Risk factors, treatment and survival rates of late-onset acquired haemophilia A: A cohort study from the Shizuoka Kokuho Database.

INTRODUCTION: Acquired haemophilia A (AHA) is a rare disease. The risk factors have yet to be studied. AIM: We aimed to identify risk factors for late-onset AHA in Japan. METHODS: A population-based cohort study was conducted using data from the Shizuoka Kokuho Database. The study population was defined as individuals aged ≥60 years. Cause-specific Cox regression analysis was performed to calculate hazard ratios. RESULTS: Of 1,160,934 registrants, there were 34 patients with newly diagnosed AHA. The mean follow-up period was 5.6 years, and the incidence of AHA was 5.21 per million person-years. Myocardial infarction, diabetes mellitus, solid tumors, antimicrobial agents, phenytoin and anti-dementia drugs, which showed significant differences in the univariate analysis, were excluded from the multivariable analysis because of the small number of cases. Multivariable regression analysis showed that the presence of Alzheimer's disease (hazard ratio [HR]:4.28, 95% confidence interval [CI]:1.67-10.97) and rheumatic disease (HR:4.65, 95% CI:1.79-12.12) increased the risk of AHA development. CONCLUSION: We found that comorbid Alzheimer's disease is a risk factor of AHA incidence in the general population. Our findings provide insight into the etiology of AHA, and the proof of the coexistence of Alzheimer's disease may support the recent notion that Alzheimer disease is an autoimmune disease.

Potential role of transforming growth factor-beta 1/Smad signaling in secondary lymphedema after cancer surgery.

Secondary lymphedema often develops after cancer surgery, and over 250 million patients suffer from this complication. A major symptom of secondary lymphedema is swelling with fibrosis, which lowers the patient's quality of life, even if cancer does not recur. Nonetheless, the pathophysiology of secondary lymphedema remains unclear, with therapeutic approaches limited to physical or surgical therapy. There is no effective pharmacological therapy for secondary lymphedema. Notably, the lack of animal models that accurately mimic human secondary lymphedema has hindered pathophysiological investigations of the disease. Here, we developed a novel rat hindlimb model of secondary lymphedema and showed that our rat model mimics human secondary lymphedema from early to late stages in terms of cell proliferation, lymphatic fluid accumulation, and skin fibrosis. Using our animal model, we investigated the disease progression and found that transforming growth factor-beta 1 (TGFB1) was produced by macrophages in the acute phase and by fibroblasts in the chronic phase of the disease. TGFB1 promoted the transition of fibroblasts into myofibroblasts and accelerated collagen synthesis, resulting in fibrosis, which further indicates that myofibroblasts and TGFB1/Smad signaling play key roles in fibrotic diseases. Furthermore, the presence of myofibroblasts in skin samples from lymphedema patients after cancer surgery emphasizes the role of these cells in promoting fibrosis. Suppression of myofibroblast-dependent TGFB1 production may therefore represent an effective pharmacological treatment for inhibiting skin fibrosis in human secondary lymphedema after cancer surgery.

The Role of Animal Models in Elucidating the Etiology and Pathology of Abdominal Aortic Aneurysms: Development of a Novel Rupture Mechanism Model.

Existing animal models do not replicate all aspects of abdominal aortic aneurysms (AAAs), including the rupture mechanisms. From histopathological analyses conducted in humans, it has been found that the vasa vasorum of the AAA wall is the starting point of circulatory failure and that bulging and dilatation of the abdominal aorta occurs through inflammation and tissue degeneration. We created a new animal model (the hypoperfusion-induced model) of AAAs. In this study, we describe the current animal models of AAAs and present the utility of our new model of AAAs.

[Spatiotemporal regulation of thrombus formation and dissolution].

Recent advances in real-time imaging techniques have greatly contributed to the full understanding of the role of functional molecules and distinctive cells. When the vascular wall is injured, platelet thrombus is immediately formed, and the subsequent fibrin formation strengthens the thrombus to prevent bleeding. These sequential reactions are well coordinated; therefore, vascular occlusion is less likely to occur as an unnecessary thrombus is not formed or quickly lysed. Real-time imaging analyses in a vascular injury mouse model and in vitro human platelet-containing plasma clot model enabled us to visualize dynamic behaviors of individual factors. In addition to impaired hemostasis, immature lysis due to impaired fibrinolysis inhibition seemed to be involved in the pathological changes in hemophilic arthropathy in hemophilia. Further imaging analysis could elucidate the spatiotemporal crosstalk regulatory mechanisms in more detail with regard to thrombus formation and dissolution.

The Effect of a High-Fat Diet on the Development of Abdominal Aortic Aneurysm in a Vascular Hypoperfusion-Induced Animal Model.

Abdominal aortic aneurysm (AAA) is a vascular disease characterized by chronic inflammation in the infrarenal aorta. Most cases of AAA remain asymptomatic until rupture, and the mortality rate of patients with AAA rupture is very high. Currently, the relation between dietary habits and AAA development remains unknown. In this study, we evaluated the effects of a high-fat diet on the development of AAA in a vascular hypoperfusion-induced animal model. The risk of AAA rupture and AAA diameter in the high-fat group significantly increased compared with those in the control group. The number and size of adipocytes in the vascular wall in the high-fat group significantly increased as compared with those in the control group. Additionally, the collagen-positive sections in the areas with adipocytes significantly decreased as compared with those without adipocytes. The protein levels of matrix metalloproteinase (MMP)-2, MMP-9, and MMP-12, and macrophage-positive areas in the parts with adipocytes also significantly increased as compared with those without adipocytes. These data suggested that AAA rupture risk increased through accelerating chronic inflammation due to the accumulation of adipocytes in the vascular wall in the high-fat group.

Ectopic Expression of PCSK9 by Smooth Muscle Cells Contributes to Aortic Dissection.

BACKGROUND: Acute aortic dissection (AAD) is a common disease among the elderly. Although several risk factors of AAD have been reported, the molecular mechanism underlying AAD development remains to be elucidated. Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases low-density lipoprotein cholesterol levels in blood by preventing its clearance. Therefore, PCSK9 inhibition is a promising therapeutic approach to treat cardiovascular diseases (CVDs). The objective of this study was to elucidate the role of PCSK9 in the pathogenesis of AAD. METHODS: We used fluorescence immunohistochemistry to assess PCSK9 expression in aortic tissues resected from 10 AAD patients and in the normal aorta from 5 autopsy samples as well as in spontaneously hyperlipidemic apolipoprotein E-deficient mice used as an experimental AD model. RESULTS: We revealed a characteristic distribution pattern of PCSK9 in atherosclerotic plaques and the degenerated tunica media in AAD tissues, which was rarely observed in normal aortic tissues. Furthermore, PCSK9 was notably expressed around calcification areas formed by vascular smooth muscle cells, especially those of the synthetic phenotype. The results obtained in the animal model were consistent with PCSK9 expression in AAD tissues. CONCLUSIONS: Our findings suggest that PCSK9 overexpression in the aorta may promote AAD. This study adds to the growing body of evidence supporting the use of PCSK9 inhibitors for the management of CVDs.

Elevated Plasma Levels of LDL Cholesterol Promote Dissecting Thoracic Aortic Aneurysms in Angiotensin II-Induced Mice.

BACKGROUND: Plasma low-density lipoprotein (LDL) cholesterol is implicated in abdominal aorta (AA) and aortic dissection (AD); however, its role in the pathogenesis of AA and AD, a disease with a high mortality rate, is unknown. The existing animal models such as apolipoprotein E-deficient (Apoe-/-) mice cannot reproduce all the conditions of AA/AD, including elevated LDL-cholesterol levels and spontaneous atheroma formation; therefore, a more reliable in vivo model is required. Here, we analyzed angiotensin II (Ang II)-induced mice with combined deficiency of the LDL receptor and the catalytic component of the apolipoprotein B-edisome complex (Ldlr-/-/Apobec1-/- [WKO]) to understand AA formation and AD occurrence in relation to plasma lipid composition. METHODS: AAs and ADs were created in 18- to 22- week-old male Apoe-/- and Ldlr-/-/Apobec1-/- mice by Ang II infusion. Immunostaining allowed assessment of smooth muscle cells and mural monocytes/macrophages. RESULTS: Ldlr-/-/Apobec1-/- mice had elevated LDL-cholesterol levels characteristic for human type IIa hyperlipidemia, resulting in atherogenesis, which promoted mortality, AA formation, and AD development. Interestingly, variations in the distribution of atheromas and inflammatory sites between Apoe-/- and Ldlr-/-/Apobec1-/- mice depending on lipid profiles resulted in differences in AA formation and AD occurrence in the thoracic aorta. CONCLUSIONS: Our results indicate the presence of a pathogenic pathway involving serum lipid composition that plays a key role in AA formation and AD occurrence in Ang II-induced mice.

The incidence and prognosis of thromboembolism associated with oral contraceptives: Age-dependent difference in Japanese population.

AIM: We analyzed the incidence and prognosis of thromboembolism associated with combined oral contraceptives (COCs) by age groups in Japan. METHODS: A total of 581 events of venous thromboembolism (VTE) and arterial thromboembolism (ATE) associated with COCs were analyzed from the Pharmaceuticals and Medical Devices Agency database from 2004 to 2013. In a statistical analysis, a good-prognosis group included recovery cases and a poor-prognosis group involved unrecovered cases with some sequela and fatal cases. The significant difference between these two groups was calculated by Pearson's chi-square test, and the age-specific tendency and the trend of differences in prognosis according to different hormonal contraceptives were examined by Cochran-Armitage trend test. RESULTS: A total of 543 events were analyzed except 38 events due to unknown age, in which DVT only was the most frequent, followed by cerebral infarction, PE with DVT, PE only, cerebral vein thromboses. ATE ratio for overall thromboembolism tended to increase with advancing age (P = 0.0041). Good-prognosis group was common (291 cases in VTE and 83 cases in ATE), followed by poor-prognosis group (46 cases in VTE and 34 cases in ATE). All ATE cases had a significantly poorer prognosis in comparison with all VTE cases (P < 0.0001). Types of progestin and age difference, however, showed no trend in the differences between good-prognosis group and poor-prognosis group (P = 0.3548 and P = 0.6097). CONCLUSION: Thromboembolic events were the most frequent in the 40s. The ATE ratio for overall thromboembolism tended to increase with advancing age. All ATE cases had a significantly poorer prognosis in comparison with all VTE cases.

Platelets Regulate the Migration of Keratinocytes via Podoplanin/CLEC-2 Signaling during Cutaneous Wound Healing in Mice.

Podoplanin is an endogenous ligand for C-type lectin-like receptor 2 (CLEC-2), which is expressed on platelets. Recent evidence indicates that this specific marker of lymphatic endothelial cells is also expressed by keratinocytes at the edge of wounds. However, whether podoplanin or platelets play a role in keratinocyte activity during wound healing remains unknown. We evaluated the effect of podoplanin expression levels on keratinocyte motility using cultured primary normal human epidermal keratinocytes (NHEKs). Down-regulation of podoplanin in NHEKs via transfection with podoplanin siRNA inhibited their migration, indicating that podoplanin plays a mandatory role in this process. In addition, down-regulation of podoplanin was correlated with up-regulation of E-cadherin, suggesting that podoplanin-mediated stimulation of keratinocyte migration is associated with a loss of E-cadherin. Both the addition of platelets and treatment with CLEC-2 inhibited the migration of NHEKs. The down-regulation of RhoA activity and the up-regulation of E-cadherin in keratinocytes were also induced by CLEC-2. In conclusion, these results suggest that podoplanin/CLEC-2 signaling regulates keratinocyte migration via modulating E-cadherin expression through RhoA signaling. Altering the regulation of keratinocyte migration by podoplanin might be a novel therapeutic approach to improve wound healing.

Lysophosphatidylcholine Acyltransferase-3 Expression Is Associated with Atherosclerosis Progression.

Free arachidonic acid (AA) is an important precursor of lipid mediators such as leukotrienes and prostaglandins that induces inflammation and is associated with atherosclerosis progression. Recent studies have shown that lysophosphatidylcholine acyltransferase-3 (LPCAT3) converts lysophosphatidylcholine (LPC) and free AA into phosphatidylcholine (PC)-containing AA (arachidonyl-PC) and thereby can regulate intracellular free-AA levels. However, the association between LPCAT3 and atherosclerosis remains to be established. In this study, we analyzed human and mouse atherosclerotic tissues to gain insight into the arachidonyl-PC metabolism involving LPCAT3 using imaging mass spectrometry. The data revealed a complementary distribution of arachidonyl-PC and LPC in human atherosclerotic tissues with arachidonyl-PC decreasing and LPC increasing as atherosclerosis progressed. Furthermore, we found a homologous distribution of LPCAT3 expression and arachidonyl-PC based on atherosclerotic progression. In contrast, in ApoE-deficient mice, atherosclerosis increased both arachidonyl-PC accumulation and LPCAT3 expression. Taken together, these findings suggest that the regulation of LPCAT3 expression might be associated with atherosclerotic progression in humans.

The preventive effect of fish oil on abdominal aortic aneurysm development.

Abdominal aortic aneurysm (AAA) is a vascular disease involving gradual dilation of the abdominal aorta and high rupture-related mortality rates. AAA is histologically characterized by oxidative stress, chronic inflammation, and extracellular matrix degradation in the vascular wall. We previously demonstrated that aortic hypoperfusion could cause the vascular inflammation and AAA formation. However, the preventive method for hypoperfusion-induced AAA remains unknown. In this study, we evaluated the effect of fish oil on AAA development using a hypoperfusion-induced AAA animal model. Dilation of the abdominal aorta in the fish oil administration group was smaller than in the control group. Collagen destruction and oxidative stress were suppressed in the fish oil administration group than in the control group. These results suggested that fish oil could prevent the development of AAA induced by hypoperfusion.

[Recent progress and perspectives in the field of fibrinolysis].

Employing newly developed imaging techniques, the spatio-temporal regulatory mechanism of fibrinolysis by platelets and vascular endothelial cells has been clarified. Upon activation, platelets release polyphosphate and inhibitors of fibrinolysis, and modify fibrin fibers which are relatively resistant to fibrinolysis. Platelets, in turn, initiate fibrinolysis. The details of the mechanism by which vascular endothelial cells (VECs) maintain high fibrinolytic potential, as well as how VECs develop anti-fibrinolytic activity, have also been clarified. These mechanisms as well as their modifications in response to inflammation are discussed herein. Newly developed drugs targeting fibrinolytic components are also introduced.

In vivo imaging visualizes discoid platelet aggregations without endothelium disruption and implicates contribution of inflammatory cytokine and integrin signaling.

The mechanism by which thrombotic vessel occlusion occurs independently of plaque development or endothelial cell (EC) disruption remains unclear, largely because of an inability to visualize the formation of thrombus, especially at the single-platelet level in real time. Here we demonstrate that rapidly developing thrombi composed of discoid platelets can be induced in the mesenteric capillaries, arterioles, and large-sized arteries of living mice, enabling characterization of the kinetics of thrombosis initiation and the multicellular interrelationships during thrombus development. Platelet aggregation without EC disruption was triggered by reactive oxygen species (ROS) photochemically induced by moderate power laser irradiation. The inflammatory cytokines TNF-α and IL-1 could be key components of the EC response, acting through regulation of VWF mobilization to the cell surface. Thrombus formation was then initiated by the binding of platelet GPIbα to endothelial VWF in our model, and this effect was inhibited by the ROS scavenger N-acetylcysteine. Actin linker talin-dependent activation of alphaIIb-beta3 integrin or Rac1 in platelets was required for late-phase thrombus stability. Our novel imaging technology illustrates the molecular mechanism underlying inflammation-based thrombus formation by discoid platelets on undisrupted ECs and suggests control of ROS could be a useful therapeutic target for the prevention of thrombotic diseases.

[Basic knowledge on coagulation and fibrinolysis to understand thrombus-formation machinery].

Recent advances in genetic engineering and optical instrumentation have largely contributed to a better understanding of nicely organized spatiotemporal regulations of coagulation and fibrinolysis. In-vivo real time imaging analyses revealed how only partially activated platelets interact with vascular endothelial cells under blood flow, and how platelets are differently activated to form stable thrombus having limited size. Interactions between plasma components and vascular endothelial cells(VECs) are also analyzed by real time imaging, and the mechanism to keep high fibrinolytic potential on VECs is well documented. In this article, we review these recent progresses in basic research on coagulation and fibrinolysis, which were mainly achieved by imaging technique.

Evaluation of thrombomodulin/thrombin activatable fibrinolysis inhibitor function in plasma using tissue-type plasminogen activator-induced plasma clot lysis time.

Background: Thrombin activatable fibrinolysis inhibitor (TAFI) is one of the most important physiological fibrinolysis inhibitors. Its inhibitory efficacy under physiological conditions remains uncertain. Objectives: Elucidate the role of soluble thrombomodulin (sTM)/TAFI axis in the regulation of fibrinlysis. Methods: Since thrombin is required to generate activated TAFI (TAFIa) that targets the C-terminal lysine of partially digested fibrin, a clot lysis assay is suitable for evaluating its function. Using tissue-type plasminogen activator-induced plasma clot lysis time (tPA-PCLT) together with TAFIa inhibitor and recombinant sTM (rsTM), we evaluated the specific function of TM/TAFI in the plasma milieu. Results: tPA-PCLT values were significantly shortened by the TAFIa inhibitor. rsTM supplementation prolonged tPA-PCLT, which was shortened by the TAFIa inhibitor to a time similar to that obtained without rsTM and with the TAFIa inhibitor. Plasma obtained from patients treated with rsTM showed prolonged tPA-PCLT, which was shortened by the TAFIa inhibitor but not further prolonged by rsTM. However, no significant correlation was observed between tPA-PCLT and parameters of TM/TAFI system in the plasma. Conclusion: The role of the TM/TAFI system in regulating fibrinolysis was successfully evaluated using TAFIa inhibitor and rsTM. Trace amounts of soluble TM in normal plasma appeared sufficient to activate TAFI and inhibit fibrinolysis. Further, a therapeutic dose of rsTM appeared sufficient to activate TAFI and regulate fibrinolysis in the plasma milieu.

Surface-retained tPA is essential for effective fibrinolysis on vascular endothelial cells.

In a previous study, we demonstrated unique secretory dynamics of tissue plasminogen activator (tPA) in which tPA was retained on the cell surface in a heavy chain-dependent manner after exocytosis from secretory granules in vascular endothelial cells. Here, we examined how retained tPA expresses its enzymatic activity. Retained tPA effectively increased the lysine binding site-dependent binding of plasminogen on the cell surface and pericellular area; this was abolished by inhibition of enzymatic activity of either tPA or plasmin, which suggests that de novo generation of carboxyl-terminal lysine as a consequence of degradation of surface/pericellular proteins by plasmin is essential. Retained tPA initiated zonal clot lysis of a fibrin network that had been formed on vascular endothelial cells, which was preceded by the binding of plasminogen to the lysis front. Our results provide evidence that secreted and retained tPA is essential for maintaining both high fibrinolytic activity and effective clot lysis on the vascular endothelial cell surface.

Antithrombotic properties of water-soluble carbon monoxide-releasing molecules.

OBJECTIVE: We compared the antithrombotic effects in vivo of 2 chemically different carbon monoxide-releasing molecules (CORM-A1 and CORM-3) on arterial and venous thrombus formation and on hemostatic parameters such as platelet activation, coagulation, and fibrinolysis. The hypotensive response to CORMs and their effects on whole blood gas analysis and blood cell count were also examined. METHODS AND RESULTS: CORM-A1 (10-30 µmol/kg, i.v.), in a dose-dependent fashion, significantly decreased weight of electrically induced thrombus in rats, whereas CORM-3 inhibited thrombosis only at the highest dose used (30 µmol/kg). CORM-A1 showed a direct and stronger inhibition of platelet aggregation than CORM-3 in healthy rats, both in vitro and in vivo. The antiaggregatory effect of CORM-A1, but not CORM-3, correlated positively with weight of the thrombus. Concentration of active plasminogen activator inhibitor-1 in plasma also decreased in response to CORM-A1, but not to CORM-3. Neither CORM-A1 nor CORM-3 had an effect on plasma concentration of active tissue plasminogen activator. CORM-3, but not CORM-A1, decreased the concentration of fibrinogen, fibrin generation, and prolonged prothrombin time. Similarly, laser-induced venous thrombosis observed intravitally via confocal system in green fluorescent protein mice was significantly decreased by CORMs. Although both CORM-A1 and CORM-3 (30 µmol/kg) decreased platelets accumulation in thrombus, only CORM-A1 (3-30 µmol/kg) inhibited platelet activation to phosphatidylserine on their surface. CONCLUSIONS: CORM-3 and CORM-A1 inhibited thrombosis in vivo, however CORM-A1, which slowly releases carbon monoxide, and displayed a relatively weak hypotensive effect had a more pronounced antithrombotic effect associated with a stronger inhibition of platelet aggregation associated with a decrease in active plasminogen activator inhibitor-1 concentration. In contrast, the fast CO releaser CORM-3 that displayed a more pronounced hypotensive effect inhibited thrombosis primarily through a decrease in fibrin generation, but had no direct influence on platelet aggregation and fibrynolysis.

Visualization of Domain- and Concentration-Dependent Impact of Thrombomodulin on Differential Regulation of Coagulation and Fibrinolysis.

BACKGROUND: Thrombomodulin (TM) functions as a dual modulator-anticoagulant and antifibrinolytic potential-by the thrombin-dependent activation of protein C and thrombin-activatable fibrinolysis inhibitor (TAFI). Activated TAFI cleaves the C-terminal lysine of partially degraded fibrin and inhibits both plasminogen binding and its activation on the fibrin surface. We have reported previously that activated platelets initiate fibrin network formation and trigger fibrinolysis after the accumulation of tissue-type plasminogen activator and plasminogen. OBJECTIVE: To analyze the effects of domain-deletion variants of TM on coagulation and fibrinolysis at different concentrations. METHODS: Domain-deletion variants of TM, such as D123 (all extracellular regions), E3456 (minimum domains for thrombin-dependent activation of protein C and TAFI), and E456 (minimum domains for that of protein C but not TAFI), were used at 0.25 to 125 nM for turbidimetric assay to determine the clotting time and clot lysis time and to visualize fibrin network formation and lysis in platelet-containing plasma. RESULTS AND CONCLUSIONS: A low concentration of either D123 or E3456, but not of E456, prolonged clot lysis time, and delayed the accumulation of fluorescence-labeled plasminogen at the activated platelets/dense fibrin area due to effective TAFI activation. Conversely, only the highest concentrations of all three TM variants delayed the clotting time, though fibrin network formation in the vicinity of activated platelets was almost intact. TAFI activation might be affected by attenuation in thrombin activity after the clot formation phase. These findings suggest that the spatiotemporal balance between the anticoagulant and antifibrinolytic potential of TM is controlled in domain- and concentration-dependent manners.

Administration of Tranexamic Acid After Burr Hole Craniotomy Reduced Postoperative Recurrence of Chronic Subdural Hematoma in a Japanese Regional Population.

BACKGROUND: Although tranexamic acid (TXA) has occasionally been used to prevent postoperative recurrence of chronic subdural hematoma (CSDH) after burr hole craniotomy (BC), robust evidence of its efficacy has been lacking. OBJECTIVE: To assess the efficacy and safety of postoperative oral administration of TXA after BC for CSDH among the elderly. METHODS: This retrospective, propensity score-matched cohort study was carried out with a large Japanese local population-based longitudinal cohort in the Shizuoka Kokuho Database between April 2012 and September 2020. Patients included were age 60 years or older and had undergone BC for CSDH but were not undergoing dialysis. Covariates were collected from records of the preceding 12 months from the month of first BC, and patients were followed up for 6 months after surgery. The primary outcome was repeat surgery, and the secondary outcome was death or the onset of thrombosis. Data on postoperative TXA administration were collected and compared with controls using propensity score matching. RESULTS: Of the 8544 patients who underwent BC for CSDH, 6647 were included, with 473 placed in the TXA group and 6174 placed in the control group. After 1:1 matching, repeated BC was found to have been performed in 30 of 465 patients (6.5%) in the TXA group and in 78 of 465 patients (16.8%) in the control group (relative risk, 0.38; 95% CI, 0.26-0.56). No significant difference was observed for death or the onset of thrombosis. CONCLUSION: Oral administration of TXA reduced the occurrence of repeat surgery after BC for CSDH.