Dc Crossman

Microbubble-enhanced ultrasound for vascular gene delivery

Progress in cardiovascular gene therapy has been hampered by concerns over the safety and practicality of viral vectors and the inefficiency of current nonviral transfection techniques. We have previously reported that ultrasound exposure (USE) enhances transgene expression in vascular cells by up to 10-fold after naked DNA transfection, and enhances lipofection by up to three-fold. We report here that performing USE in the presence of microbubble echocontrast agents enhances acoustic cavitation and is associated with approximately 300-fold increments in transgene expression after naked DNA transfections. This approach also enhances by four-fold the efficiency of polyplex transfection, yielding transgene expression levels approximately 3000-fold higher than after naked DNA alone. These data indicate an important role for acoustic cavitation in the effects of USE. Ultrasound can be focused upon almost any organ and hence this approach holds promise as a means to deliver targeted gene therapy in cardiovascular conditions such as such angioplasty restenosis and in many other clinical situations.

Interleukin-1 Receptor Antagonist Gene Polymorphism and Coronary Artery Disease

BACKGROUND Cytokine gene variations are contributory factors in inflammatory pathology. Allele frequencies of interleukin (IL)-1 cluster genes [IL-1A(-889), IL-1B(-511), IL-1B(+3953), IL-1RN Intron 2 VNTR] and tissue necrosis factor (TNF)-alpha gene [TNFA(-308)] were measured in healthy blood donors (healthy control subjects), patients with angiographically normal coronary arteries (patient control subjects), single-vessel coronary disease (SVD), and those with multivessel coronary disease (MVD). METHODS AND RESULTS Five hundred fifty-six patients attending for coronary angiography in Sheffield were studied: 130 patient control subjects, 98 SVD, and 328 MVD. Significant associations were tested in an independent population (London) of 350: 57 SVD, 191 MVD, and 102 control subjects. IL-1RN*2 frequency in Sheffield patient control subjects was the same as in 827 healthy control subjects. IL-1RN*2 was significantly overrepresented in Sheffield SVD patients (34% vs 23% in patient control subjects); IL-1RN*2 homozygotes in the SVD population (chi2 carriage=8.490, 1 df, P=0.0036). This effect was present though not quite significant in the London population (P=0. 0603). A summary trend test of the IL-1RN SVD genotype data for Sheffield and London showed a significant association with *2 (P=0. 0024). No significant effect of genotype at IL-1RN was observed in the Sheffield or London MVD populations. Genotype distribution analysis comparing the SVD and MVD populations at IL-1RN showed a highly significant trend (P=0.0007) with the use of pooled data. No significant associations were seen for the other polymorphisms. CONCLUSIONS IL-1RN*2 was significantly associated with SVD. A difference in genetic association between SVD and MVD was also apparent.

Ultrasound Enhances Reporter Gene Expression After Transfection of Vascular Cells In Vitro

BACKGROUND Restenosis after percutaneous coronary intervention remains a serious clinical problem. Progress in local gene therapy to prevent restenosis has been hindered by concerns over the safety and efficacy of viral vectors and the limited efficiency of nonviral techniques. This study investigates the use of adjunctive ultrasound to enhance nonviral gene delivery. METHODS AND RESULTS Cultured porcine vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) were transfected with naked or liposome-complexed luciferase reporter plasmid for 3 hours. Ultrasound exposure (USE) for 60 seconds at 1 MHz, 0.4 W/cm2, 30 minutes into this transfection period enhanced luciferase activity 48 hours later by 7.5-fold and 2. 4-fold, respectively. Luciferase activity after lipofection of ECs was similarly enhanced 3.3-fold by adjunctive USE. USE had no effect on cell viability, although it inhibited VSMC but not EC proliferation. CONCLUSIONS Adjunctive USE was associated with enhanced transgene expression in VSMCs and ECs and reduced VSMC but not EC proliferation in vitro, which suggests that ultrasound-assisted local gene therapy has potential as an antirestenotic therapy.

A Genomewide Scan for Early-Onset Coronary Artery Disease in 438 Families: The GENECARD Study

A family history of coronary artery disease (CAD), especially when the disease occurs at a young age, is a potent risk factor for CAD. DNA collection in families in which two or more siblings are affected at an early age allows identification of genetic factors for CAD by linkage analysis. We performed a genomewide scan in 1,168 individuals from 438 families, including 493 affected sibling pairs with documented onset of CAD before 51 years of age in men and before 56 years of age in women. We prospectively defined three phenotypic subsets of families: (1) acute coronary syndrome in two or more siblings; (2) absence of type 2 diabetes in all affected siblings; and (3) atherogenic dyslipidemia in any one sibling. Genotypes were analyzed for 395 microsatellite markers. Regions were defined as providing evidence for linkage if they provided parametric two-point LOD scores >1.5, together with nonparametric multipoint LOD scores >1.0. Regions on chromosomes 3q13 (multipoint LOD = 3.3; empirical P value <.001) and 5q31 (multipoint LOD = 1.4; empirical P value <.081) met these criteria in the entire data set, and regions on chromosomes 1q25, 3q13, 7p14, and 19p13 met these criteria in one or more of the subsets. Two regions, 3q13 and 1q25, met the criteria for genomewide significance. We have identified a region on chromosome 3q13 that is linked to early-onset CAD, as well as additional regions of interest that will require further analysis. These data provide initial areas of the human genome where further investigation may reveal susceptibility genes for early-onset CAD.

Apoptosis and Cell Proliferation After Porcine Coronary Angioplasty

BACKGROUND Angioplasty initiates a number of responses in the vessel wall including cellular migration, proliferation, and matrix accumulation, all of which contribute to neointima formation and restenosis. Cellular homeostasis within a tissue depends on the balance between cell proliferation and apoptosis. METHODS AND RESULTS Profiles of apoptosis and proliferation were therefore examined in a porcine PTCA injury model over a 28-day period. Forty-two arteries from 21 pigs, harvested at the site of maximal injury at 1, 6, and 18 hours, and 3, 7, 14, and 28 days after PTCA, were examined (n=3 animals per time point). Uninjured arteries were used as controls. Apoptosis was demonstrated by the terminal uridine nick-end labeling (TUNEL) method, transmission electron microscopy (TEM), and DNA fragmentation. Cells traversing the cell cycle were identified by immunostaining for proliferating cell nuclear antigen (PCNA). Apoptosis was not detected in control vessels at all time points nor at 28 days after PTCA. Apoptotic cells were identified at all early time points with a peak at 6 hours (5.1+/-0.26%; compared to uninjured artery, P<0.001) and confirmed by characteristic DNA ladders and TEM findings. Regional analysis showed apoptosis within the media, adventitia, and neointima peaked at 18 hours, 6 hours, and 7 days after PTCA, respectively. In comparison, PCNA staining peaked at 3 days after PTCA (7.16+/-0.29%; compared to 1.78+/-0.08% PCNA-positive cells in the uninjured artery, P<0.001). Profiles of apoptosis and cell proliferation after PTCA were discordant in all layers of the artery except the neointima. These profiles also differed between traumatized and nontraumatized regions of the arterial wall. Immunostaining with cell-type specific markers and TEM analysis revealed that apoptotic cells included vascular smooth muscle cells (VSMCs), inflammatory cells, and adventitial fibroblasts. CONCLUSIONS These results suggest that the profile of apoptosis and proliferation after PTCA is regional and cell specific, and attempts to modulate either of these events for therapeutic benefit requires recognition of these differences.

Brain inflammation is induced by co-morbidities and risk factors for stroke

Highlights ► Risk factors for stroke include atherosclerosis, obesity, diabetes and hypertension. ► Stroke risk factors are associated with peripheral inflammation. ► Corpulent rats and atherogenic mice show increased inflammation in the brain. ► Pilot data show that patients at risk of stroke may also develop brain inflammation. ► Chronic peripheral inflammation can drive inflammatory changes in the brain.

Simultaneous intravital imaging of macrophage and neutrophil behaviour during inflammation using a novel transgenic zebrafish

The zebrafish is an outstanding model for intravital imaging of inflammation due to its optical clarity and the ability to express fluorescently labelled specific cell types by transgenesis. However, although several transgenic labelling myeloid cells exist, none allow distinction of macrophages from neutrophils. This prevents simultaneous imaging and examination of the individual contributions of these important leukocyte subtypes during inflammation. We therefore used Bacterial Artificial Chromosome (BAC) recombineering to generate a transgenic Tg(fms:GAL4.VP16)i186 , in which expression of the hybrid transcription factor Gal4-VP16 is driven by the fms (CSF1R) promoter. This was then crossed to a second transgenic expressing a mCherry-nitroreductase fusion protein under the control of the Gal4 binding site (the UAS promoter), allowing intravital imaging of mCherry-labelled macrophages. Further crossing this compound transgenic with the neutrophil transgenic Tg(mpx:GFP)i114 allowed clear distinction between macrophages and neutrophils and simultaneous imaging of their recruitment and behaviour during inflammation. Compared with neutrophils, macrophages migrate significantly more slowly to an inflammatory stimulus. Neutrophil number at a site of tissue injury peaked around 6 hours post injury before resolving, while macrophage recruitment increased until at least 48 hours. We show that macrophages were effectively ablated by addition of the prodrug metronidazole, with no effect on neutrophil number. Crossing with Tg(Fli1:GFP)y1 transgenic fish enabled intravital imaging of macrophage interaction with endothelium for the first time, revealing that endothelial contact is associated with faster macrophage migration. Tg(fms:GAL4.VP16)i186 thus provides a powerful tool for intravital imaging and functional manipulation of macrophage behaviour during inflammation.

Peakwide Mapping on Chromosome 3q13 Identifies the Kalirin Gene as a Novel Candidate Gene for Coronary Artery Disease

A susceptibility locus for coronary artery disease (CAD) has been mapped to chromosome 3q13-21 in a linkage study of early-onset CAD. We completed an association-mapping study across the 1-LOD-unit-down supporting interval, using two independent white case-control data sets (CATHGEN, initial and validation) to evaluate association under the peak. Single-nucleotide polymorphisms (SNPs) evenly spaced at 100-kb intervals were screened in the initial data set (N=468). Promising SNPs (P<.1) were then examined in the validation data set (N=514). Significant findings (P<.05) in the combined initial and validation data sets were further evaluated in multiple independent data sets, including a family-based data set (N=2,954), an African American case-control data set (N=190), and an additional white control data set (N=255). The association between genotype and aortic atherosclerosis was examined in 145 human aortas. The peakwide survey found evidence of association in SNPs from multiple genes. The strongest associations were found in three SNPs from the kalirin (KALRN) gene, especially in patients with early-onset CAD (P=.00001-00028 in the combined CATHGEN data sets). In-depth investigation of the gene found that an intronic SNP, rs9289231, was associated with early-onset CAD in all white data sets examined (P<.05). In the joint analysis of all white early-onset CAD cases (N=332) and controls (N=546), rs9289231 was highly significant (P=.00008), with an odds-ratio estimate of 2.1. Furthermore, the risk allele of this SNP was associated with atherosclerosis burden (P=.03) in 145 human aortas. KALRN is a protein with many functions, including the inhibition of inducible nitric oxide synthase and guanine-exchange-factor activity. KALRN and two other associated genes identified in this study (CDGAP and MYLK) belong to the Rho GTPase-signaling pathway. Our data suggest the importance of the KALRN gene and the Rho GTPase-signaling pathway in the pathogenesis of CAD.

Coronary artery stretch versus deep injury in the development of in-stent neointima

Objective: To investigate the relative importance of stent induced arterial stretch and deep injury to the development of in-stent neointima. Setting: Normal porcine coronary arteries Methods: 30 BiodivYsio stents (Biocompatibles) were deployed at a stent to artery ratio of 1.25:1 (a moderate injury) and harvested at 28 days. Multiple serial cross sections were analysed morphometrically and the neointimal areas were correlated with the type and degree of injury. Results: Arterial stretch occurred in 78% of struts (77% of sections) and produced moderate neointimal growth (neointimal area 1.93 (0.13) mm2). Deep injury (rupture of the internal elastic lamina) occurred in 20% of struts (23% of sections) and produced a 1.7-fold increase in neointimal area (3.33 (0.41) mm2) compared with stretch only (p = 0.0002). With even deeper injury (rupture of the external elastic lamina), there was a 2.6-fold increase in neointimal area (5.01 (0.48) mm2) compared with stretch only (p = 0.02). A new injury score, incorporating both stretch and deep injury, correlated with neointimal area (r = 0.60, p < 0.001). Conclusions: Stretch of the coronary artery in a stent is common, and a major contributor to neointima formation, even in the absence of deep injury. Deep injury is, however, a more potent stimulus to neointima formation than stretch. Greater degrees of stretch are associated with thicker neointima. Where neither deep injury nor stretch are seen, the stent has no effect upon the development of neointima.

Modeling Cardiovascular Disease in the Zebrafish

The zebrafish possesses a host of advantages that have established it as an excellent model of vertebrate development. These include ease of genetic manipulation and transgenesis, optical clarity, and small size and cost. Biomedical researchers are increasingly exploiting these advantages to model human disease mechanisms. Here we review the use of the zebrafish for cardiovascular research. We summarize previous studies with the use of this organism to model such processes as thrombosis, collateral vessel development, inflammation, cardiomyopathy, and cardiac regeneration, evaluate its promise for novel drug discovery, and consider where the zebrafish fits into the framework of existing cardiovascular models.