Pathogenic Basis of Thromboinflammation and Endothelial Injury in COVID-19 : Current Findings and Therapeutic Implications

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic with a great impact on social and economic activities, as well as public health. In most patients, the symptoms of COVID-19 are a high-grade fever and a dry cough, and spontaneously resolve within ten days. However, in severe cases, COVID-19 leads to atypical bilateral interstitial pneumonia, acute respiratory distress syndrome, and systemic thromboembolism, resulting in multiple organ failure with high mortality and morbidity. SARS-CoV-2 has immune evasion mechanisms, including inhibition of interferon signaling and suppression of T cell and B cell responses. SARS-CoV-2 infection directly and indirectly causes dysregulated immune responses, platelet hyperactivation, and endothelial dysfunction, which interact with each other and are exacerbated by cardiovascular risk factors. In this review, we summarize current knowledge on the pathogenic basis of thromboinflammation and endothelial injury in COVID-19. We highlight the distinct contributions of dysregulated immune responses, platelet hyperactivation, and endothelial dysfunction to the pathogenesis of COVID-19. In addition, we discuss potential therapeutic strategies targeting these mechanisms

Inhibition of activated factor X by rivaroxaban attenuates neointima formation after wire-mediated vascular injury

Accumulating evidence suggests that activated factor X (FXa), a key coagulation factor, plays an important role in the development of vascular inflammation through activation of many cell types. Here, we investigated whether pharmacological blockade of FXa attenuates neointima formation after wire-mediated vascular injury. Transluminal femoral artery injury was induced in C57BL/6 mice by inserting a straight wire. Rivaroxaban (5 mg/kg/day), a direct FXa inhibitor, was administered from one week before surgery until killed. At four weeks after surgery, rivaroxaban significantly attenuated neointima formation in the injured arteries compared with control (P<0.01). Plasma lipid levels and blood pressure were similar between the rivaroxaban-treated group and non-treated group. Quantitative RT-PCR analyses demonstrated that rivaroxaban reduced the expression of inflammatory molecules (e.g., IL-1β and TNF-α) in injured arteries at seven days after surgery (P<0.05, respectively). In vitro experiments using mouse peritoneal macrophages demonstrated that FXa increased the expression of inflammatory molecules (e.g., IL-1β and TNF-α), which was blocked in the presence of rivaroxaban (P<0.05). Also, in vitro experiments using rat vascular smooth muscle cells (VSMC) demonstrated that FXa promoted both proliferation and migration of this cell type (P<0.05), which were blocked in the presence of rivaroxaban. Inhibition of FXa by rivaroxaban attenuates neointima formation after wire-mediated vascular injury through inhibition of inflammatory activation of macrophages and VSMC

P2Y12阻害薬チカグレロールはアポリポ蛋白E欠損マウスの血管障害を軽減して動脈硬化形成を抑制する

Background and aims: Ticagrelor reduces cardiovascular events in patients with acute coronary syndrome (ACS). Recent studies demonstrated the expression of P2Y12 on vascular cells including endothelial cells, as well as platelets, and suggested its contribution to atherogenesis. We investigated whether ticagrelor attenuates vascular dysfunction and inhibits atherogenesis in apolipoprotein E-deficient (apoe-/-) mice. Methods: Eight-week-old male apoe-/- mice were fed a western-type diet (WTD) supplemented with 0.1% ticagrelor (approximately 120 mg/kg/day). Non-treated animals on WTD served as control. Atherosclerotic lesions were examined by en-face Sudan IV staining, histological analyses, quantitative RT-PCR analysis, and western blotting. Endothelial function was analyzed by acetylcholine-dependent vasodilation using aortic rings. Human umbilical vein endothelial cells (HUVEC) were used for in vitro experiments. Results: Ticagrelor treatment for 20 weeks attenuated atherosclerotic lesion progression in the aortic arch compared with control (p < 0.05). Ticagrelor administration for 8 weeks attenuated endothelial dysfunction (p < 0.01). Ticagrelor reduced the expression of inflammatory molecules such as vascular cell adhesion molecule-1, macrophage accumulation, and lipid deposition. Ticagrelor decreased the phosphorylation of JNK in the aorta compared with control (p < 0.05). Ticagrelor and a JNK inhibitor ameliorated impairment of endothelium-dependent vasodilation by adenosine diphosphate (ADP) in wild-type mouse aortic segments. Furthermore, ticagrelor inhibited the expression of inflammatory molecules which were promoted by ADP in HUVEC (p < 0.001). Ticagrelor also inhibited ADP-induced JNK activation in HUVEC (p < 0.05). Conclusions: Ticagrelor attenuated vascular dysfunction and atherogenesis through the inhibition of inflammatory activation of endothelial cells. These effects might be a potential mechanism by which ticagrelor decreases cardiovascular events in patients with ACS

DPP-4阻害薬のひとつリナグリプチンは，正常血糖アポリポ蛋白E欠損マウスにおける血管内皮機能を改善し，動脈硬化を抑制する

Background: Dipeptidyl peptidase-4 (DPP-4) inhibitors have vasoprotective effects. This study investigated whether a recently approved DPP-4 inhibitor, linagliptin (Lina), suppresses atherogenesis in non-diabetic apolipoprotein-E deficient (ApoE−/−) mice, and examined its effects on endothelial function. Methods and results: Lina (10 mg/kg/day) was administered orally to ApoE−/− mice for 20 weeks. Lina reduced atherogenesis without alteration of metabolic parameters including blood glucose level compared with control (P b 0.05). Results of immunohistochemical analyses and quantitative RT-PCR demonstrated that Lina significantly decreased inflammatory molecule expression and macrophage infiltration in the atherosclerotic aorta. Lina administration to ApoE−/− mice for 9 weeks ameliorated endothelium-dependent vasodilation compared with that in untreated mice. Plasma active glucagon-like peptide-1 (GLP-1) level was significantly higher in the treated group (P b 0.05). Exendin-4 (Ex-4), a GLP-1 analog, ameliorated endothelium-dependent vasodilation impaired by palmitic acid (PA) in wild-type mouse aortic segments. Ex-4 promoted phosphorylation of eNOSSer1177 and Akt, both of which were abrogated by PA, in human umbilical vein endothelial cells. In addition, Lina administration to ApoE−/− mice decreased oxidative stress, as determined by urinary 8-OHdG secretion and NADPH oxidase subunit expression in the abdominal aorta. Conclusion: Lina inhibited atherogenesis in non-diabetic ApoE−/− mice. Amelioration of endothelial dysfunction associated with a reduction of oxidative stress by GLP-1 contributes to the atheroprotective effects of Lina

TLR9 and Blood Flow Recovery

Background: Peripheral artery disease causes significant functional disability and results in impaired quality of life. Ischemic tissue injury releases various endogenous ligands for Toll-like receptors (TLRs), suggesting the involvement of TLRs in blood flow recovery. However, the role of TLR9, which was originally known as a sensor for bacterial DNA, remains unknown. This study investigated the role of TLR9 in blood flow recovery in the ischemic limb using a mouse hind-limb ischemia model. Methods and Results: Unilateral femoral artery ligation was performed in TLR9-deficient (Tlr9−/−) mice and wild-type mice. In wild-type mice, femoral artery ligation significantly increased mRNA expression of TLR9 in the ischemic limb (P < 0.001) and plasma levels of cell-free DNA (cfDNA) as determined by single-stranded DNA (ssDNA) (P < 0.05) and double-stranded DNA (dsDNA) (P < 0.01), which are endogenous ligands for TLR9, compared with the sham-operated group. Laser Doppler perfusion imaging demonstrated significantly improved ratio of blood flow in the ischemic to non-ischemic limb in Tlr9−/− mice compared with wild-type mice at 2 weeks after ligation (P < 0.05). Tlr9−/− mice showed increased capillary density and reduced macrophage infiltration in ischemic limb. Genetic deletion of TLR9 reduced the expression of TNF-α, and attenuated NF-kB activation in ischemic muscle compared with wild-type mice (P < 0.05, respectively) at 3 days after the surgery. ODN1826, a synthetic agonistic oligonucleotide for TLR9, or plasma obtained from mice with ischemic muscle promoted the expression of TNF-α in wild-type macrophages (P < 0.05), but not in Tlr9−/− macrophages. ODN1826 also activated NF-kB signaling as determined by the degradation of IkBα in wild-type macrophages (P < 0.05), but not in Tlr9−/− macrophages. In vitro experiments using human umbilical vein endothelial cells demonstrated that TNF-α, or conditioned medium obtained from wild-type macrophages treated with ODN1826 accelerated cell death as determined by MTS assay (P < 0.05 and P < 0.01, respectively). Conclusion: Our results suggest that ischemic muscle releases cfDNA, which activates TLR9 and enhances inflammation, leading to impairment of blood flow recovery in the ischemic limb. cfDNA-TLR9 signaling may serve as a potential therapeutic target in ischemic limb disease

Activation of Toll-Like Receptor 9 Impairs Blood Flow Recovery After Hind-Limb Ischemia

Background: Peripheral artery disease causes significant functional disability and results in impaired quality of life. Ischemic tissue injury releases various endogenous ligands for Toll-like receptors (TLRs), suggesting the involvement of TLRs in blood flow recovery. However, the role of TLR9, which was originally known as a sensor for bacterial DNA, remains unknown. This study investigated the role of TLR9 in blood flow recovery in the ischemic limb using a mouse hind-limb ischemia model.Methods and Results: Unilateral femoral artery ligation was performed in TLR9-deficient (Tlr9−/−) mice and wild-type mice. In wild-type mice, femoral artery ligation significantly increased mRNA expression of TLR9 in the ischemic limb (P &lt; 0.001) and plasma levels of cell-free DNA (cfDNA) as determined by single-stranded DNA (ssDNA) (P &lt; 0.05) and double-stranded DNA (dsDNA) (P &lt; 0.01), which are endogenous ligands for TLR9, compared with the sham-operated group. Laser Doppler perfusion imaging demonstrated significantly improved ratio of blood flow in the ischemic to non-ischemic limb in Tlr9−/− mice compared with wild-type mice at 2 weeks after ligation (P &lt; 0.05). Tlr9−/− mice showed increased capillary density and reduced macrophage infiltration in ischemic limb. Genetic deletion of TLR9 reduced the expression of TNF-α, and attenuated NF-κB activation in ischemic muscle compared with wild-type mice (P &lt; 0.05, respectively) at 3 days after the surgery. ODN1826, a synthetic agonistic oligonucleotide for TLR9, or plasma obtained from mice with ischemic muscle promoted the expression of TNF-α in wild-type macrophages (P &lt; 0.05), but not in Tlr9−/− macrophages. ODN1826 also activated NF-κB signaling as determined by the degradation of IκBα in wild-type macrophages (P &lt; 0.05), but not in Tlr9−/− macrophages. In vitro experiments using human umbilical vein endothelial cells demonstrated that TNF-α, or conditioned medium obtained from wild-type macrophages treated with ODN1826 accelerated cell death as determined by MTS assay (P &lt; 0.05 and P &lt; 0.01, respectively).Conclusion: Our results suggest that ischemic muscle releases cfDNA, which activates TLR9 and enhances inflammation, leading to impairment of blood flow recovery in the ischemic limb. cfDNA-TLR9 signaling may serve as a potential therapeutic target in ischemic limb disease

肥満によって遊離する脂肪細胞由来のDNA断片が脂肪組織の炎症とインスリン抵抗性を引き起こす

Obesity stimulates chronic inflammation in adipose tissue, which is associated with insulin resistance, although the underlying mechanism remains largely unknown. Here we showed that obesity-related adipocyte degeneration causes release of cell-free DNA (cfDNA), which promotes macrophage accumulation in adipose tissue via Toll-like receptor 9 (TLR9), originally known as a sensor of exogenous DNA fragments. Fat-fed obese wild-type mice showed increased release of cfDNA, as determined by the concentrations of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in plasma. cfDNA released from degenerated adipocytes promoted monocyte chemoattractant protein-1 (MCP-1) expression in wild-type macrophages, but not in TLR9-deficient (Tlr9−/−) macrophages. Fat-fed Tlr9−/− mice demonstrated reduced macrophage accumulation and inflammation in adipose tissue and better insulin sensitivity compared with wild-type mice, whereas bone marrow reconstitution with wild-type bone marrow restored the attenuation of insulin resistance observed in fat-fed Tlr9−/− mice. Administration of a TLR9 inhibitory oligonucleotide to fat-fed wild-type mice reduced the accumulation of macrophages in adipose tissue and improved insulin resistance. Furthermore, in humans, plasma ssDNA level was significantly higher in patients with computed tomography–determined visceral obesity and was associated with homeostasis model assessment of insulin resistance (HOMA-IR), which is the index of insulin resistance. Our study may provide a novel mechanism for the development of sterile inflammation in adipose tissue and a potential therapeutic target for insulin resistance

Role of Toll-Like Receptor 9 in Atherogenesis

Background Toll-like receptor (TLR) 9 recognizes bacterial DNA, activating innate immunity, whereas it also provokes inflammation in response to fragmented DNA released from mammalian cells. We investigated whether TLR9 contributes to the development of vascular inflammation and atherogenesis using apolipoprotein E–deficient (Apoe-/-) mice. Methods and Results Tlr9-deficient Apoe-/- (Tlr9-/-Apoe-/-) mice and Apoe-/- mice on a Western-type diet received subcutaneous angiotensin II infusion (1000 ng/kg per minute) for 28 days. Angiotensin II increased the plasma level of double-stranded DNA, an endogenous ligand of TLR9, in these mice. Genetic deletion or pharmacologic blockade of TLR9 in angiotensin II–infused Apoe-/- mice attenuated atherogenesis in the aortic arch (P<0.05), reduced the accumulation of lipid and macrophages in atherosclerotic plaques, and decreased RNA expression of inflammatory molecules in the aorta with no alteration of metabolic parameters. On the other hand, restoration of TLR9 in bone marrow in Tlr9-/-Apoe-/- mice promoted atherogenesis in the aortic arch (P<0.05). A TLR9 agonist markedly promoted proinflammatory activation of Apoe-/- macrophages, partially through p38 mitogen-activated protein kinase signaling. In addition, genomic DNA extracted from macrophages promoted inflammatory molecule expression more effectively in Apoe-/- macrophages than in Tlr9-/-Apoe-/- macrophages. Furthermore, in humans, circulating double-stranded DNA in the coronary artery positively correlated with inflammatory features of coronary plaques determined by optical coherence tomography in patients with acute myocardial infarction (P<0.05). Conclusions TLR9 plays a pivotal role in the development of vascular inflammation and atherogenesis through proinflammatory activation of macrophages. TLR9 may serve as a potential therapeutic target for atherosclerosis