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Numerous studies have investigated the relationship between polymorphisms, in particular 677C-T and 1298A-C, of the methylene-tetrahydrofolate reductase (MTHFR) gene and coronary artery disease (CAD) with conflicting results. This study investigates the potential association of two point mutations in MTHFR, 677C-T and 1793G-A, along with other risk factors, with CAD. This is the first hospital-based study to investigate 1793G-A in this context. Genotype analysis was performed on 729 Caucasians and 66 African Americans undergoing coronary angiography using a novel PCR-based assay involving formation of Holliday junctions. Allelic frequencies for 677C-T were 66.2% C and 33.8% T for Caucasians and 90.9% C and 9.1% T for African Americans. With respect to the 1793G-A polymorphism, allelic frequencies were 94.7% G and 5.3% A for Caucasians and 99.2% G and 0.8% A for African Americans. Disease associations were examined in the Caucasian patients due to their greater genotype variability and larger number in the patient cohort. Results suggest that neither 677CT heterozygotes (OR-1.36; 95% CI 0.95 to 1.96) nor mutant homozygotes (OR-0.73; 95% CI 0.44 to 1.20) have either an increased or decreased risk for CAD compared to the 677CC genotype. Likewise, the 1793GA genotype did not demonstrate a statistically significant association with CAD compared to 1793GG patients (OR-0.79; 95% CI 0.47 to 1.33). Mean homocysteine levels (μmol/L) increased from normal to mutant for 677C-T (677CC: 10.2; 677CT: 11.0; 677TT: 11.6) and normal to heterozygous in 1793G-A (1793GG: 10.7; 1793GA: 11.5). These MTHFR polymorphisms did not contribute to the prediction of clinically defined CAD in Caucasians.

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VE‐cadherin, the major endothelial adhesion molecule of AJs, forms hemophilic adhesion and induces re‐organization of actin and microtubule (MT) cytoskeleton. In the current study, we have addressed the role of MT end binding (EB) protein 3 in regulating stability of VE‐cadherin adhesion and lung tissue‐fluid homeostasis. Depletion of EB3 significantly attenuated inositol 1,4,5‐thriphosphate (IP3)‐gated release of calcium from intracellular stores and cell shape change in response to serine protease thrombin. Using acceptor‐photobleaching FRET and pulldown experiments we demonstrated that EB3 interacted with inositol 1,4,5‐thriphosphate receptor type 3 (IP3R3) both in cells and in cell‐free system. Immunofluorescent staining of endogenous IP3R3 and live‐cell imaging of exogenously‐expressed IP3R3‐GFP in EB3 depleted cells showed that the interaction between EB3 and IP3R3 at the MT tip is required for IP3R3 clustering. Rescue with EB3 mutant lacking the last four amino acids did not restored IP3R3 clustering suggesting that presence of acidic tail is critical for interaction with IP3R3. Pre‐treatment of cells with membrane permeant variant of IP3R3‐derivative peptide (IPRP), containing the EBs consensus motif, significantly inhibited the interaction between EB3 and IP3R3 as well as attenuated thrombin‐induced release of calcium from intracellular stores. Furthermore, infusion of IPRP in lung microvasculature significantly inhibited increase in lung vascular liquid permeability (Kf,c) in response to activation of Protease‐activated receptors‐1 with an agonist peptide. These results identify a novel function of EB3 in regulating calcium signaling and tissue‐fluid homeostasis in lung.

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