Harinder S. Juneja

Prospective Study of Fibrinolytic Factors and Incident Coronary Heart Disease: The Atherosclerosis Risk in Communities (ARIC) Study

Abstract— The fibrinolytic system may play a role in the pathogenesis of coronary heart disease (CHD), but existing prospective studies have not consistently shown an independent association between fibrinolytic factors and CHD. None has reported an association between plasminogen and CHD incidence. In the prospective Atherosclerosis Risk in Communities (ARIC) Study of middle-aged adults, we examined the association of incident CHD with several fibrinolytic factors: tissue plasminogen activator antigen, plasminogen activator inhibitor-1, plasminogen, and fibrin fragment D-dimer as well as a marker of coagulation activation (prothrombin fragment F1.2). We measured these in stored baseline plasma samples of 326 subjects who developed CHD and, for comparison, a stratified random sample of the entire cohort (n=720). Tissue plasminogen activator and plasminogen activator inhibitor-1 antigen levels were associated positively with CHD incidence in analyses adjusted for age, race, and sex but were not associated with CHD after adjustment for other risk factors. Plasminogen and D-dimer levels were associated positively and independently with CHD incidence; the multivariable-adjusted relative risks (95% CIs) for the highest versus lowest quintiles were 2.20 (1.2 to 4.2) for plasminogen and 4.21 (1.9 to 9.6) for D-dimer. F1.2 was not associated with CHD incidence. Our findings lend support for a link between fibrinolytic factors and CHD incidence. A positive association between plasminogen and CHD is seemingly opposite the direction expected but may reflect a compensatory response to impaired plasminogen activation in subjects prone to CHD.

Soluble thrombomodulin as a predictor of incident coronary heart disease and symptomless carotid artery atherosclerosis in the Atherosclerosis Risk in Communities (ARIC) Study: a case-cohort study

BACKGROUND Small amounts of soluble thrombomodulin in plasma are thought to reflect endothelial damage. In a case-cohort study, we examined whether soluble thrombomodulin is a predictor of incident coronary heart disease and carotid artery atherosclerosis. METHODS The study population consisted of 14,170 black and white participants from the Atherosclerosis Risk in Communities (ARIC) study, who did not have cardiovascular disease at the start of the study and who we followed up for 6 years. After appropriate exclusions, we analysed 258 cases of incident coronary heart disease and 449 cases of carotid atherosclerosis. A stratified random sample of 753 individuals from the ARIC cohort was used as the comparison group. We used EIA to measure soluble thrombomodulin in plasma samples from both groups. For the analysis, we used quintiles of soluble thrombomodulin concentrations (< 24.7, 24.8-30.6, 30.7-40.2, 40.3-55.2, and > or = 55.3 ng/mL). FINDINGS Soluble thrombomodulin showed a strong, graded, inverse association with incident coronary heart disease (p=0.005). The adjusted rate ratio of the highest quintile of soluble thrombomodulin compared with the lowest quintile was 0.29 (95% CI 0.15-0.57). The association with carotid atherosclerosis, however, tended to be positive, especially among white participants (odds ratio 2.94 [1.15-7.51] for highest vs lowest quintile). The relation of soluble thrombomodulin to incident coronary heart disease and carotid atherosclerosis was dependent on factor VIII coagulant activity (p=0.06 and 0.003, respectively). INTERPRETATION The prospective association of soluble thrombomodulin with incident coronary heart disease differs from its cross-sectional association with carotid atherosclerosis. In healthy people, plasma concentrations of soluble thrombomodulin may reflect endothelial expression of thrombomodulin. High concentration of soluble thrombomodulin may be associated with decreased risk of coronary heart disease.

Detrimental effects of adenosine signaling in sickle cell disease

Hypoxia can act as an initial trigger to induce erythrocyte sickling and eventual end organ damage in sickle cell disease (SCD). Many factors and metabolites are altered in response to hypoxia and may contribute to the pathogenesis of the disease. Using metabolomic profiling, we found that the steady-state concentration of adenosine in the blood was elevated in a transgenic mouse model of SCD. Adenosine concentrations were similarly elevated in the blood of humans with SCD. Increased adenosine levels promoted sickling, hemolysis and damage to multiple tissues in SCD transgenic mice and promoted sickling of human erythrocytes. Using biochemical, genetic and pharmacological approaches, we showed that adenosine A2B receptor (A2BR)-mediated induction of 2,3-diphosphoglycerate, an erythrocyte-specific metabolite that decreases the oxygen binding affinity of hemoglobin, underlies the induction of erythrocyte sickling by excess adenosine both in cultured human red blood cells and in SCD transgenic mice. Thus, excessive adenosine signaling through the A2BR has a pathological role in SCD. These findings may provide new therapeutic possibilities for this disease.

Thrombomodulin Ala455Val Polymorphism and Risk of Coronary Heart Disease

Background —Thrombomodulin (TM) is expressed on the endothelial surface and plays an important role in vasoprotection. A common polymorphism of TM at amino acid position 455 with an alanine (A) to valine (V) transition was previously reported to be associated cross-sectionally with acute myocardial infarction. Whether this single nucleotide polymorphism predicts risk of developing coronary heart disease (CHD) is unclear. Methods and Results —Within a large cohort study, we identified 467 incident CHD cases during an average of 5 years of follow-up. We determined TM-455 genotypes on 376 CHD cases (23% black, 77% white) and a reference sample of 461. The AA genotype was significantly more prevalent in noncases than in cases (P =0.016). The prevalences of the AA genotype in noncase blacks and whites were 93% and 67%, respectively. The AA genotype frequency was significantly reduced in black cases versus noncases (P =0.018). It was also lower in white cases than in noncases, but the difference was not statistically significant (P =0.066). Weighted proportional hazards regression analysis after adjustment for age, sex, and other CHD risk factors showed that having the V allele increased risk of CHD by 6.1-fold (risk ratio 6.1, 95% CI 1.7 to 22.9) in blacks but did not significantly increase the risk in whites. Conclusions —The TM A455V polymorphism predicts risk of developing CHD in blacks.

Interaction Between Soluble Thrombomodulin and Intercellular Adhesion Molecule-1 in Predicting Risk of Coronary Heart Disease

Background—Results from previous ARIC (Atherosclerosis Risk In Communities) analyses indicate that soluble intercellular adhesive molecule-1 (sICAM) and soluble thrombomodulin (sTM) levels are associated with risk of coronary heart disease (CHD) in an opposite direction. A high sICAM level increases the risk of CHD, whereas a high level of sTM has a lower risk of CHD. It was unclear whether there was an interaction between sTM and sICAM. Methods and Results—Using a nested case-cohort design, we measured sTM and sICAM in 317 incident CHD cases and 726 non-cases from the ARIC participants. Consistent with our previous reports, sICAM values in the upper versus the lower tertile increased the risk of CHD event by ≈2-fold (95% confidence interval [CI], 1.46 to 2.87) whereas sTM values in the lower versus the upper tertile increased CHD risk by ≈4-fold (95% CI, 2.80 to 5.74). Interaction between these 2 parameters was determined by weighted Cox proportional hazard regression. A significant interaction (P =0.038) was noted. Combinatorial analysis shows a significant increase in CHD risk ratio (RR) (4.66, 95% CI, 1.89 to 11.46) of the lower sTM/upper sICAM group versus the upper sTM/lower sICAM group. Individuals whose sTM values were in the upper tertile had a RR below 1, even when sICAM were in the upper tertile. The RR of lower tertile sTM was increased by sICAM in a tertile-dependent manner. Conclusion—Weighted Cox proportional hazard analysis shows a significant interaction between sTM and sICAM in predicting risk of CHD event. Combinatorial analysis reveals that an upper tertile sICAM had a significant increase in the risk of a CHD event only when sTM was in the lower tertile.

Elevated sphingosine-1-phosphate promotes sickling and sickle cell disease progression

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates multicellular functions through interactions with its receptors on cell surfaces. S1P is enriched and stored in erythrocytes; however, it is not clear whether alterations in S1P are involved in the prevalent and debilitating hemolytic disorder sickle cell disease (SCD). Here, using metabolomic screening, we found that S1P is highly elevated in the blood of mice and humans with SCD. In murine models of SCD, we demonstrated that elevated erythrocyte sphingosine kinase 1 (SPHK1) underlies sickling and disease progression by increasing S1P levels in the blood. Additionally, we observed elevated SPHK1 activity in erythrocytes and increased S1P in blood collected from patients with SCD and demonstrated a direct impact of elevated SPHK1-mediated production of S1P on sickling that was independent of S1P receptor activation in isolated erythrocytes. Together, our findings provide insights into erythrocyte pathophysiology, revealing that a SPHK1-mediated elevation of S1P contributes to sickling and promotes disease progression, and highlight potential therapeutic opportunities for SCD.

Platelet Pl(A2) allele and incidence of coronary heart disease: results from the Atherosclerosis Risk In Communities (ARIC) Study.

BackgroundThe major platelet integrin glycoprotein IIb-IIIa plays a primary role in platelet aggregation and acute thrombus formation at the site of vascular injury. A genetic polymorphism of glycoprotein IIb-IIIa (PlA) has recently been proposed as a potential genetic factor linking to platelet hyperaggregability and increased risk of myocardial infarction. Despite numerous, mostly nonprospective studies, the role of this polymorphism as a clinically relevant, inherited risk factor for coronary heart disease (CHD) is still controversial. The purpose of this study was to determine whether PlA2 is a risk factor for incident CHD and whether it is correlated with increased platelet activation in a case-cohort study nested within a prospective epidemiologic investigation. Methods and ResultsBlood samples were collected and processed from the Atherosclerosis Risk in Communities Study cohort at the baseline examination (1987 to 1989). They were stored at −80°C. PlA1/A2 genotype and plasma &bgr;-thromboglobulin levels were determined in 439 incident CHD cases and a reference cohort sample of 544 (of whom 18 were also CHD cases). The prevalence of the PlA2 allele was not different in cases versus noncases. No significant correlation between CHD risk factors and the PlA2 allele was noted either. Platelet activation, as measured by plasma &bgr;-thromboglobulin levels, was not enhanced in individuals with the PlA2 allele. ConclusionsThis prospective study indicates that healthy individuals carrying the PlA2 allele do not have an increased risk of CHD.

Aspirin and salicylate inhibit colon cancer medium- and VEGF-induced endothelial tube formation: correlation with suppression of cyclooxygenase-2 expression

Summary.  To determine whether aspirin and salicylate suppress colon cancer cell‐mediated angiogenesis, we evaluated the effects of aspirin and sodium salicylate on endothelial tube formation on Matrigel. Aspirin and sodium salicylate concentration‐dependently inhibited human endothelial cell (EC) tube formation induced by conditioned medium collected from DLD‐1, HT‐29 or HCT‐116 colon cancer cells. Aspirin and sodium salicylate at pharmacological concentrations were equally effective in blocking tube formation. Neutralizing antivascular endothelial growth factor (VEGF) antibodies blocked colon cancer medium‐induced tube formation. VEGF receptor 2 but not receptor 1 antibodies inhibited tube formation to a similar extent as anti‐VEGF antibodies. These results indicate that VEGF interaction with VEGF receptor 2 is the primary mechanism underlying colon cancer‐induced angiogenesis. Aspirin or sodium salicylate inhibited VEGF‐induced tube formation in a concentration‐dependent manner comparable to that of inhibition of colon cancer medium‐induced endothelial tube formation. It has been shown that cyclooxygenase‐2 (COX‐2) is pivotal in cancer angiogenesis. We found that colon cancer medium‐induced COX‐2 protein expression in EC and aspirin or sodium salicylate suppressed the cancer‐induced COX‐2 protein levels at concentrations correlated with those that suppressed endothelial tube formation. Furthermore, aspirin and sodium salicylate inhibited COX‐2 expression stimulated by VEGF. These findings indicate that aspirin and other salicylate drugs at pharmacological concentrations inhibit colon cancer‐induced angiogenesis which is correlated with COX‐2 suppression.

Cross-sectional association of soluble thrombomodulin with mild peripheral artery disease; the ARIC study

Thrombomodulin, an endothelial membrane glycoprotein, is an essential part of the protein C anti-coagulant pathway. It may also have a role in the regulation of fibrinolysis. We carried out a cross-sectional study to assess the association of soluble thrombomodulin (sTM) with peripheral artery disease (PAD) in a stratified random sample (n=863) of otherwise healthy black and white participants of the Atherosclerosis Risk in Communities (ARIC) Study. PAD was more common in black than in white participants and associated with classical risk factors in an expected manner; positively with age, smoking, hypertension, diabetes (P=0.05), and LDL-cholesterol, and inversely with HDL-cholesterol. Significant positive associations were observed also with fibrinogen and white blood cell count. Overall, the sTM concentration was not a significant predictor of PAD. The association was, however, modified by the level of factor VIII:C in whites (P=0.002 for the interaction), but not in blacks. Protein C was inversely associated with PAD prevalence (odds ratio 0.33, 95% CI 0.18--0.61, P=0.0004). sTM was inversely associated with plasminogen, but no associations with t-PA, PAI-1, or D-dimer were seen. In conclusion, the present results provide some additional evidence on the role of thrombomodulin-protein C pathway in atherosclerotic disease and support our earlier observation on interaction between sTM and factor VIII:C.

Elevated Adenosine Signaling Via Adenosine A2B Receptor Induces Normal and Sickle Erythrocyte Sphingosine Kinase 1 Activity

Erythrocyte possesses high sphingosine kinase 1 (SphK1) activity and is the major cell type supplying plasma sphingosine-1-phosphate, a signaling lipid regulating multiple physiological and pathological functions. Recent studies revealed that erythrocyte SphK1 activity is upregulated in sickle cell disease (SCD) and contributes to sickling and disease progression. However, how erythrocyte SphK1 activity is regulated remains unknown. Here we report that adenosine induces SphK1 activity in human and mouse sickle and normal erythrocytes in vitro. Next, using 4 adenosine receptor-deficient mice and pharmacological approaches, we determined that the A2B adenosine receptor (ADORA2B) is essential for adenosine-induced SphK1 activity in human and mouse normal and sickle erythrocytes in vitro. Subsequently, we provide in vivo genetic evidence that adenosine deaminase (ADA) deficiency leads to excess plasma adenosine and elevated erythrocyte SphK1 activity. Lowering adenosine by ADA enzyme therapy or genetic deletion of ADORA2B significantly reduced excess adenosine-induced erythrocyte SphK1 activity in ADA-deficient mice. Finally, we revealed that protein kinase A-mediated extracellular signal-regulated kinase 1/2 activation functioning downstream of ADORA2B underlies adenosine-induced erythrocyte SphK1 activity. Overall, our findings reveal a novel signaling network regulating erythrocyte SphK1 and highlight innovative mechanisms regulating SphK1 activity in normal and SCD.