Mechanisms and management of thrombosis in cancer: focus on gastrointestinal malignancies.

Cancer patients have an increased risk of venous thromboembolism (VTE) which is their second cause of death after disease progression itself. Several thrombotic risk factors coexist in cancer patients, including the ability of both cancer and tumoral microenvironment's cells to directly or indirectly activate platelets and the enzymes of the coagulation cascade, resulting in a hyper-coagulable state of blood. This narrative review gives an overview of the main mechanisms leading to VTE in cancer patients, including the role that platelets and the clotting proteins may have in tumor growth and metastasis. Noteworthy, the haemostatic balance is altered in cancer patients who may, next to a thrombosis tendency, also have an increased risk of bleeding. To highlight the complexity and the precariousness of the haemostatic balance of these patients, we discuss two specific gastrointestinal malignancies: hepatocellular carcinoma, which is frequently associated with liver cirrhosis, a condition that causes profound alterations of haemostasis, and colorectal cancer, which is characterized by a fragile mucosa that is prone to bleeding. Understanding the molecular mechanisms of cancer-associated thrombosis may give a unique opportunity to develop new innovative drugs, acting differently on distinct pathways and potentially allowing to reduce the risk of bleeding related to antithrombotic therapies. Significance Statement The topic is significant because understanding the molecular mechanisms leading to cancer associated thrombosis and bleeding, focusing on gastrointestinal malignancies, enables the development of more rationale and innovative antithrombotic strategies for cancer associated thrombosis. Eventually, this will support an improved and patient-tailored antithrombotic management in vulnerable oncologic patients.

Skin erythematous migrant lesions consistent with histologically confirmed dermal arteriolar thrombosis connected to APS.

Antiphospholipid syndrome (APS) is an acquired thrombophilic disorder related to the presence of antiphospholipid antibodies (LAC, anticardiolipin, anti Beta2-glycoprotein) known to cause venous and arterial thrombosis and recurrent pregnancy loss. Skin disorder is a frequent finding usually due to vascular thrombosis involving the dermal layer and can be either localized or widespread causing necrosis and ulceration of the skin, without histological evidence of vasculitis. We present a case of a woman with APS with both arterial and venous thrombotic involvement associated with an atypical dermatological manifestation histologically consistent with a pauci-inflammatory intermediate-deep dermal arteriolar platelet-mediated thrombosis that appeared despite anticoagulation with warfarin and responding to the addition of antiplatelet therapy.

Traditional Korean diet high in one-carbon nutrients increases global DNA methylation: implication for epigenetic diet.

PURPOSE: DNA methylation is a major epigenetic phenomenon through which diet affects health and disease. This study aimed to determine the epigenetic influence of the traditional Korean diet (K-diet) on global DNA methylation via one-carbon metabolism. METHODS: A crossover study was conducted on 52 women. Two diets, a K-diet, high in plant foods and low in calories and animal fat, and a control diet, similar to the diet currently consumed in Korea, were provided to all subjects alternately for 4 weeks with a 4-week washout period. Clinical parameters were measured before and after each dietary intervention. Nutrient intake was calculated by using a computer-aided nutritional analysis program. One-carbon metabolites in the serum and global DNA methylation in peripheral mononuclear cells were determined using ultra-performance liquid chromatography-tandem mass spectrometry. RESULTS: The K-diet group consumed more folate (669.9 ± 6.7 µg vs. 502.7 ± 3.0, p < 0.001), B6, B12, serine, and choline, and less methionine (992.6 ± 63 vs. 1048.3 mg ± 34.1, p < 0.0001) than the control group did. In the K-diet group, the increment of plasma 5-methyltetrahydrofolate (0.08 µg/mL ± 0.11 vs 0.02 ± 0.10, p < 0.009) and decrement of L-homocysteine (- 70.7 ± 85.0 vs - 39.3 ± 69.4, p < 0.0168) were greater than those of the control group. Global DNA methylation was significantly increased in the K-diet group (6.70 ± 3.02% to 9.45 ± 3.69, p < 0.0001) but not in the control group. CONCLUSIONS: A K-diet high in one-carbon nutrients can enhance the global DNA methylation status, suggesting an epigenetic mechanism by which the K-diet conveys health effects. Trial registration Korean Clinical Trial Registry (trial number: KCT0005340, 24/08/2020, retrospectively registered).

Lifestyle and dietary factors, iron status and one-carbon metabolism polymorphisms in a sample of Italian women and men attending a Transfusion Medicine Unit: a cross-sectional study.

Iron (Fe) status among healthy male and female blood donors, aged 18-65 years, is estimated. General characteristics and lifestyle factors, dietary habits and major one-carbon metabolism-related polymorphisms were also investigated. An explorative cross-sectional study design was used to examine a sample of blood donors attending the Transfusion Medicine Unit of the Verona University Hospital, Italy. From April 2016 to May 2018, 499 subjects were enrolled (255 men, 244 women, 155 of whom of childbearing age). Major clinical characteristics including lifestyle, dietary habits and Fe status were analysed. The MTHFR 677C > T, cSHMT 1420C > T, DHFR 19bp ins/del and RFC1 80G > A polymorphisms were also assayed. Mean plasma concentrations of Fe and ferritin were 16·6 µmol/l (95 % CI 16·0, 17·2) and 33·8 µg/l (95 % CI 31·5, 36·2), respectively. Adequate plasma Fe concentrations (> 10·74 µmol/l) were detected in 84·3 % and adequate ferritin concentrations (20-200 µg/l) was found in 72·5 % of the whole cohort. Among the folate-related polymorphisms analysed, carriers of the DHFR 19bp del/del mutant allele showed lower ferritin concentration when compared with DHFR 19bp ins/del genotypes. In a sample of Italian healthy blood donors, adequate plasma concentrations of Fe and ferritin were reached in a large proportion of subjects. The relationship of Fe status with lifestyle factors and folate-related polymorphisms requires more investigation to clarify further gene-nutrient interactions between folate and Fe metabolism.

B vitamin blood concentrations and one-carbon metabolism polymorphisms in a sample of Italian women and men attending a unit of transfusion medicine: a cross-sectional study.

PURPOSE: To define blood status of folate, vitamin B12, vitamin B6, homocysteine, and major one-carbon metabolism-related polymorphisms in healthy, males and females blood donors, aged 18-65 years were evaluated. General characteristics and lifestyle factors were also investigated. METHODS: An explorative cross-sectional study design was used to evaluate a sample of blood donors attending the Unit of Transfusion Medicine of the Verona University Hospital, Italy. From April 2016 to May 2018, 499 subjects were enrolled (255 men, 244 women of whom 155 of childbearing age). Major clinical characteristics including lifestyle and dietary habits, B vitamins and homocysteine were analyzed. The MTHFR 677 C>T, cSHMT 1420 C>T, DHFR 19 bp ins/del, RFC1 80 G>A polymorphisms were also determined. RESULTS: Mean plasma concentrations of folate, vitamin B12, vitamin B6 and homocysteine were 14.2 nmol/L (95% CI 13.7-14.8), 271.9 pmol/L (95% CI 262.6-281.5), 51.0 nmol/L (95% CI 48.7-53.4) and 13.5 µmol/L (95% CI 13.1-14.0), respectively. Plasma folate, was adequate (> 15 nmol/L) in 44.7% of all subjects, 39.0% of males and 42.5% of women < 45 years. Similarly, vitamin B12 was adequate (> 350 pmol/L) in 25.1% of all subjects and in 20.3% of men ≥ 45 years. The rare allele frequencies were 0.21 for MTHFR 677TT, 0.11 for cSHMT 1420TT, 0.18 for DHFR 19 bp del/del, 0.20 for RFC1 80AA, and a gene-nutrient interaction was confirmed for folate concentrations according to MTHFR 677C>T and DHFR 19 bp del/del. CONCLUSION: An Italian sample of healthy blood donors shows that an adequate concentration of plasma folate and vitamin B12 is reached only in a limited percentage of subjects, thus encouraging consideration for specific public health strategies.

Increased Incidence of Ischemic Cerebrovascular Events in Cardiovascular Patients With Elevated Apolipoprotein CIII.

Background and Purpose- Apo CIII (apolipoprotein CIII), a crucial regulator of lipoprotein metabolism, has been associated with increased activity of coagulation factors and thrombin generation and, in turn, with an increased risk of thromboembolic events in both arterial and venous districts. Thus, we hypothesized that it may affect the risk of acute ischemic cerebrovascular events in cardiovascular patients. Methods- We systematically checked medical records and quantified cerebral ischemic events in a cohort of 950 subjects (median age 65 with interquartile range, 55-79 years; 30.7% females) with or without angiographically defined coronary artery disease (CAD: 774 CAD and 176 CAD-free, respectively). All the subjects, enrolled between May 1999 and December 2006, were prospectively followed until death or July 31, 2018. Assessments of complete plasma lipid and apolipoprotein profiles, including Apo A-I, B, CIII, and E, were available for all subjects at enrollment. Results- After a median follow-up of 130 months (interquartile range, 69-189), 95 subjects (10%) suffered ischemic stroke/transient ischemic attack (TIA) events. Stroke/TIA subjects had higher Apo CIII plasma concentration (11.4; interquartile range: 9.3-14.4 mg/dL) at enrollment than those without stroke/TIA (10.4, interquartile range: 8.7-13.0 mg/dL). Subjects with Apo CIII levels above the median value (10.6 mg/dL) exhibited an ≈2-fold increased risk of stroke/TIA, even after adjustment for potential confounders, including sex, age, CAD diagnosis, hypertension, atrial fibrillation, oral anticoagulant treatment, and all plasma lipid parameters (hazard ratio: 2.23 [95% CI, 1.21-4.13]). This result was confirmed in CAD and CAD-free populations, separately, and even by a propensity score matching method, in which 98 CAD and 98 CAD-free subjects were one-to-one matched for all clinical and laboratory characteristics. Conclusions- These findings suggest that a high Apo CIII plasma concentration may predict an increased risk of ischemic stroke/TIA in cardiovascular patients.

The Positive Association between Plasma Myristic Acid and ApoCIII Concentrations in Cardiovascular Disease Patients Is Supported by the Effects of Myristic Acid in HepG2 Cells.

BACKGROUND: In the settings of primary and secondary prevention for coronary artery disease (CAD), a crucial role is played by some key molecules involved in triglyceride (TG) metabolism, such as ApoCIII. Fatty acid (FA) intake is well recognized as a main determinant of plasma lipids, including plasma TG concentration. OBJECTIVES: The aim was to investigate the possible relations between the intakes of different FAs, estimated by their plasma concentrations, and circulating amounts of ApoCIII. METHODS: Plasma samples were obtained from 1370 subjects with or without angiographically demonstrated CAD (mean ± SD age: 60.6 ± 11.0 y; males: 75.8%; BMI: 25.9 ± 4.6 kg/m2; CAD: 73.3%). Plasma lipid, ApoCIII, and FA concentrations were measured. Data were analyzed by regression models adjusted for FAs and other potential confounders, such as sex, age, BMI, diabetes, smoking, and lipid-lowering therapies. The in vitro effects of FAs were tested by incubating HepG2 hepatoma cells with increasing concentrations of selected FAs, and the mRNA and protein contents in the cells were quantified by real-time RT-PCR and LC-MS/MS analyses. RESULTS: Among all the analyzed FAs, myristic acid (14:0) showed the most robust correlations with both TGs (R = 0.441, P = 2.6 × 10-66) and ApoCIII (R = 0.327, P = 1.1 × 10-31). By multiple regression analysis, myristic acid was the best predictor of both plasma TG and ApoCIII variability. Plasma TG and ApoCIII concentrations increased progressively at increasing concentrations of myristic acid, independently of CAD diagnosis and gender. Consistent with these data, in the in vitro experiments, an ∼2-fold increase in the expression levels of the ApoCIII mRNA and protein was observed after incubation with 250 µM myristic acid. A weaker effect (∼30% increase) was observed for palmitic acid, whereas incubation with oleic acid did not affect ApoCIII protein or gene expression. CONCLUSIONS: Plasma myristic acid is associated with increased ApoCIII concentrations in cardiovascular patients. In vitro experiments indicated that myristic acid stimulates ApoCIII expression in HepG2 cells.

Hepcidin and DNA promoter methylation in hepatocellular carcinoma.

BACKGROUND: The liver hormone hepcidin regulates iron homoeostasis that is often altered in hepatocellular carcinoma (HCC). Epigenetic phenomena control gene expression through a dynamic fashion; therefore, considering the plasticity of both iron homoeostasis and epigenetic mechanisms and their role in liver carcinogenesis, we investigated whether hepcidin gene (HAMP) expression is modulated by DNA methylation, thus affecting iron status in human HCC. MATERIALS AND METHODS: Thirty-two patients affected by nonviral HCC were enrolled, and their main clinical and biochemical characteristics were obtained. Neoplastic and homologous non-neoplastic liver tissues were analysed for HAMP promoter DNA methylation, for HAMP gene expression and for iron content. An in vitro demethylation assay with a human hepatocarcinoma cell line was performed to evaluate the role of DNA methylation on HAMP transcriptional repression. RESULTS: Gene expression and DNA methylation analyses on tissues showed that HAMP was transcriptionally repressed in HCC tissues consensually with a promoter hypermethylation. Furthermore, patients with HCC had low serum hepcidin concentrations, and HCC tissues had relative iron depletion as compared to non-neoplastic liver tissues. The cell culture model showed the functional role of DNA hypermethylation by downregulating HAMP gene expression. Through a quantitative methylation analysis on HCC tissues, we then proved that methylation at definite CpG sites within consensus sequences for specific transcription factors is possibly the mechanism underlying HAMP repression. CONCLUSIONS: This study highlights a novel role for HAMP downregulation through DNA promoter hypermethylation and emphasises the significance of epigenetics in the regulation of iron metabolism in HCC.

Hepatic DNA hydroxymethylation is site-specifically altered by chronic alcohol consumption and aging.

PURPOSE: Global DNA hydroxymethylation is markedly decreased in human cancers, including hepatocellular carcinoma, which is associated with chronic alcohol consumption and aging. Because gene-specific changes in hydroxymethylcytosine may affect gene transcription, giving rise to a carcinogenic environment, we determined genome-wide site-specific changes in hepatic hydroxymethylcytosine that are associated with chronic alcohol consumption and aging. METHODS: Young (4 months) and old (18 months) male C57Bl/6 mice were fed either an ethanol-containing Lieber-DeCarli liquid diet or an isocaloric control diet for 5 weeks. Genomic and gene-specific hydroxymethylcytosine patterns were determined through hydroxymethyl DNA immunoprecipitation array in hepatic DNA. RESULTS: Hydroxymethylcytosine patterns were more perturbed by alcohol consumption in young mice than in old mice (431 differentially hydroxymethylated regions, DhMRs, in young vs 189 DhMRs in old). A CpG island ~2.5 kb upstream of the glucocorticoid receptor gene, Nr3c1, had increased hydroxymethylation as well as increased mRNA expression (p = 0.015) in young mice fed alcohol relative to the control group. Aging alone also altered hydroxymethylcytosine patterns, with 331 DhMRs, but alcohol attenuated this effect. Aging was associated with a decrease in hydroxymethylcytosine ~1 kb upstream of the leptin receptor gene, Lepr, and decreased transcription of this gene (p = 0.029). Nr3c1 and Lepr are both involved in hepatic lipid homeostasis and hepatosteatosis, which may create a carcinogenic environment. CONCLUSIONS: These results suggest that the location of hydroxymethylcytosine in the genome is site specific and not random, and that changes in hydroxymethylation may play a role in the liver's response to aging and alcohol.

Global DNA methylation and hydroxymethylation differ in hepatocellular carcinoma and cholangiocarcinoma and relate to survival rate.

UNLABELLED: In addition to DNA methylation, hydroxymethylation of DNA is recognized as a novel epigenetic mark. Primary liver cancers, i.e., hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC), are highly prevalent but epigenetically poorly characterized, so far. In the present study we measured global methylcytosine (mCyt) and hydroxymethylcytosine (hmCyt) in HCC and CC tissues and in peripheral blood mononuclear cell (PBMC) DNA to define mCyt and hmCyt status and, accordingly, the survival rate. Both mCyt and hmCyt were measured by a liquid chromatography/tandem mass spectrometry method in neoplastic and homologous nonneoplastic tissues, i.e., liver and gallbladder, and in PBMCs of 31 HCC and 16 CC patients. Content of mCyt was notably lower in HCC than in CC tissues (3.97% versus 5.26%, respectively; P < 0.0001). Significantly reduced mCyt was also detected in HCC compared to nonneoplastic tissue (3.97% versus 4.82% mCyt, respectively; P < 0.0001), but no such difference was found for CC versus homologous nonneoplastic tissue. Hydroxymethylation was significantly decreased in HCC versus nonneoplastic liver tissue (0.044 versus 0.128, respectively; P < 0.0001) and in CC versus both liver and gallbladder nonneoplastic tissue (0.030 versus 0.124, P = 0.026, and 0.030 versus 0.123, P = 0.006, respectively). When the survival rate was evaluated according to mCyt PBMC content by Kaplan-Meier analysis, patients with mCyt ≥5.59% had a significantly higher life expectancy than those with mCyt <5.59% (P = 0.034) at a follow-up period up to 48 months. CONCLUSION: A significant DNA hypomethylation distinguishes HCC from CC, while DNA hypo-hydroxymethylation characterizes both HCC and CC, and a PBMC DNA mCyt content ≥5.59% relates to a favorable outcome in primary liver cancers.