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Computed Tomography Coronary Angiography (CTCA)is a technique for imaging coronary arteries with increasing indications in clinical cardiology. AIMS 1.Develop a heart rate (HR) lowering regime for CTCA and to measure its association with image quality. 2.Examine the diagnostic accuracy of 64 slice CTCA (CTCA64) in patients with known coronary artery disease (CAD). 3.Examine the diagnostic accuracy of CTCA64 for assessment of stent restenosis 4.Demonstrate utility of CTCA as an endpoint in assessment of novel diagnostic biomarkers of CAD. METHODS I developed a HR reducing strategy using metoprolol and assessed its effectiveness for improving CTCA64 image quality. The diagnostic value of CTCA in patients with suspected angina was evaluated by comparison with invasive coronary angiography. The diagnostic value of CTCA for quantifying stent restenosis was evaluated by comparison with intravascular ultrasound. The utility of CTCA for evaluating the diagnostic value of B-type natriuretic peptide (BNP) and high sensitivity cardiac troponin I (hs- TnI) was evaluated by blood sampling in patients with suspected angina who subsequently underwent CTCA. RESULTS 1.In 121 patients undergoing CTCA, 75 required rate control. This was achieved (rate ≤60 bpm) in 83% using a systematic regimen of oral and IV metoprolol (n=71) or verapamil (n=4). I demonstrated a significant relation between HR reduction and graded image quality (p<0.001). 2.80 patients underwent CTCA64 and invasive coronary angiography. 724 coronary arterial segments were available for analysis. The sensitivity and specificity of CTCA for significant luminal stenosis was 83.3% (95% CI 67.1-92.5%) and 96.7% (95% CI 95.1-97.9%), respectively, but the positive predictive value was only 63.5% (95% CI 50.4-75.3%). 3.80 patients with 125 stented segments underwent CTCA64 and invasive coronary angiography. Additional intravascular ult rasound (IVUS) examination of stented segments was performed in 48 patients. Using IVUS as the gold-standard for stent restenosis, CTCA and invasive coronary angiography had comparable diagnostic specificities for binary stent restenosis: 82.7% (95% confidence intervals 69.7- 91.84%)and 78.9% (95% confidence intervals 65.3-88.9%), respectively. Sensitivities were lower, particularly the sensitivity of CTCA which was only 11.8% (95% confidence intervals 1.5-36.4%) compared with 58.8% (95% confidence intervals 32.9-81.6%) for invasive coronary angiography. 4. In 93 patients with suspected angina CTCA64 provided a useful endpoint for assessing the diagnostic value of novel circulating biomarkers. BNP levels were higher in the 13 patients shown to have significant (≥50% stenosis) coronary artery disease compared with patients who had unobstructed coronary arteries (18.08pg/ml (IQR 22) vs 9.14pg/ml (IQR 12.62), p=0.024) and increased significantly with exercise, particularly in the group with anatomic coronary artery disease (2.73 ± 5.69 pg/ml vs 1.27±3.29 pg/ml, p=0.16). Conversely I found no association between hs-TnI and the presence of CAD. CONCLUSION Image quality of CTCA64 is enhanced by heart rate reduction below 60 bpm which can be achieved safely by a regimen of oral and intravenous metoprolol. Although CTCA64 is a useful non-invasive method for diagnosis of coronary artery disease, it has a low positive predictive value for identifying severe (≥50%) luminal stenosis which limits its clinical value. Its value for assessment of stent restenosis is even more limited but it finds useful application as an endpoint for diagnostic evaluation of novel biomarkers, allowing confirmation of an association between circulating BNP levels and stable coronary artery disease.

Restenosis is one of the major adverse outcomes of Percutaneous Coronary Intervention (PCI). Previous studies have shown conflicting reports for homocysteine as a predictor of restenosis following PCI. The conflicting reports may be due to oxidative factors (stimulation of polymorphonuclear leukocyte [PMNL]-induced reactive oxygen species generation, xanthine- xanthine oxidase, and arachidonic acid metabolism) other than homocysteine which could cause endothelial cell dysfunction leading to restenosis. Malondialdehyde (MDA), a lipid peroxidation product, is a marker for oxidative stress and is related to all oxidative factors. Therefore, it is possible that serum MDA may be a better predictor of restenosis than plasma homocysteine. The purpose of this study is to determine whether or not the pre-procedural serum MDA and plasma homocysteine levels are elevated in patients who develop restenosis post PCI. <p>The study included fifty-one patients undergoing elective PCI who consented to participate in a protocol that was approved by the Ethics Committee of the University of Saskatchewan. Homocysteine and malondialdehyde were measured in the plasma and serum respectively. Blood samples were collected pre-procedural, 0 time, 8 hours, 24 hours, and 6 months post-procedure. Exercise tolerance tests were performed at two weeks, and six months post-procedure to determine if there was any evidence of restenosis. <p>The results of the study showed that pre-procedural values of plasma homocysteine in the restenosis and non-restenosis groups were 10.37 ± 0.46 and 10.73 ± 0.49 respectively. These values were not significantly different (p=0.60) between the groups. The pre-procedural levels of plasma homocysteine were not significantly different (p=0.08) from the post-PCI values of those patients who did not develop restenosis at the 6-month time interval. However, the pre-procedural levels of plasma homocysteine were significantly different from the post-PCI values of those patients in the restenosis group at the 24hr (p=0.04) and 6-month (p=0.002) time intervals. In the restenosis group there was a significant increase (24%) after six months in the values of homocysteine from the pre-procedural levels. Thus, this indicates that restenosis is associated with higher post-PCI levels of homocysteine. <p>The pre-procedural levels of serum MDA in the restenosis and non-restenosis groups were 0.124± 0.16 and 0.147± 0.02 respectively. There was no significant difference (p=0.60) between the two groups. There was also no significant difference (p=0.053) between the pre-procedural values and the 6-month post-PCI values in those patients who did not develop restenosis. However, there was a significant difference (p=0.001) between the pre-procedural values and the 6-month post-PCI values in those patients who developed restenosis. The levels of serum MDA in patients with restenosis at 6-months increased by 109% and were significantly different (p=0.001) in the restenosis group. <p>The results suggest that pre-procedural levels of plasma homocysteine and serum MDA were not predictors of restenosis following PCI. However, the post-PCI six-month levels of both homocysteine and MDA are predictors of restenosis. Moreover, the post-PCI levels of MDA were better predictors of restenosis than the post-PCI levels of homocysteine because the increase in MDA levels were greater at six months than the rise in homocysteine levels at the same time interval.

Restenosis is one of the major adverse outcomes of Percutaneous Coronary Intervention (PCI). Previous studies have shown conflicting reports for homocysteine as a predictor of restenosis following PCI. The conflicting reports may be due to oxidative factors (stimulation of polymorphonuclear leukocyte [PMNL]-induced reactive oxygen species generation, xanthine- xanthine oxidase, and arachidonic acid metabolism) other than homocysteine which could cause endothelial cell dysfunction leading to restenosis. Malondialdehyde (MDA), a lipid peroxidation product, is a marker for oxidative stress and is related to all oxidative factors. Therefore, it is possible that serum MDA may be a better predictor of restenosis than plasma homocysteine. The purpose of this study is to determine whether or not the pre-procedural serum MDA and plasma homocysteine levels are elevated in patients who develop restenosis post PCI. <p>The study included fifty-one patients undergoing elective PCI who consented to participate in a protocol that was approved by the Ethics Committee of the University of Saskatchewan. Homocysteine and malondialdehyde were measured in the plasma and serum respectively. Blood samples were collected pre-procedural, 0 time, 8 hours, 24 hours, and 6 months post-procedure. Exercise tolerance tests were performed at two weeks, and six months post-procedure to determine if there was any evidence of restenosis. <p>The results of the study showed that pre-procedural values of plasma homocysteine in the restenosis and non-restenosis groups were 10.37 ± 0.46 and 10.73 ± 0.49 respectively. These values were not significantly different (p=0.60) between the groups. The pre-procedural levels of plasma homocysteine were not significantly different (p=0.08) from the post-PCI values of those patients who did not develop restenosis at the 6-month time interval. However, the pre-procedural levels of plasma homocysteine were significantly different from the post-PCI values of those patients in the restenosis group at the 24hr (p=0.04) and 6-month (p=0.002) time intervals. In the restenosis group there was a significant increase (24%) after six months in the values of homocysteine from the pre-procedural levels. Thus, this indicates that restenosis is associated with higher post-PCI levels of homocysteine. <p>The pre-procedural levels of serum MDA in the restenosis and non-restenosis groups were 0.124± 0.16 and 0.147± 0.02 respectively. There was no significant difference (p=0.60) between the two groups. There was also no significant difference (p=0.053) between the pre-procedural values and the 6-month post-PCI values in those patients who did not develop restenosis. However, there was a significant difference (p=0.001) between the pre-procedural values and the 6-month post-PCI values in those patients who developed restenosis. The levels of serum MDA in patients with restenosis at 6-months increased by 109% and were significantly different (p=0.001) in the restenosis group. <p>The results suggest that pre-procedural levels of plasma homocysteine and serum MDA were not predictors of restenosis following PCI. However, the post-PCI six-month levels of both homocysteine and MDA are predictors of restenosis. Moreover, the post-PCI levels of MDA were better predictors of restenosis than the post-PCI levels of homocysteine because the increase in MDA levels were greater at six months than the rise in homocysteine levels at the same time interval.

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No description available.

Percutaneous coronary intervention with stent deployment is the dominant form of revascularisation for patients with coronary artery disease. Although drug-eluting stents have reduced the incidence of instent restenosis, they are associated with late problems related to delayed vascular healing including late stent thrombosis. The use of gene-eluting stents offers the potential to deliver localised gene therapy to the vascular wall with the aim of both reducing restenosis and promoting endothelialisation. Two candidate genes were investigated. Connective tissue growth factor (CTGF) promotes smooth muscle cell apoptosis and stimulates endothelial growth in vitro, and has an integral role in wound healing. Fibromodulin (FMOD) is involved in collagen metabolism and is a key mediator of scarless wound healing. Both genes have previously been shown to suppress restenosis in an ex vivo vein graft model. Plasmids containing these two genes were constructed with an expression cassette specially designed to maximise transgene expression in vascular smooth muscle cells. These plasmids were coated onto coronary stents with a polymer and the effects of these gene-eluting stents were investigated in an in vivo pig coronary artery model. Previous work by our group has suggested that systemic -blockade can affect the degree of transgene expression from viral vectors, and experiments were also performed to investigate the effect of β-blockers on plasmid-mediated gene expression. At 28 days there was no significant difference in angiographic late loss or neointimal hyperplasia between the groups treated with stents coated with FMOD or CTGF and the group treated with stents coated with the marker gene lacZ. This lack of efficacy appeared to be as a result of extremely poor transgene expression rather than due to a genuine failure of the transgenes to elicit a relevant biological effect. There was no difference in in vivo gene expression demonstrated as a result of β-blockade, but again this result was probably due to limited transgene expression. The potential causes of poor transgene expression in this study are reviewed and future directions for research on plasmid-mediated gene therapy are considered.

The first genetic CAD risk locus to be identified by genome-wide association studies, single nucleotide polymorphisms (SNPs) at 9p21.3 predispose to increased risk of CAD. By bioinformatics scan analysis of the 9p21.3 locus; we interrogated the 59 linked SNPs over the 53,202bp to identify putative transcription factor-binding consensus sequences. We hypothesize that some genetic polymorphisms at the 9p21.3 locus are functional and will disrupt specific regulatory sequences within enhancers. Here, I investigated how polymorphisms affect TEAD-dependent regulation at the 9p21.3 locus, and also how polymorphisms affect GATA factor-dependent regulation at the 9p21.3 locus, using cultured HEK293 and primary human aortic smooth muscle cells (HAoSMCs) to transfect the pGL3-promoter plasmid constructs containing the reference or risk variant sequences (rs10611656, rs4977757, rs10757269, rs9632885). We showed by luciferase reporter assay that the risk allele of the SNPs disrupt activation by various TEAD transcription factors. We also performed electrophoretic mobility shift assay (EMSA) to test for allele-specific transcription factor binding that affect the family of TEAD transcription factors and the GATA factors. EMSA showed binding of TEAD3 and TEAD4, and differential binding for both GATA genotypes, and luciferase reporter assay confirmed that TEAD3 and TEAD4 activate the non-risk but not the risk allele, and for GATA factors no significant activation was shown. Our investigations lead us to conclude that rs10811656 and rs4977757 are functional and disrupt specific TEAD regulatory sequences within enhancers

Coronary artery disease (CAD) remains the leading cause of death in both the United States and the world at large. This is primarily due to the extreme difficulty associated with preemptive diagnosis of CAD. Currently, only about 20% of all patients are diagnosed with CAD prior to the occurrence of a heart attack. This is the result of limitations in current techniques, which are either noninvasive, extremely expensive, or have very poor correlation with the actual disease state of the patient. Phonoangiography is an alternative approach to the diagnosis of CAD that relies upon detection of the sound generated by turbulent flow downstream from occlusions. Although the technique is commonly used for the carotid arteries, in the case of the coronary arteries the technique is hampered by signal-to-noise problems as well as uncertainty regarding the spectral characteristics associated with CAD. To date, these signal processing difficulties have prevented the use of the technique clinically. This research introduces an alternative approach to the processing of phonoangiographic data based upon knowledge of the acoustic transfer within the chest. The validity of the proposed approach was examined using transfer functions which were calculated for 14 physiologically relevant locations within the chest using a 2-D Finite Element Model (FEM) generated from physiologic data. These transfer functions were then used to demonstrate the technique using test cases generated with the FEM. Finally, the vulnerability of the technique to noise was quantified through calculation of matrix condition numbers for the chest acoustic transfer at each frequency. These results show that while in general the technique is susceptible to noise; noise tolerance is greatly improved within the frequency range most likely to correspond to an occlusion. Taken together, these results suggest that the proposed technique has the potential to make phonoangiography viable as a screening technique for CAD. Such a technique would greatly reduce the cost of CAD, measured in terms of both financial cost as well as lives. / Master of Science

Toxicity preventing activity (TxPA) is a recently identified substance in serum which counteracts the toxic effect of very low density lipoproteins upon endothelial cells in vitro. In two clinical studies, TxPA was low in individuals with angiographically demonstrable coronary artery disease. An atherogenic index which combines TxPA with lipoprotein cholesterol values classifies individuals with coronary artery disease with an accuracy of greater than 93%. TxPA precipitates with 0.15 M trichloroacetic acid and above 3 M (NH4)2SO4. Activity is present in Cohn fractions IV4 and V and is stabilized by antioxidants. TxPA co-lutes with the albumin peak on gel filtration chromatography and as a subcomponent of albumin on ion-exchange chromatography. Isoelectric focusing resolves albumin into two major peaks with pI values of 4.8 and 5.6. The TxPA is identified as the pI 5.6 albumin peak.