Histopathological Features of Cancer-Associated Venous Thromboembolism: Presence of Intrathrombus Cancer Cells and Prothrombotic Factors.

BACKGROUND: Cancer-associated venous thromboembolism (VTE) is a critical complication in patients with cancer. However, the pathological findings of VTE are limited. Here, we investigated the histopathological features of cancer-associated VTE in human autopsy cases. METHODS: We clinically examined the autopsy cases of VTE with (n=114) and without cancer (n=66) and immunohistochemically analyzed the expression of prothrombotic factors in intrathrombus cancer cells, the thrombus contents of erythrocytes, fibrin, platelets, citrullinated histone H3, and degree of organization. RESULTS: Vascular wall invasion or small cell clusters of cancer cells was observed in thrombi in 27.5% of deep vein thrombosis and 25.9% of pulmonary embolism cases. The majority of the cancer cells in deep vein thrombi appeared to be invading the vessel wall, whereas the majority of pulmonary thrombi had cancer cell clusters, consistent with embolization via blood flow. These cancer cells were immunohistochemically positive for TF (tissue factors) or podoplanin in up to 88% of VTE cases. The frequency of TF-positive monocyte/macrophages in thrombi was higher in cancer-associated VTE than that in VTE without cancer. Citrullinated histone H3 was predominantly observed in the early stages of organizing thrombi. There was no significant difference in thrombus components between VTE with cancer and without cancer groups. CONCLUSIONS: Vascular wall invasion or cancer cell clusters in thrombi might influence thrombogenesis of cancer-associated VTE. TF and podoplanin in cancer cells and in monocyte/macrophages may induce coagulation reactions and platelet aggregation. Neutrophil extracellular traps may play a role in the early stages of VTE, regardless of cancer status.

Massive platelet-rich thrombus formation in small pulmonary vessels in amniotic fluid embolism: An autopsy study.

OBJECTIVE: To identify pulmonary/uterine thrombus formation in amniotic fluid embolism (AFE). DESIGN: Retrospective, observational. SETTING: Nationwide. POPULATION: Eleven autopsy cases of AFE and control cases. METHODS: We assessed pulmonary and uterine thrombus formation and thrombus area in AFE and pulmonary thromboembolism (PTE) as a control. The area of platelet glycoprotein IIb/IIIa, fibrin, neutrophil elastase, citrullinated histone H3 (a neutrophil extracellular trap marker) and mast cell chymase immunopositivity was measured in 90 pulmonary emboli, 15 uterine thrombi and 14 PTE. MAIN OUTCOME MEASURES: Pathological evidence of thrombus formation and its components in AFE. RESULTS: Amniotic fluid embolism lung showed massive thrombus formation, with or without amniotic emboli in small pulmonary arteries and capillaries. The median pulmonary thrombus size in AFE (median, 0.012 mm2 ; P < 0.0001) was significantly smaller than that of uterine thrombus in AFE (0.61 mm2 ) or PTE (29 mm2 ). The median area of glycoprotein IIb/IIIa immunopositivity in pulmonary thrombi in AFE (39%; P < 0.01) was significantly larger than that of uterine thrombi in AFE (23%) and PTE (15%). The median area of fibrin (0%; P < 0.001) and citrullinated histone H3 (0%; P < 0.01) immunopositivity in pulmonary thrombi in AFE was significantly smaller than in uterine thrombi (fibrin: 26%; citrullinated histone H3: 1.1%) and PTE (fibrin: 42%; citrullinated histone H3: 0.4%). No mast cells were identified in pulmonary thrombi. CONCLUSIONS: Amniotic fluid may induce distinct thrombus formation in the uterus and lung. Pulmonary and uterine thrombi formation may contribute to cardiorespiratory collapse and/or consumptive coagulopathy in AFE.

Underlying mechanisms of thrombus formation/growth in atherothrombosis and deep vein thrombosis.

Thrombosis remains a leading cause of death worldwide despite technological advances in prevention, diagnosis, and treatment. The traditional view of arterial thrombus formation is that it is a platelet-dependent process, whereas that of venous thrombus formation is a coagulation-dependent process. Current pathological and basic studies on atherothrombosis and venous thrombosis have revealed the diverse participation of platelet and coagulation activation mechanisms in both thrombus initiation and growth processes during clinical thrombotic events. Atherosclerotic plaque cell-derived tissue factor contributes to fibrin formation and platelet aggregation. The degree of plaque disruption and a blood flow alteration promote atherothrombotic occlusion. While blood stasis/turbulent flow due to luminal stenosis itself initiates venous thrombus formation. The coagulation factor XI-driven propagation phase of blood coagulation plays a major role in venous thrombus growth, but a minor role in hemostasis. These lines of evidence indicate that atherothrombosis onset is affected by the thrombogenic potential of atherosclerotic plaques, the plaque disruption size, and an alteration in blood flow. Upon onset of venous thrombosis, enhancement of the propagation phase of blood coagulation under blood stasis and a hypercoagulable state contribute to large thrombus formation.

Association between fetal vascular malperfusion and gestational diabetes.

AIM: Diabetes mellitus (DM) is a major complication in pregnancy. Placental lesions with DM remain unclear and controversial. Recently, the terms of placental pathological findings, such as maternal and fetal vascular malperfusions (MVM and FVM, respectively) were introduced by the Amsterdam Placental Workshop Group Consensus Statement (APWGCS). FVM cases were classified as the partial obstruction type (global FVM) and the complete obstruction type (segmental FVM). The aim of this study was to clarify the pathological characteristics of the placenta with pregestational DM/gestational DM; GDM according to APWGCS. METHODS: We studied the placentas of 182 DM women (27 pregestational DM and 155 GDM) and control placentas of 460 women without DM during 2011-2018. We excluded cases of intrauterine fetal death or multiple pregnancies. We reviewed microscopical findings including, MVM, FVM, chorioamnionitis with the slides according to the APWGCS. RESULTS: Microscopically, the incidence of FVM was significantly higher in GDM patients than control (17% vs. 10%, p = 0.0138), but not significant in pregestational DM (11%, p = 0.7410). Segmental FVM (complete obstruction) was significantly more observed in GDM than control group (5% vs. 0.4%, p = 0.0013). Segmental FVM in GDM showed high incidence of light-for-dates infant (three of seven cases, 43%, p = 0.0288). In addition, several segmental FVM findings (villous stromal-vascular karyorrhexis and stem vessel occlusion) were frequently noted in 2 or 3 points positive of 75 g oral glucose tolerance test than 1 point positive GDM. CONCLUSION: Our placental findings suggest disorder of carbohydrate metabolism might affect the fetal vascular damage, especially complete fetal vascular obstruction.

Tissue factor expression and tumor-infiltrating T lymphocytes in ovarian carcinomas and their association with venous thromboembolism.

Ovarian cancer is a known risk factor of venous thromboembolism (VTE). Thrombogenic factor expression and lymphocytic infiltrate have been reported in endometriosis and ovarian cancers. We reviewed 30 cases of ovarian carcinomas (high grade serous carcinoma, 10; endometrioid carcinoma, 10; clear cell carcinoma (CCC), 10) and 16 endometriotic lesions. We immunohistochemically investigated the expressions of tissue factor (TF), podoplanin, P-selectin, and number of CD4 and CD8 positive lymphocytes in cancer tissue and endometriotic lesions, along with their relationship with VTE. The expression of TF was higher in CCC. The TF expression and the number of CD8 positive cells were higher in cancer tissues with VTE than in those without VTE. The podoplanin or P-selectin expression did not differ among histological types or between cases with and without VTE. Our results demonstrated a high TF expression and intraepithelial CD8 cells in CCC, which were associated with VTE. The results suggest that infiltrating lymphocytes may affect TF expression that, in turn, influences VTE.

Interaction between cardiac myosin-binding protein C and formin Fhod3.

Mutations in cardiac myosin-binding protein C (cMyBP-C) are a major cause of familial hypertrophic cardiomyopathy. Although cMyBP-C has been considered to regulate the cardiac function via cross-bridge arrangement at the C-zone of the myosin-containing A-band, the mechanism by which cMyBP-C functions remains unclear. We identified formin Fhod3, an actin organizer essential for the formation and maintenance of cardiac sarcomeres, as a cMyBP-C-binding protein. The cardiac-specific N-terminal Ig-like domain of cMyBP-C directly interacts with the cardiac-specific N-terminal region of Fhod3. The interaction seems to direct the localization of Fhod3 to the C-zone, since a noncardiac Fhod3 variant lacking the cMyBP-C-binding region failed to localize to the C-zone. Conversely, the cardiac variant of Fhod3 failed to localize to the C-zone in the cMyBP-C-null mice, which display a phenotype of hypertrophic cardiomyopathy. The cardiomyopathic phenotype of cMyBP-C-null mice was further exacerbated by Fhod3 overexpression with a defect of sarcomere integrity, whereas that was partially ameliorated by a reduction in the Fhod3 protein levels, suggesting that Fhod3 has a deleterious effect on cardiac function under cMyBP-C-null conditions where Fhod3 is aberrantly mislocalized. Together, these findings suggest the possibility that Fhod3 contributes to the pathogenesis of cMyBP-C-related cardiomyopathy and that Fhod3 is critically involved in cMyBP-C-mediated regulation of cardiac function via direct interaction.

Pathophysiology of atherothrombosis: Mechanisms of thrombus formation on disrupted atherosclerotic plaques.

Atherothrombosis is a leading cause of cardiovascular mortality and morbidity worldwide. The underlying mechanisms of atherothrombosis comprise plaque disruption and subsequent thrombus formation. Arterial thrombi are thought to mainly comprise aggregated platelets as a result of high blood velocity. However, thrombi that develop on disrupted plaques comprise not only aggregated platelets, but also large amounts of fibrin, because plaques contain large amount of tissue factor that activate the coagulation cascade. Since not all thrombi grow large enough to occlude the vascular lumen, the propagation of thrombi is also critical in the onset of adverse vascular events. Various factors such as vascular wall thrombogenicity, local hemorheology, systemic thrombogenicity and fibrinolytic activity modulate thrombus formation and propagation. Although the activation mechanisms of platelets and the coagulation cascade have been intensively investigated, the underlying mechanisms of occlusive thrombus formation on disrupted plaques remain obscure. Pathological findings derived from humans and animal models of human atherothrombosis have uncovered pathophysiological processes during thrombus formation and propagation after plaque disruption, and novel factors have been identified that modulate the activation of platelets and the coagulation cascade. These findings have also provided insights into the development of novel drugs for atherothrombosis.

Chondroitin sulfate N-acetylgalactosaminyltransferase-2 deletion alleviates lipoprotein retention in early atherosclerosis and attenuates aortic smooth muscle cell migration.

Glycosaminoglycans (GAGs) play an integral role in low-density lipoprotein (LDL) retention in the vascular intimal layer and have emerged as attractive therapeutic targets for atherosclerosis. GAG biosynthesis involves the cooperation of numerous enzymes. Chondroitin sulfate N-acetylgalactosaminyltransferase-2 (ChGn-2) is a vital Golgi transferase that participates in enzymatic elongation of GAGs. Here, we investigated the effects of ChGn-2 gene deletion on the development of atherosclerosis. Partial carotid artery ligation was performed on ChGn-2-/-/LDLr-/- and ChGn-2+/+/LDLr-/- mice to induce diffuse intimal thickening (DIT). Aortic smooth muscle cells (ASMCs) were isolated to investigate cellular LDL binding and migration. Histological analysis of human coronary artery sections revealed that ChGn-2 was expressed in early and advanced atherosclerotic lesions. Deletion of the ChGn-2 gene significantly reduced LDL retention in the DIT mouse model. Furthermore, LDL binding, visualized using rhodamine-labeled LDLs, was dramatically reduced. Interestingly, a functional assay of ASMCs prepared from ChGn-2-/- mice displayed abrogation of platelet-derived growth factor (PDGF)-mediated migration via reduced PDGF receptor phosphorylation. Taken together, these findings indicate that ChGn-2 is functionally involved in the progression of atherosclerosis both in its early and advanced stages. Therefore, ChGn-2 may serve as a plausible target to treat atherosclerotic-related diseases in the future.

Loss of apoptosis regulator through modulating IAP expression (ARIA) protects blood vessels from atherosclerosis.

Atherosclerosis is the primary cause for cardiovascular disease. Here we identified a novel mechanism underlying atherosclerosis, which is provided by ARIA (apoptosis regulator through modulating IAP expression), the transmembrane protein that we recently identified. ARIA is expressed in macrophages present in human atherosclerotic plaque as well as in mouse peritoneal macrophages. When challenged with acetylated LDL, peritoneal macrophages isolated from ARIA-deficient mice showed substantially reduced foam cell formation, whereas the uptake did not differ from that in wild-type macrophages. Mechanistically, loss of ARIA enhanced PI3K/Akt signaling and consequently reduced the expression of acyl coenzyme A:cholesterol acyltransferase-1 (ACAT-1), an enzyme that esterifies cholesterol and promotes its storage, in macrophages. Inhibition of PI3K abolished the reduction in ACAT-1 expression and foam cell formation in ARIA-deficient macrophages. In contrast, overexpression of ARIA reduced Akt activity and enhanced foam cell formation in RAW264.7 macrophages, which was abrogated by treatment with ACAT inhibitor. Of note, genetic deletion of ARIA significantly reduced the atherosclerosis in ApoE-deficient mice. Oil red-O-positive lipid-rich lesion was reduced, which was accompanied by an increase of collagen fiber and decrease of necrotic core lesion in atherosclerotic plaque in ARIA/ApoE double-deficient mice. Analysis of bone marrow chimeric mice revealed that loss of ARIA in bone marrow cells was sufficient to reduce the atherosclerogenesis in ApoE-deficient mice. Together, we identified a unique role of ARIA in the pathogenesis of atherosclerosis at least partly by modulating macrophage foam cell formation. Our results indicate that ARIA could serve as a novel pharmacotherapeutic target for the treatment of atherosclerotic diseases.

CD163 macrophage and erythrocyte contents in aspirated deep vein thrombus are associated with the time after onset: a pilot study.

BACKGROUND: Thrombolytic therapy is effective in selected patients with deep vein thrombosis (DVT). Therefore, identification of a marker that reflects the age of thrombus is of particular concern. This pilot study aimed to identify a marker that reflects the time after onset in human aspirated DVT. METHODS: We histologically and immunohistochemically analyzed 16 aspirated thrombi. The times from onset to aspiration ranged from 5 to 60 days (median of 13 days). Paraffin sections were stained with hematoxylin and eosin and antibodies for fibrin, glycophorin A, integrin α2bß3, macrophage markers (CD68, CD163, and CD206), CD34, and smooth muscle actin (SMA). RESULTS: All thrombi were immunopositive for glycophorin A, fibrin, integrin α2bß3, CD68, CD163, and CD206, and contained granulocytes. Almost all of the thrombi had small foci of CD34- or SMA-immunopositive areas. CD68- and CD163-immunopositive cell numbers were positively correlated with the time after onset, while the glycophorin A-immunopositive area was negatively correlated with the time after onset. In double immunohistochemistry, CD163-positive cells existed predominantly among the CD68-immunopositive macrophage population. CD163-positive macrophages were closely localized with glycophorin A, CD34, or SMA-positive cell-rich areas. CONCLUSIONS: These findings indicate that CD163 macrophage and erythrocyte contents could be markers for evaluation of the age of thrombus in DVT. Additionally, CD163 macrophages might play a role in organization of the process of venous thrombus.

Human coronary thrombus formation is associated with degree of plaque disruption and expression of tissue factor and hexokinase II.

BACKGROUND: Atherosclerotic plaque thrombogenicity is a critical factor that affects thrombus formation and the onset of acute myocardial infarction (AMI). The aim of this study was to identify the vascular factors involved in thrombus formation and AMI onset. METHODS AND RESULTS: Culprit lesions in 40 coronary arteries with thrombi at autopsy after lethal AMI and non-cardiac death (asymptomatic plaque disruption) were analyzed on histology. Thrombus size, ratio of thrombus to lumen area, length of plaque disruption, and immunopositive areas for tissue factor (TF) and hexokinase (HK)-II were significantly larger in coronary arteries with AMI than with asymptomatic plaque disruption. The size of coronary thrombus positively correlated with the length of plaque disruption (r=0.80) and with immunopositive areas for TF (r=0.38) and HK-II (r=0.40). Because both M1 and M2 macrophages express TF and HK-II in symptomatic plaques, we assessed TF and HK-II expression in M1- and M2-polarized macrophages. The expression of TF was increased and that of HK-II was decreased in M2-, compared with M1-polarized THP-1 macrophages. Inhibiting glycolysis enhanced TF expression in the macrophages partly via hypoxia inducible factor-1α. CONCLUSIONS: The degree of plaque disruption and expression of TF and HK-II appear to be important vascular factors for AMI onset, and polarized macrophages make a distinct contribution to thrombogenicity and glucose metabolism.

Human apolipoprotein A-II protects against diet-induced atherosclerosis in transgenic rabbits.

OBJECTIVE: Apolipoprotein (apo) A-II is the second major apo of high-density lipoproteins, yet its pathophysiological roles in the development of atherosclerosis remain unknown. We aimed to examine whether apo A-II plays any role in atherogenesis and, if so, to elucidate the mechanism involved. METHODS AND RESULTS: We compared the susceptibility of human apo A-II transgenic (Tg) rabbits to cholesterol diet-induced atherosclerosis with non-Tg littermate rabbits. Tg rabbits developed significantly less aortic and coronary atherosclerosis than their non-Tg littermates, while total plasma cholesterol levels were similar. Atherosclerotic lesions of Tg rabbits were characterized by reduced macrophages and smooth muscle cells, and apo A-II immunoreactive proteins were frequently detected in the lesions. Tg rabbits exhibited low levels of plasma C-reactive protein and blood leukocytes compared with non-Tg rabbits, and high-density lipoproteins of Tg rabbit plasma exerted stronger cholesterol efflux activity and inhibitory effects on the inflammatory cytokine expression by macrophages in vitro than high-density lipoproteins isolated from non-Tg rabbits. In addition, ß-very-low-density lipoproteins of Tg rabbits were less sensitive to copper-induced oxidation than ß-very-low-density lipoproteins of non-Tg rabbits. CONCLUSIONS: These results suggest that enrichment of apo A-II in high-density lipoprotein particles has atheroprotective effects and apo A-II may become a target for the treatment of atherosclerosis.

Progressive worsening of aortic regurgitation due to detachment of the aortic valve commissure with multimodality imaging to elucidate pathogenesis: a case report.

Background: Aortic regurgitation (AR) associated with detachment of the aortic valve commissure is extremely rare. We present a case of progressively worsening severe chronic AR due to detachment of the aortic valve commissure during hospitalization that was confirmed with multimodality imaging. Case summary: A 50-year-old male with Marfan syndrome visited our hospital to receive treatment for cholelithiasis. Pre-operative examination revealed severe AR and aortic root aneurysm. Because the patient was asymptomatic, it was decided that cholecystectomy should be performed first. However, the patient's heart failure worsened acutely when his blood pressure increased just before induction of anaesthesia. The patient required intubation and management of heart failure. Five days later, the patient underwent cholecystectomy. He was treated for heart failure and underwent open heart surgery on the 35th hospital day. Intraoperative transoesophageal echocardiography revealed that his AR was caused by both enlargement of the aortic root and localized dissection of the aortic valve commissure, which was supported by intraoperative findings and histopathological evaluation. Aortic regurgitation was exacerbated by a new localized dissection, resulting in acute worsening of heart failure. Discussion: Aortic valve commissure detachment can easily lead to sudden onset of severe AR, deteriorating haemodynamics, and acute pulmonary oedema. Since delayed medical treatment leads to poor clinical outcomes, prompt and accurate diagnosis and appropriately timed surgical intervention are essential. This very rare case of severe AR worsening due to spontaneous aortic valve commissure dissection was evaluated with multiple modalities during hospitalization. Understanding this clinical condition will help cardiologists provide better medical care.

Elevated plasma levels of factor VIII enhance arterial thrombus formation on erosive smooth muscle cell-rich neointima but not normal intima in rabbits.

BACKGROUND: Plaque erosion, a type of coronary atherothrombosis, involves superficial injury to smooth muscle cell (SMC)-rich plaques. Elevated levels of coagulation factor VIII (FVIII) correlate with an increased ischemic heart disease risk. FVIII may contribute to thrombus formation on eroded plaques. AIMS: We aimed to elucidate the role of elevated FVIII in arterial thrombus formation within SMC-rich neointima in rabbits. METHODS AND RESULTS: We assessed the effect of recombinant human FVIII (rFVIII) on blood coagulation in vitro and platelet aggregation ex vivo. An SMC-rich neointima was induced through balloon injury to the unilateral femoral artery. Three weeks after the first balloon injury, superficial erosive injury and thrombus formation were initiated with a second balloon injury of the bilateral femoral arteries 45 min after the administration of rFVIII (100 IU/kg) or saline. The thrombus area and contents were histologically measured 15 min after the second balloon injury. rFVIII administration reduced the activated partial thromboplastin time and augmented botrocetin-induced, but not collagen- or adenosine 5'-diphosphate-induced, platelet aggregation. While rFVIII did not influence platelet-thrombus formation in normal intima, it increased thrombus formation on SMC-rich neointima post-superficial erosive injury. Enhanced immunopositivity for glycoprotein IIb/IIIa and fibrin was observed in rFVIII-administered SMC-rich neointima. Neutrophil count in the arterial thrombus on the SMC-rich neointima correlated positively with thrombus size in the control group, unlike the rFVIII group. CONCLUSIONS: Increased FVIII contributes to thrombus propagation within erosive SMC-rich neointima, highlighting FVIII's potential role in plaque erosion-related atherothrombosis.

Intralesional pentraxin 3 increases with atherosclerotic disease progression, but may protect from thrombosis: Friend or foe?

AIM: To investigate the role of pentraxin 3 (PTX3) in atherosclerotic disease progression and plaque destabilization, as well as in coronary restenosis after directional coronary atherectomy (DCA). MATERIALS AND METHODS: PTX3 contents of early and advanced atherosclerotic lesions of the aorta obtained at autopsy were determined by ELISA and Western blot. Also, coronary plaques of patients with acute coronary syndrome (ACS) or stable angina pectoris (SAP) obtained by DCA were analyzed by immunohistochemistry for PTX3. The effects of PTX3 on smooth muscle cells (SMCs) and thrombogenesis were investigated with cultured human coronary artery SMCs and a flow chamber system, respectively. RESULTS: Advanced atherosclerotic lesions contained a significantly larger amount of PTX3 than early lesions (ELISA: 9.96 ± 2.77 ng/100 mg tissue, n = 8 vs 0.24 ± 0.18 ng/100 mg tissue, n = 6, P = 0.0097). Also, ACS plaques contained a significantly larger amount of PTX3 than SAP plaques (PTX3 immunohistochemistry-positive area percentage: 2.88 ± 0.53 %, n = 22 vs 0.67 ± 0.27 %, n = 23, P = 0.0009). Curiously, the patients who would remain free of post-DCA restenosis (n = 19) had plaques with a significantly higher PTX3 immunohistochemistry-positive area percentage than those who would develop restenosis (n = 12) (2.32 ± 0.49 % vs 0.49 ± 0.17 %, P = 0.002). In the mechanistic part of the study, PTX3 inhibited SMC proliferation and migration. PTX3 also inhibited platelet thrombus formation in the condition simulating arterial blood flow. CONCLUSIONS: PTX3 is increased in advanced (vs early) atherosclerotic lesions and unstable (vs stable) coronary plaques. The inhibitory effects of PTX3 on SMCs and thrombogenesis suggest that intraplaque PTX3 might have atheroprotective effects.