K. Raman Purushothaman

Plaque Neovascularization Is Increased in Ruptured Atherosclerotic Lesions of Human Aorta Implications for Plaque Vulnerability

Background—Growth of atherosclerotic plaques is accompanied by neovascularization from vasa vasorum microvessels extending through the tunica media into the base of the plaque and by lumen-derived microvessels through the fibrous cap. Microvessels are associated with plaque hemorrhage and may play a role in plaque rupture. Accordingly, we tested this hypothesis by investigating whether microvessels in the tunica media, the base of the plaque, and the fibrous cap are increased in ruptured atherosclerotic plaques in human aorta. Methods and Results—Microvessels, defined as CD34-positive tubuloluminal capillaries recognized in cross-sectional and longitudinal profiles, were quantified in 269 advanced human plaques by bicolor immunohistochemistry. Macrophages/T lymphocytes and smooth muscle cells were defined as CD68/CD3-positive and &agr;-actin–positive cells. Total microvessel density was increased in ruptured plaques when compared with nonruptured plaques (P=0.0001). Furthermore, microvessel density was increased in lesions with severe macrophage infiltration at the fibrous cap (P=0.0001) and at the shoulders of the plaque (P=0.0001). In addition, microvessel density was also increased in lesions with intraplaque hemorrhage (P=0.04) and in thin-cap fibroatheromas (P=0.038). Logistic regression analysis identified plaque base microvessel density (P=0.003) as an independent correlate to plaque rupture. Conclusions—Thus, neovascularization as manifested by the localized appearance of microvessels is increased in ruptured plaques in the human aorta. Furthermore, microvessel density is increased in lesions with inflammation, with intraplaque hemorrhage, and in thin-cap fibroatheromas. Microvessels at the base of the plaque are independently correlated with plaque rupture, suggesting a contributory role for neovascularization in the process of plaque rupture.

Intimomedial Interface Damage and Adventitial Inflammation Is Increased Beneath Disrupted Atherosclerosis in the Aorta Implications for Plaque Vulnerability

Background—Atherosclerotic plaque progression is frequently accompanied by compensatory enlargement to preserve the lumen. These enlarging plaques develop features of vulnerability, however, leading to disruption and lumen obstruction. This complex transition from compensatory expansion to plaque disruption may not derive solely from progressive intimal disease. Concurrent changes at the intimomedial interface and within the tunica media and adventitia may play a role in plaque instability. We tested this hypothesis by investigating whether interface changes, including internal elastic lamina (IEL) rupture, and medial and adventitial changes, including inflammation, fibrosis, and atrophy, more frequently accompany disrupted than nondisrupted atherosclerotic plaques. Methods and Results—Computerized planimetry and ocular micrometry were used for systematic quantification of intimal, medial, and adventitial histopathological features in 598 human aortic plaques according to the AHA classification. Disrupted plaques exhibited larger plaque and lipid pool areas (P =0.0001) and a higher incidence of rupture of the IEL (P =0.0001). Medial and adventitial inflammation (P =0.01), medial fibrosis (P =0.0001), and medial atrophy (P =0.0001) were also higher in disrupted plaques. Furthermore, medial thickness was reduced in disrupted plaques (P =0.0001). Logistic regression analysis identified rupture of the IEL as an independent predictor for fibrous cap disruption (P =0.0001). Conclusions—Compared with nondisrupted plaques, disrupted plaques have an increased incidence of IEL rupture, medial and adventitial inflammation, medial fibrosis, and medial atrophy. These intimomedial interface and adventitial changes may play a role in the natural history of complex atherosclerotic lesions. The interaction between medial and adventitial pathology and the intimal atherosclerotic process deserves further investigation.

Haptoglobin Genotype Is a Determinant of Iron, Lipid Peroxidation, and Macrophage Accumulation in the Atherosclerotic Plaque

Objective—Intraplaque hemorrhage increases the risk of plaque rupture and thrombosis. The release of hemoglobin (Hb) from extravasated erythrocytes at the site of hemorrhage leads to iron deposition, which may increase oxidation and inflammation in the atherosclerotic plaque. The haptoglobin (Hp) protein is critical for protection against Hb-induced injury. Two common alleles exist at the Hp locus and the Hp 2 allele has been associated with increased risk of myocardial infarction. We have demonstrated decreased anti-oxidative and anti-inflammatory activity for the Hp 2 protein. We tested the hypothesis that the Hp 2-2 genotype is associated with increased oxidative and macrophage accumulation in atherosclerotic plaques. Methods and Results—The murine Hp gene is a type 1 Hp allele. We created a murine type 2 Hp allele and targeted its insertion to the Hp locus by homologous recombination. Atherosclerotic plaques from C57Bl/6 ApoE−/− Hp 2-2 mice were associated with increased iron (P=0.008), lipid peroxidation (4-hydroxynonenal and ceroid) and macrophage accumulation (P=0.03) as compared with plaques from C57Bl/6 ApoE−/− Hp 1-1 mice. Conclusions—Increased iron, lipid peroxidation and macrophage accumulation in ApoE−/− Hp 2-2 plaques suggests that the Hp genotype plays a critical role in the oxidative and inflammatory response to intraplaque hemorrhage.

Downregulation of the Hemoglobin Scavenger Receptor in Individuals With Diabetes and the Hp 2-2 Genotype Implications for the Response to Intraplaque Hemorrhage and Plaque Vulnerability

In individuals with diabetes mellitus (DM), the haptoglobin (Hp) genotype is a major determinant of susceptibility to myocardial infarction. We have proposed that this is because of DM and Hp genotype-dependent differences in the response to intraplaque hemorrhage. The macrophage hemoglobin scavenging receptor CD163 plays an essential role in the clearance of hemoglobin released from lysed red blood cells after intraplaque hemorrhage. We sought to test the hypothesis that expression of CD163 is DM and Hp genotype-dependent. CD163 was quantified in plaques by immunohistochemistry, on peripheral blood monocytes (PBMs) by FACS, and as soluble CD163 (sCD163) in plasma by ELISA. In DM plaques, despite an increase in macrophage infiltration, CD163 immunoreactivity was lower, resulting in a dramatic reduction in the percentage of macrophages expressing CD163 (27±2% versus 70±2%, P=0.0001). In individuals with DM as compared with individuals without DM, the percentage of PBMs expressing CD163 was reduced (3.7±0.6% versus 7.1±0.9%, P<0.002) whereas soluble plasma CD163 was increased (2.6±1.1 &mgr;g/mL versus 1.6±0.8 &mgr;g/mL, P<0.0005). Among DM individuals, the Hp 2-2 genotype was associated with a decrease in the percentage of PBMs expressing CD163 (2.3±0.5% versus 5.6±1.3%, P=0.01) and an increase in plasma soluble CD163 (3.0±0.2 &mgr;g/mL versus 2.3±0.2 &mgr;g/mL, P=0.04). Taken together, these results demonstrate an impaired hemoglobin clearance capacity in Hp 2-2 DM individuals and may provide the key insight explaining the increased incidence of myocardial infarction in this population.

Haptoglobin Genotype Is a Major Determinant of the Amount of Iron in the Human Atherosclerotic Plaque

OBJECTIVES We sought to test the hypothesis that haptoglobin (Hp) genotype is a determinant of the amount of iron in the atherosclerotic plaque. BACKGROUND In atherosclerotic lesions, intraplaque hemorrhage releases free hemoglobin (Hb), whose incorporated iron can act as an oxidant and inflammatory stimulus. These effects are antagonized by Hp, which binds free Hb and facilitates its clearance from the plaque. The Hp gene has 2 alleles (1 and 2), giving rise to 3 genotypes: Hp 1-1, Hp 2-1, and Hp 2-2. We previously hypothesized that Hp 2-2 individuals with diabetes mellitus (DM) have impaired clearance of Hb and its iron cargo from the plaque. METHODS We identified the presence or absence of Perls iron stain in 189 plaques obtained from 37 decedents at autopsy. RESULTS Among DM, the prevalence of Perls iron stain was increased in Hp 2-2 compared with that seen in Hp 1-1 or 2-1 (46.2% vs. 11.8%). After accounting for the within-decedent correlation of plaques, the prevalence ratio of Perls iron stain associated with Hp 2-2 was 3.97 (95% confidence interval: 1.38 to 11.5; p = 0.025). In non-DM plaques, there was a nonsignificant trend towards a higher prevalence of iron staining in Hp 2-2 compared with that in Hp 1-1 or 2-1 (26.8% vs. 11.1%; prevalence ratio =2.40 [95% confidence interval: 0.81 to 7.09]; p = 0.114). CONCLUSIONS These data support an impaired clearance of Hb from plaques in Hp 2-2 individuals, particularly in DM. The higher prevalence of plaque iron in Hp 2-2 DM individuals may contribute to the increased incidence of atherothrombotic events in these patients.

Increased neovascularization in advanced lipid-rich atherosclerotic lesions detected by gadofluorine-M-enhanced MRI: implications for plaque vulnerability.

Background—Inflammation and neovascularization may play a significant role in atherosclerotic plaque progression and rupture. We evaluated gadofluorine-M–enhanced MRI for detection of plaque inflammation and neovascularization in an animal model of atherosclerosis. Methods and Results—Sixteen rabbits with aortic plaque and 6 normal control rabbits underwent gadofluorine-M–enhanced MRI. Eight rabbits had advanced atherosclerotic lesions, whereas the remaining 8 had early lesions. Magnetic resonance atherosclerotic plaque enhancement was meticulously compared with plaque inflammation and neovessel density as assessed by histopathology. Advanced plaques and early atheroma were enhanced after gadofluorine-M injection. Control animals displayed no enhancement. After accounting for the within-animal correlation of observations, mean contrast-to-noise ratio was significantly higher in advanced plaques than compared with early atheroma (4.29±0.21 versus 3.00±0.32; P=0.004). Macrophage density was higher in advanced plaques in comparison to early atheroma (geometric mean=0.50 [95% CI, 0.19 to 1.03] versus 0.25 [0.07 to 0.42]; P=0.05). Furthermore, higher neovessel density was observed in advanced plaques (1.83 [95% CI, 1.51 to 2.21] versus 1.29 [0.99 to 1.69]; P=0.05). The plaque accumulation of gadofluorine-M correlated with increased neovessel density as shown by linear regression analysis (r=0.67; P<0.001). Confocal and fluorescence microscopy revealed colocalization of gadofluorine-M with plaque areas containing a high density of neovessels. Conclusion—Gadofluorine-M–enhanced MRI is effective for in vivo detection of atherosclerotic plaque inflammation and neovascularization in an animal model of atherosclerosis. These findings suggest that gadofluorine-M enhancement reflects the presence of high-risk plaque features believed to be associated with plaque rupture. Gadofluorine-M plaque enhancement may therefore provide functional assessment of atherosclerotic plaque in vivo.

Increased macrophage infiltration and neovascularization in congenital bicuspid aortic valve stenosis

OBJECTIVES Patients with congenital bicuspid aortic valves have aortic valve stenosis at a relatively young age compared with patients with tricuspid aortic valves. We hypothesize that aortic valve stenosis evolves from a more aggressive inflammatory process, with increased macrophage/T-cell and neovessel content in congenital bicuspid aortic valveswhen compared with that seen in tricuspid valves. METHODS Fifty-one severely stenotic aortic valves were obtained at the time of aortic valve replacement. A total of 17 bicuspid and 34 tricuspid aortic valves were evaluated. Macrophage/T-cell infiltration (CD68 plus CD3) and neovessel density (CD34) were evaluated with immunohistochemical staining. Leaflet calcification and ossification were also quantified. Real-time polymerase chain reaction was used to assess expression of chondromodulin 1 and vascular endothelial growth factor. RESULTS The density of macrophages/T cells was greater in congenital bicuspid aortic valves than in tricuspid valves (51 ± 31 vs 23 ± 13 cells/mm(2), P = .002). Neovascularization was more frequently noted in congenital bicuspid aortic valves when compared with tricuspid valves (31 ± 10 vs 21 ± 9 vessels/mm(2), P = .0005), and calcification was more severe (P = .03). Expression of chondromodulin 1 demonstrated a 6-fold downregulation (P = .0003) and expression of vascular endothelial growth factor demonstrated a 2-fold increase (P = .02) in congenital bicuspid aortic valves compared with that seen in tricuspid valves. Multivariable analyses demonstrated significant associations between bicuspid aortic valve anatomy and increased inflammatory cell infiltration (β = 25.8, P = .0007) and neovascularization (β = 9.4, P = .001), despite adjusting for measured covariates. CONCLUSIONS The pathogenesis of aortic valve stenosis in bicuspid aortic valves is associated with a more aggressive inflammatory process with increased macrophage infiltration and neovascularization when compared with that seen in tricuspid valves.

Coronary atherosclerosis in sudden cardiac death: an autopsy study.

BACKGROUND The incidence of ischemic heart disease (IHD) has markedly increased in India over the past few years. Considering the variations in racial, dietary and lifestyle patterns in our population, it is essential to study the biology of coronary atherosclerosis in our patients. Vulnerable plaques have a large number of foam cells, extracellular lipid, thin fibrous caps and clusters of inflammatory cells and are more prone to rupture. These plaques are nourished by the microvessels arising from the vasa vasorum of the blood vessels and by lumen-derived microvessels through the fibrous cap. This autopsy study was designed to analyse the coronary arterial tree in cases of sudden cardiac death, classify coronary atherosclerotic plaques and to assess the factors contributing to vulnerability of the plaques including inflammation, calcification and microvascular density. MATERIALS AND METHODS Seven cases of sudden cardiac death were included in the study. The hearts were perfusion-fixed and the coronary arteries along with their main branches were dissected and studied. The location of the plaques, type of plaques, presence of inflammation and calcification were assessed. The cap thickness and microvessel density per 1000 um 2 were assessed. The statistical significance was estimated. RESULTS AND CONCLUSIONS Extensive high-grade coronary atherosclerotic disease was seen in all sudden cardiac death cases. Majority of the plaques were vulnerable. High-grade inflammation was seen in most of the vulnerable and ruptured plaques. All the ruptured plaques were uncalcified indicating that calcification probably stabilizes the plaques and protects against rupture. Increased microvessel density was noted in ruptured plaques compared to vulnerable plaques. However, it was not statistically significant.

831-1 Ruptured diabetic atherosclerotic plaques have more inflammation and neovascularization than ruptured plaques from patients without diabetes

Va sc ul ar D is ea se , H yp er te ns io n, a nd P re ve nt io n (23%). Over the two study periods, a smaller proportion of patients on bosentan than on placebo required epoprostenol therapy (2.8% versus 4.3%), additional therapy for PAH (28.6% versus 38.5%), or hospitalization (4.2% versus 13.0% for PAH-related and 16.0% versus 23.2% for all causes of hospitalization). Conclusion: The benefit achieved in exercise capacity, dyspnea, and reduced need for additional therapy or hospitalization demonstrated in the initial 16-week BREATHE-1 study appears to be sustained when patients are followed for 28 weeks.