesions that in summary determined the maladaptive vascular regeneration process in wild type compared to TLR2−/− mice exhibited 3 weeks after injury. In this regard, it is of limitation to the present study that the degree of homing of bone marrow-derived cells to sites of arterial injury in carotid arteries was not assessed during the early phase post injury (i.e. between 1 and 3 weeks after the insult), while 3 weeks after injury the identification of bone marrow-derived cells in lesions was unlikely [3]. We can thus only speculate that the increased numbers of intimal smooth muscle cells in wild type mice 3 weeks after injury were the result of a differential initial homing or pro-inflammatory phenotype of bone marrow-derived cells in wild type vs. TLR2−/− mice. Histomorphological analysis of neointimal lesions in the present study revealed that smooth muscle cells made up for almost half of the cellular components in intimal lesions in wild type mice while lesions of TLR2−/− mice exhibited only scarce distribution of α-smooth muscle actin-positive cells in the intima. These results are in line with studies in porcine models that evaluated the cellular components of neointimal lesion developing after self-expanding stent implantations and percutaneous transluminal coronary angioplasty (PTCA) [19]. Smooth muscle cells normally reside in the medial layer of the vessel wall and exhibit a mostly quiescent and contractile phenotype. Early responses to luminal injury of the vessel wall such as platelet activation and thrombus formation, endothelial activation, endothelial dysfunction or even denudation and leukocyte recruitment can induce the local expression of matrix metalloproteinases. These matrix metalloproteinases facilitate breakdown of the laminal elastica interna and thus trigger smooth muscle cell migration into the intimal layer [1]. Ferric chloride, 5
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Fig. 2. Generation of WT/TLR2−/− chimeric mice reverted the phenotype of vascular lesion development. A Representative graphics depicting the transplantation modalities mice had undergone prior to arterial injury and a schematic depiction of the morphologic changes detected in the carotid arteries (upper chart). In the lower chart, representative hematoxylin/eosin-stained carotid artery sections after generation of chimeric mice or controls are shown. BM indicates bone marrow, SMC smooth muscle cells. White arrows in histological sections indicate neointimal thickness. Bar indicates 10 μm. B, C, D, E Quantitative summary of the histomorphological assessment of n = 6 mice/group. Mean and SEM, One-way ANOVA/Bonferroni, *P < 0.05 and **P < 0.01 and ***P < 0.001 as indicated, n = 6 mice/group.
Despite various efforts to understand the pathophysiology underlying neointima formation, therapeutic options for the prevention or treatment of in-stent stenosis or restenosis following arterial injury are not available to date. TLR2 antagonists have been suggested for the treatment of cardiovascular disease [26] and we have previously shown that they can mimic the beneficial effects of genetic TLR2 ablation on vascular regeneration and function [16,27]. In this study, we tested the
monocytic characteristics and determine the fate of vascular remodeling in a paracrine manner [3]. Based on this hypothesis and in line with our previous investigations, TLR2 deficiency on these cell types or pharmacological TLR2 blockade would thus support vascular remodeling by resulting in a less inflammatory, more regeneration-facilitating panel of mediators that limits scar formation and thus luminal stenosis after vascular injury. 6
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Fig. 2. (continued)
5. Conclusion
hypothesis that pharmacological blockade of TLR2 initially after chemically induced arterial injury can reduce neointimal hyperplasia and luminal stenosis. Using an established and previously tested TLR2-targeting antibody and an intracardiac route of application on POD 2 and 4 after arterial injury, vascular remodeling 3 weeks after injury revealed reduced lesion formation and luminal stenosis in wild type mice that were comparable to those exhibited in TLR2-deficient mice. Future studies have to evaluate whether blockade of innate immune receptors in the early phase after vascular insults may display a promising therapeutic tool to augment vascular regeneration and patient outcome. Chronic exposure to TLR2 antagonists in contrast appears to less likely become an option for the treatment of cardiovascular disease, since functional TLR2 has also been reported to be an important cornerstone of collateral arterial remodeling and vascular regeneration [28–30].
In summary, this study provides evidence that TLR2 genetic deficiency or pharmacological blockade beneficially modulates neointimal hyperplasia in response to arterial injury and that these effects are primarily mediated by bone marrow-derived circulating cells. Lack of TLR2 signaling in these cells may facilitate arterial regeneration in a paracrine manner. Future studies will have to dissect whether blockade of innate immune receptor signaling can reduce maladaptive vascular regenerative processes and scar formation in the vessel wall by limiting the effects of danger associated molecular patterns released upon tissue injury. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lfs.2019.117189.
Fig. 3. Effects of WT and TLR2-deficient CD117-positive bone marrow cells on smooth muscle cell migration. A Representative images of co-culture of CD117-positive bone marrow cells with smooth muscle cells. Depicted are smooth muscle cell layer scratch wounds 12 h after scratching in the presence of either bone marrow-derived cells from wild type animals (WT) or TLR2-deficient (TLR2−/−) mice or media only serving as vehicle control (ctrl). Bar indicates 100 μm. B Quantitative summary of n = 7 independent experiments. Mean and SEM, One-way ANOVA/Bonferroni, *P < 0.05 and **P < 0.01 and ***P < 0.001 vs. ctrl or as indicated. C Effects of blockade or stimulation of TLR2 on bone marrow derived cells from wild type mice on SMC migration. AB antibody, LTA lipoteichoic acid. Mean and SEM, One-way ANOVA/Bonferroni, *P < 0.05 and ***P < 0.001 vs. ctrl or as indicated.
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Funding
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