The vulnerable artery: early and rapid deposition of lipid in coronary arteries is associated with subsequent development of thin-cap fibroatheromas.

AIMS: We aimed to determine whether intravascular ultrasound (IVUS) and near infrared spectroscopy (NIRS) could identify arteries which would subsequently develop a thin-cap fibroatheroma (TCFA). METHODS AND RESULTS: Three-vessel angiography, IVUS and NIRS evaluations were performed at three, six and nine months after induction of diabetes mellitus and hypercholesterolaemia in 13 Yorkshire pigs (n=37 arteries). In vivo total arterial plaque plus media (P+M) area, echo-attenuated plaque (AP) area by IVUS, and lipid core burden index (LCBI) by NIRS were compared to histology at nine months. P+M mean area increased over time (3 vs. 6 months p<0.01; 6 vs. 9 months p<0.01), as did the AP area and mean LCBI between three and six months (p<0.01). There were 69 TCFAs within 18 arteries. The mean LCBI at six months was greater in arteries containing a TCFA (77.8±17.4 vs. 34.3±11.4; p=0.04) as was the ∆LCBI from three to six months (55.3±16.9 vs. 3.3±16.0; p=0.03). Arteries which contained TCFA at nine months had greater AP area by IVUS at six months (p=0.007). CONCLUSIONS: The early and persistent accumulation of total arterial lipid detected by NIRS was associated with the future development of TCFAs.

Atherosclerotic plaque inflammation varies between vascular sites and correlates with response to inhibition of lipoprotein-associated phospholipase A2.

BACKGROUND: Despite systemic exposure to risk factors, the circulatory system develops varying patterns of atherosclerosis for unclear reasons. In a porcine model, we investigated the relationship between site-specific lesion development and inflammatory pathways involved in the coronary arteries (CORs) and distal abdominal aortas (AAs). METHODS AND RESULTS: Diabetes mellitus (DM) and hypercholesterolemia (HC) were induced in 37 pigs with 3 healthy controls. Site-specific plaque development was studied by comparing plaque severity, macrophage infiltration, and inflammatory gene expression between CORs and AAs of 17 DM/HC pigs. To assess the role of lipoprotein-associated phospholipase A2 (Lp-PLA2) in plaque development, 20 DM/HC pigs were treated with the Lp-PLA2 inhibitor darapladib and compared with the 17 DM/HC untreated pigs. DM/HC caused site-specific differences in plaque severity. In the AAs, normalized plaque area was 4.4-fold higher (P<0.001) and there were more fibroatheromas (9 of the 17 animals had a fibroatheroma in the AA and not the COR, P=0.004), while normalized macrophage staining area was 1.5-fold higher (P=0.011) compared with CORs. DM/HC caused differential expression of 8 of 87 atherosclerotic genes studied, including 3 important in inflammation with higher expression in the CORs. Darapladib-induced attenuation of normalized plaque area was site-specific, as CORs responded 2.9-fold more than AAs (P=0.045). CONCLUSIONS: While plaque severity was worse in the AAs, inflammatory genes and inflammatory pathways that use Lp-PLA2 were more important in the CORs. Our results suggest fundamental differences in inflammation between vascular sites, an important finding for the development of novel anti-inflammatory therapeutics.

Porcine models of accelerated coronary atherosclerosis: role of diabetes mellitus and hypercholesterolemia.

Animal models of atherosclerosis have proven to be an invaluable asset in understanding the pathogenesis of the disease. However, large animal models may be needed in order to assess novel therapeutic approaches to the treatment of atherosclerosis. Porcine models of coronary and peripheral atherosclerosis offer several advantages over rodent models, including similar anatomical size to humans, as well as genetic expression and development of high-risk atherosclerotic lesions which are similar to humans. Here we review the four models of porcine atherosclerosis, including the diabetic/hypercholesterolemic model, Rapacz-familial hypercholesterolemia pig, the (PCSK9) gain-of-function mutant pig model, and the Ossabaw miniature pig model of metabolic syndrome. All four models reliably represent features of human vascular disease.

Subsequent development of fibroatheromas with inflamed fibrous caps can be predicted by intracoronary near infrared spectroscopy.

OBJECTIVE: To prospectively evaluate whether the development of fibroatheromas exhibiting features of potential instability can be detected and predicted by serial invasive imaging. METHODS AND RESULTS: Multivessel intravascular ultrasound and near infrared spectroscopy (NIRS) were performed in diabetic/hypercholesterolemic pigs 3, 6, and 9 months after induction. Animals were euthanized at 9 months and histological/immunohistochemical evaluation of the arteries was performed (n=304 arterial segments). Intravascular ultrasound demonstrated, over time, a progressive increase in plaque + media and necrotic core areas and positive vascular remodeling. By histology, NIRS+ lesions were significantly more likely to be a high-risk fibroatheroma (P=0.0001) containing larger plaque (P<0.0001) and necrotic core areas (P<0.0019) and thinner fibrous caps (P=0.04). NIRS + fibroatheromas possessed a greater concentration of inflammatory cells demonstrating protease activity (P=0.006), and proliferating (P=0.016), and apoptotic cells (P=0.04) within the fibrous cap. Eighty-eight percent of NIRS+ lesions at 3 and 6 months subsequently developed into a fibroatheroma at 9 months (P<0.01). By multivariate analysis NIRS positivity at 6 months predicted the subsequent presence of a fibroatheroma at 9 months (P=0.005; odds ratio, 2.71). CONCLUSIONS: The future development of inflamed fibroatheromas with thinner fibrous caps, greater plaque, and necrotic core areas, and posessing characteristics of increased plaque instability were detected by intravascular ultrasound/NIRS imaging.

Vascular responses to drug-eluting and bare metal stents in diabetic/hypercholesterolemic and nonatherosclerotic porcine coronary arteries.

BACKGROUND: Animal models used to gain insight into the vascular response to drug-eluting stents are generally juvenile and nonatherosclerotic, whereas stents are placed in patients with complex atherosclerosis and comorbidities. Hence, models reflecting these complexities are needed to help elucidate the vascular effects of drug-eluting stents. We compared the vascular responses with bare metal stent (BMS) and paclitaxel-eluting stent (PES) implantation in a diabetic/hypercholesterolemic (DM/HC) porcine model of advanced coronary atherosclerosis with the standard juvenile porcine model. METHODS AND RESULTS: Two studies using similar stent procedural protocols were performed in either DM/HC (n=20) or domestic swine (non-DM/HC, n=20). Animals pretreated with dual-antiplatelet therapy, underwent BMS or PES implantation (1/artery, 2 stents per animal) and were euthanized 30 or 90 days later. DM/HC resulted in a 24% increase in platelet aggregation (P=0.05 versus baseline), whereas dual-antiplatelet therapy reduced platelet aggregation in both groups (P<0.0001). DM/HC pigs developed substantially greater neointimal area versus non-DM/HC pigs, regardless of stent type, (P=0.004 for BMS at 30 days and P=0.002 at 90 days, P=0.005 for PES at 30 days, P=0.002 at 90 days). Compared with non-DM/HC pigs, reendothelialization was delayed in DM/HC pigs, more so after PES implantation. Increased para-strut leukocytes were observed for PES compared with BMS in the DM/HC pigs at both 30 days (P=0.023) and 90 days (P=0.04). As well, increased T-lymphocyte infiltration was seen in the DM/HC pigs. CONCLUSIONS: Stent implantation in a DM/HC swine model provides a metabolic environment closer to human disease, including hyperglycemia, hypercholesterolemia, and increased platelet aggregation. This model augmented differences in the vascular response between PES and BMS that are not as clearly evident in the non-DM/HC swine, including increased neointimal area, delayed reendothelialization, and greater, persistent vascular inflammation.

Akt pathway is hypoactivated by synergistic actions of diabetes mellitus and hypercholesterolemia resulting in advanced coronary artery disease.

Atherosclerosis is an inflammatory process leading to enhanced cellular proliferation, apoptosis, and vasa vasorum (VV) neovascularization. While both diabetes mellitus (DM) and hypercholesterolemia (HC) predispose to atherosclerosis, the precise interaction of these risk factors is unclear. Akt is a central node in signaling pathways important for inflammation, and we hypothesized that DM/HC would lead to aberrant Akt signaling and advanced, complex atherosclerosis. DM was induced in pigs by streptozotocin and HC by a high-fat diet. Animals were randomized to control (non-DM, non-HC), DM only, HC only, and DM/HC groups. Coronary artery homogenates were analyzed by immunoblotting for proteins involved in the Akt pathway, including phosphorylated (p)-Akt (Ser473), p-GSK-3beta (Ser9), activated NF-kappaB p65, and VEGF. Immunohistochemical staining for Ki67 (cell proliferation), terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) (apoptosis), and von Willebrand factor (vWF) (neovascularization) was performed. Neovascularization was visualized with micro-computerized tomography (CT). Only DM/HC animals developed advanced atherosclerosis and showed decreased p-Akt (Ser473) and p-GSK-3beta (Ser9) levels (P < 0.01 and P < 0.05, respectively). DM/HC arteries demonstrated increased cellular proliferation (P < 0.001), apoptosis (P < 0.01), and activation of NF-kappaB p65 (P < 0.05). Induction of DM/HC also resulted in significant VV neovascularization by enhanced VEGF expression (P < 0.05), increased vWF staining (P < 0.01), and increased density by micro-CT. In conclusion, DM and HC synergistically resulted in complex atherosclerosis associated with attenuated p-Akt (Ser473) levels. Aberrant Akt signaling correlated with increased inflammation, cellular proliferation, apoptosis, and VV neovascularization. Our results revealed a synergistic effect of DM and HC in triggering abnormal Akt signaling, resulting in advanced atherosclerosis.

Intracoronary delivery of mesenchymal stem cells at high flow rates after myocardial infarction improves distal coronary blood flow and decreases mortality in pigs.

OBJECTIVES: Evaluate the effects of pressure and duration of intracoronary (IC) infusion of mesenchymal stem cells (MSCs) on delivery efficiency and safety after myocardial infarction (MI). BACKGROUND: Standard IC delivery of MSCs can lead to intravascular plugging and reduced coronary blood flow. The optimal delivery pressure and duration is unknown. METHODS: Immediately after MI pigs were randomized to 1 of 3 delivery protocols of 5 x 10(7) iron-fluorescent microspheres labeled MSCs, control received 2 ml infusions at 1 ml/min (five times), very high flow rate (VHFR) a single 10 ml infusion at 60 ml/min and the high flow rate (HFR) group a single 10 ml infusion at 20 ml/min. TIMI grade flow was assessed throughout the procedure and at sacrifice (day 14). MSCs distribution was analyzed in isolated hearts by 4.7T MRI. Delivery efficiency was quantified via fluorescent microsphere recovery using a magnetic separation technique and by light microscopy. RESULTS: TIMI grade flow did not change following MI (all groups TIMI 3). However, following MSCs delivery only 18% (2/11) of control animals had TIMI 3 blood flow vs. 56% (5/9) in VHFR and 67% (4/6) in HFR (P = 0.03). As a consequence, 63% of control animals died within 24 hr, 33% in VHFR and none in HFR (P = 0.02). MSCs delivery in the infarct tissue did not differ between the groups (P = 0.06). CONCLUSIONS: A single MSCs infusion at 20 ml/min resulted in improved coronary blood flow and decreased mortality, without sacrificing delivery efficiency.

Diabetes reduces bone marrow and circulating porcine endothelial progenitor cells, an effect ameliorated by atorvastatin and independent of cholesterol.

Bone marrow derived endothelial progenitor cells (EPCs) are early precursors of mature endothelial cells which replenish aging and damaged endothelial cells. The authors studied a diabetic swine model to determine if induction of DM adversely affects either bone marrow or circulating EPCs and whether a HMG-CoA reductase inhibitor (statin) improves development and recruitment of EPCs in the absence of cholesterol lowering. Streptozotocin was administered to Yorkshire pigs to induce DM. One month after induction, diabetic pigs were treated with atorvastatin (statin, n = 10), ezetimibe (n = 10) or untreated (n = 10) and evaluated for number of bone marrow and circulating EPCs and femoral artery endothelial function. There was no effect of either medication on cholesterol level. One month after induction of DM prior to administration of drugs, the number of bone marrow and circulating EPCs significantly decreased (P < 0.0001) compared to baseline. Three months after DM induction, the mean proportion of circulating EPCs significantly increased in the atorvastatin group, but not in the control or ezetimibe groups. The control group showed progressive reduction in percentage of flow mediated vasodilatation (no dilatation at 3 months) whereas the atorvastatin group and ezetimibe exhibited vasodilatation, 6% and 4% respectively. DM results in significant impairment of bone marrow and circulating EPCs as well as endothelial function. The effect is ameliorated, in part, by atorvastatin independent of its cholesterol lowering effect. These data suggest a model wherein accelerated atherosclerosis seen with DM may, in part, result from reduction in EPCs which may be ameliorated by treatment with a statin.

Inhibition of lipoprotein-associated phospholipase A2 reduces complex coronary atherosclerotic plaque development.

Increased lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) activity is associated with increased risk of cardiac events, but it is not known whether Lp-PLA(2) is a causative agent. Here we show that selective inhibition of Lp-PLA(2) with darapladib reduced development of advanced coronary atherosclerosis in diabetic and hypercholesterolemic swine. Darapladib markedly inhibited plasma and lesion Lp-PLA(2) activity and reduced lesion lysophosphatidylcholine content. Analysis of coronary gene expression showed that darapladib exerted a general anti-inflammatory action, substantially reducing the expression of 24 genes associated with macrophage and T lymphocyte functioning. Darapladib treatment resulted in a considerable decrease in plaque area and, notably, a markedly reduced necrotic core area and reduced medial destruction, resulting in fewer lesions with an unstable phenotype. These data show that selective inhibition of Lp-PLA(2) inhibits progression to advanced coronary atherosclerotic lesions and confirms a crucial role of vascular inflammation independent from hypercholesterolemia in the development of lesions implicated in the pathogenesis of myocardial infarction and stroke.

Site-specific atherogenic gene expression correlates with subsequent variable lesion development in coronary and peripheral vasculature.

OBJECTIVE: The relationship between specific gene regulation and subsequent development and progression of atherosclerosis is incompletely understood. We hypothesized that genes in the vasculature related to cholesterol metabolism, inflammation, and insulin signaling pathways are differentially regulated in a site-specific and time-dependent manner. METHODS AND RESULTS: Expression of 59 genes obtained from coronary, carotid, and thoracic aortic arteries were characterized from diabetic (DM)/hypercholesterolemic (HC) swine (n=52) 1, 3, and 6 months after induction. Lesion development in the 3 arterial beds was quantified and characterized at 1, 3, 6, and 9 months. Progressive lesion development was observed in the coronary>thoracic aorta>>carotid arteries. Genes involved in cholesterol metabolism and insulin pathways were upregulated in coronaries>thoracic aortae>carotids. Inflammatory genes were more markedly upregulated in coronary arteries than the other 2 arteries. Genes implicated in plaque instability (eg, matrix metalloproteinase-9, CCL2 and Lp-PLA(2) mRNAs) were only upregulated at 6 months in coronary arteries. CONCLUSIONS: Variable gene expression, both in regard to the arterial bed and duration of disease, was associated with variable plaque development and progression. These findings may provide further insight into the atherosclerotic process and development of potential therapeutic targets.

A placebo controlled, dose-ranging, safety study of allogenic mesenchymal stem cells injected by endomyocardial delivery after an acute myocardial infarction.

AIMS: Although mesenchymal stem cells (MSCs) show promising signs in reducing myocardial infarct (MI) size, the safety of endomyocardial delivery and the most efficacious dose is unknown. METHODS AND RESULTS: Three days after MI, female Yorkshire swine (25-32 kg, age 2 months, n = 32) were randomized to endomyocardial delivery of one of three MSC doses (2.4 x 10(7), 2.4 x 10(8), 4.4 x 10(8) cells) or vehicle control. Animals were sacrificed at 12 weeks. There were no safety issues related to cell delivery and all animals tolerated the procedure. By magnetic resonance imaging infarct size (g) was decreased in the experimental groups and increased in the control group; 2.4 x 10(7): Delta -2.5 +/- 2.5 g, 2.4 x 10(8): -0.9 +/- 2.71 g, 4.4 x 10(8): -1.6 +/- 5.8 g, and control +3.6 +/- 3.4 g (P = 0.002, P = 0.016, and P = 0.055 compared with control, respectively). There was no effect on ejection fraction or left ventricular volumes. By histology there were no toxic effects of MSC delivery, however, few engrafted MSCs were observed. CONCLUSION: Direct MSC delivery into infarcted myocardium was safe and produced a local but not a functional effect. There was no dose-dependent effect. The effect of MSCs on infarct reduction may result from transient residence and subsequent paracrine effects.

The molecular basis of vulnerable plaque: potential therapeutic role for immunomodulation.

PURPOSE OF REVIEW: Atherosclerosis is a chronic inflammatory/immune disease involving multiple cell types including monocytes-macrophages, T-lymphocytes, mast cells, and endothelial cells. Through recent studies the role of the immune system on development of atherosclerosis and approaches to modulate this response are being elucidated. RECENT FINDINGS: The use of statins, PPARgamma agonists or lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibitors may play a role in reducing progression of atherosclerosis through immunomodulatory pathways. Oxidized LDL biases development toward the pro-inflammatory T-cell Th1 subset and recruits macrophages into the vascular wall. IFNgamma, produced by Th1 cells, inhibits PPARgamma effects. Lp-PLA2 levels correlate with an increased risk of recurrent ischemic events in patients presenting with acute coronary syndromes or myocardial infarction. SUMMARY: Recent research has shown that immune pathways play a major role in the development and progression of atherosclerosis. Commonly used medications, specifically statins and some PPARgamma agonists, have demonstrated anti-inflammatory/immune effects unrelated to their primary mode of action. Treatment of infectious agents has proven elusive in the clinical arena. Novel agents targeting immune and inflammatory pathways may prove beneficial in reducing progression and instability of the atherosclerotic plaque.

The role of infectious agents in the pathogenesis of systemic sclerosis.

Over the past few years, increasing evidence has accumulated to implicate infectious agents in the etiology of systemic sclerosis (SSc) and Raynaud phenomenon. Infection rates in patients with SSc compared with those in control populations do not provide clear support for any specific pathogen. However, increased antibody titers, a preponderance of specific strains in patients with SSc, and evidence of molecular mimicry inducing autoimmune responses suggest mechanisms by which infectious agents may contribute to the development and progression of SSc. Here we review studies examining the potential involvement of, cytomegalovirus, and parvovirus B19 in SSc pathogenesis.