K-Raman Purushothaman

Plaque neovascularization: defense mechanisms, betrayal, or a war in progress

Angiogenesis is induced from sprouting of preexisting endothelial cells leading to neovascularization. Imbalance in the angiogenic and antiangiogenic mediators triggers angiogenesis, which may be physiological in the normal state or pathological in malignancy and atherosclerosis. Physiologic angiogenesis is instrumental for restoration of vessel wall normoxia and resolution inflammation, leading to atherosclerosis regression. However, pathological angiogenesis enhances disease progression, increasing macrophage infiltration and vessel wall thickness, perpetuating hypoxia and necrosis. In addition, thin‐walled fragile neovessels may rupture, leading to intraplaque hemorrhage. Lipid‐rich red blood cell membranes and free hemoglobin are detrimental to plaque composition, increasing inflammation, lipid core expansion, and oxidative stress. In addition, associated risk factors that include polymorphysms in the haptoglobin genotype and diabetes mellitus may modulate the features of plaque vulnerability. This review will focus on physiological and pathological angiogenesis in atherosclerosis and summarizes the current status of anti‐vascular endothelial growth factor (VEGF) therapy, microvascular rarefaction, and possible statin‐mediated effects in atherosclerosis neovascularization.

Increased Neovascularization in Advanced Lipid-Rich Atherosclerotic Lesions Detected by Gadofluorine-M Enhanced Magnetic Resonance Imaging (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.

Inflammation, neovascularization and intra-plaque hemorrhage are associated with increased reparative collagen content: Implication for plaque progression in diabetic atherosclerosis

Sustained inflammation may stimulate a reparative process increasing early reparative type III collagen synthesis, promoting atherosclerotic plaque progression. We evaluated inflammation, neovascularization, intra-plaque hemorrhage (IPH), and collagen deposition in human aortic atherosclerotic plaques from patients with and without diabetes mellitus (DM). Plaques were procured at autopsy from lower thoracic and abdominal aorta from DM (n = 20) and non-DM (n = 22) patients. Inflammation and neovascularization were quantified by double-label immunochemistry and the IPH grade was scored using H&E-stained sections. Type I and type III collagens were quantified using Picro-Sirius red stain with polarization microscopy and computerized planimetry. In non-DM plaques, 27%, 40%, and 33% had mild, moderate and severe inflammation in the fibrous cap and shoulder compared with 2%, 30% and 68% in DM plaques (p < 0.001). The geometric mean neovessel count was increased in DM versus non-DM plaques (140 [95% CI: 119—165] versus 59 [95% CI: 51—70]; p < 0.001). The IPH grade was increased in DM verses non-DM plaques (0.82 ± 0.11 versus 0.29 ± 0.11; p < 0.001) (percentage grade). The density of type III was increased in DM plaques (0.16 ± 0.01 versus 0.06 ± 0.01; p < 0.001) with a non-significant reduction in type I density in DM when compared with non-DM (0.28 ± 0.03 versus 0.33 ± 0.03; p = 0.303) (content per mm2). The increase in type III collagen content correlated with total neovessel content (r = 0.58; p < 0.001) in DM plaques. In conclusion, our study suggests that enhanced type III collagen deposition was associated with inflammation, neovascularization and IPH, and may be a contributing factor in DM plaque progression.

Atherosclerosis Neovascularization and Imaging

Neovascularization in atherosclerotic plaques is particularly prominent in complicated lesions, and has been recently identified as a marker of plaque vulnerability. This observation has led to a growing interest in the development of imaging techniques with the ability to visualize and quantify the extent of plaque neovascularization. Such feature may play an important role in identifying those lesions more prone to destabilization and rupture, and in the guidance and monitoring of therapeutic interventions. Several modalities have emerged as potential candidates for imaging neovessels in atherosclerotic lesions. They include magnetic resonance imaging, x-ray computed tomography, positron emission tomography, single photon emission computed tomography, ultrasound, or near-infrared optical imaging. These techniques differ in their achievable spatial and temporal resolution, availability, cost, reproducibility, degree of intrusiveness, capability to image atherosclerotic plaques in various vascular territories and ability to discern different plaque components, specifically the presence of neovessels. Molecular imaging, a rapidly evolving multidisciplinary field devoted to the visualization of specific physiopathologic processes at the cellular or molecular level, appears particularly well suited for this purpose because of its ability to target and visualize individual molecules specific to neoangiogenesis. In this manuscript we will review current evidence on the potential application of the various modalities, with a particular emphasis in molecular imaging.

Increased expression of oxidation-specific epitopes and apoptosis are associated with haptoglobin genotype: possible implications for plaque progression in human atherosclerosis.

OBJECTIVES The purpose of this study was to test the hypothesis that increased oxidative stress is associated with apoptosis in human plaques with the haptoglobin (Hp) 2-2 genotype. BACKGROUND Intraplaque hemorrhage releases free hemoglobin (Hb). Impaired Hb clearance induces oxidative stress leading to plaque progression. The binding of Hp to Hb attenuates iron-induced oxidative reactions. METHODS Twenty-six human aortic plaques were Hp genotyped. Hp2-2 plaques (n = 13) were compared with control (Hp1-1/2-1) (n = 13). The iron grade was measured by Perls staining. Immunostaining was used to detect oxidation-specific epitopes (OSEs) reflecting oxidized phospholipids and malondialdehyde-like epitopes. The percentages of apoptotic cells and apoptotic morphological features were quantified. DNA fragmentation and active caspase-3 were measured by in situ end-labeling and immunohistochemistry, respectively. RESULTS In Hp2-2 plaques, iron content was increased (1.22 ± 0.15 vs. 0.54 ± 0.08; p < 0.0001) along with expression of oxidized phospholipid- (78.9 ± 5.8 vs. 38.8 ± 3.8; p < 0.0001), and malondialdehyde-like OSEs (93.9 ± 7.9 vs. 54.7 ± 3.9; p < 0.0001). The total percentages of apoptotic cells (11.9 ± 0.44 vs. 3.5 ± 0.28; p < 0.0001), nuclear fragmentation (11.8 ± 0.50 vs. 3.3 ± 0.26; p < 0.0001), nuclear condensation (10.9 ± 0.58 vs. 3.4 ± 0.20; p < 0.0001), chromatin margination (14.2 ± 0.57 vs. 6.5 ± 0.37; p < 0.0001), cytoplasmic blebs (1.6 ± 0.28 vs. 0.8 ± 0.14; p < 0.002), and eosinophilia (10.8 ± 0.74 vs. 4.2 ± 0.27; p < 0.0001) were increased in Hp2-2 plaques. Furthermore, DNA fragmentation (119.9 ± 1.40 vs. 57.5 ± 0.80; p < 0.001), and active caspase-3 density (84.7 ± 7.62 vs. 50.6 ± 7.49; p < 0.004) were increased in Hp2-2 plaques. Logistic regression analysis identified correlation between the percentage of apoptotic cells and the density of OSEs (r = 0.56; p < 0.003). CONCLUSIONS These findings provide insights into genetic predisposition to oxidative stress and the relationship between OSEs and macrophage apoptosis that may explain advanced atherosclerosis in human Hp2-2 plaques.

Genotype-Dependent Impairment of Hemoglobin Clearance Increases Oxidative and Inflammatory Response in Human Diabetic Atherosclerosis

Objective—Haptoglobin (Hp) protein is responsible for hemoglobin clearance after intra-plaque hemorrhage. Hp gene exists as Hp-1 and Hp-2 alleles and the phenotypes show important molecular heterogeneity. We tested the hypothesis that hemoglobin clearance may be deficient in diabetic atheroma from patients with Hp2-2, triggering increased oxidative, inflammatory, and angiogenic patterns compared with controls. Methods and Results—Forty patients with diabetes mellitus were genotyped and their peripheral plaques compared after atherectomy. Plaque hemorrhage, iron content, hemoglobin-binding protein CD163, and heme-oxygenase-1 were quantified. Oxidative, inflammatory, and angiogenic patterns were evaluated by measuring myeloperoxidase, interleukin-10, macrophages, vascular cell adhesion molecule-1, smooth muscle actin, and plaque neovascularization (CD34/CD31). Plaques with Hp2-2 (n=7) had increased hemorrhage (P<0.005), iron content (P<0.001), and reduced CD163 expression (P<0.002) compared with controls (n=14). Hp2-2 plaques had increased heme-oxygenase-1 protein (P<0.02), myeloperoxidase gene (P<0.05), and protein (P<0.0001). Anti-inflammatory interleukin-10 gene (P<0.04), and protein expressions (P<0.0001) were decreased in Hp2-2. Finally, macrophage (P<0.0001), vascular cell adhesion molecule-1 (P=0.001), smooth muscle actin (P=0.002) scores, and neovessels density (P<0.0001) were increased in Hp2-2. Conclusion—Genotype-dependent impairment of hemoglobin clearance after intra-plaque hemorrhage is associated with increased oxidative, inflammatory, and angiogenic response in human diabetic atherosclerosis.

Differential patterns of replacement and reactive fibrosis in pressure and volume overload are related to the propensity for ischaemia and involve resistin

•  Pressure overload hypertrophy is profibrotic while volume overload hypertrophy is not profibrotic. •  Fibrosis occurs in the form of replacement or reactive fibrosis. •  Replacement fibrosis in pressure overload is considered ischaemic in origin. •  There is less propensity for ischaemia in volume overload, explaining the relative lack of fibrosis. •  Reactive fibrosis pathways are more active in pressure than in volume overload. •  Local resistin expression reflects replacement fibrosis in chronic ischaemia.

Histopathological Evidence of Adventitial or Medial Injury Is a Strong Predictor of Restenosis During Directional Atherectomy for Peripheral Artery Disease

Purpose: To investigate the impact on restenosis rates of deep injury to the adventitial layer during directional atherectomy. Methods: Between 2007 and 2010, 116 consecutive patients (mean age 69.6 years; 56 men) with symptomatic femoropopliteal stenoses were treated with directional atherectomy at a single center. All patients had claudication and TASC A/B lesions in the superficial femoral or popliteal arteries. Histopathology analysis of atherectomy specimens was performed to identify adventitial injury. Clinical follow-up included physical examination and duplex ultrasound scans at 3, 6, and 12 months in all patients. The primary endpoint was the duplex-documented 1-year rate of restenosis, which was determined by a peak systolic velocity ratio <2.4. Patients were dichotomized by the presence or absence of adventitial or medial cuts as evaluated by histopathology. Results: Adventitial injury were identified in 62 (53%) of patients. There were no differences in baseline demographic and clinical features (p>0.05), lesion length (58.7±12.8 vs 56.2±13.6 mm, p=0.40), or vessel runoff (1.9±0.6 vs 2.0±0.6, p=0.37) between patients with and without adventitial injury, respectively. The overall 1-year incidence of restenosis was 57%, but the rate was significantly higher (p<0.0001) in patients with adventitial or medial injury (97%, 60/62) as compared with those without (11%, 6/54). Conclusion: Lack of adventitial injury after atherectomy for femoropopliteal stenosis is strongly related to patency at 1 year.

Expression of angiotensin-converting enzyme 2 and its end product angiotensin 1-7 is increased in diabetic atheroma: implications for inflammation and neovascularization

AIMS The angiotensin-converting enzyme 2 (ACE2) and its end product angiotensin 1-7 (Ang1-7) are key counterregulatory proteins to offset the deleterious effects of angiotensin II. ACE2 is decreased in diabetic kidney disease but overexpressed in metabolically active atheroma. We tested the hypothesis that ACE2 is increased in diabetic peripheral atheroma, concomitantly with Ang1-7, angiotensin II receptor 1 (AT1R), proinflammatory cytokines, macrophage infiltration, and plaque neovascularization. METHODS AND RESULTS Peripheral atherectomy plaques collected from 12 diabetic (DM) and 12 non-DM patients were immunostained for ACE2, Ang1-7, AT1R, and proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Macrophage infiltration and neovascularization were counted using double-label immunochemistry with CD68/CD3 and CD34, respectively. Quantification was performed blindly by randomly counting positively stained cells in 20 high-power fields using previously validated methods. Tissue content of ACE2, Ang1-7, and AT1R was increased in DM when compared to non-DM (P<.0001). IL-6 and TNF-α were also increased in DM when compared to non-DM (P<.0001), as well as macrophage infiltration score and neovessel counting (P<.0001). CONCLUSION Expression of ACE2 and its end product Ang1-7 is increased in DM atheroma, along with overexpression of AT1R, IL6, TNF-α, macrophage infiltration, and neovascularization. These results suggest that the renin-angiotensin system counterregulatory pathway may be preserved in metabolically active atheroma, offering potential targets for future therapies in diabetic atherosclerosis.

Intravascular ultrasound is an effective tool for predicting histopathology-confirmed evidence of adventitial injury following directional atherectomy for the treatment of peripheral artery disease

To the Editors: Directional atherectomy (DA) has emerged as a viable option for treatment of superficial femoral artery (SFA) occlusive disease. Despite excellent initial success, longterm patency with this therapy has had varying success. This variation in patency may be due to injury to the arterial wall during DA. Recently, we showed that histopathology evidence of deep injury to the adventitial layer of the vessel wall has a substantial negative effect on patency rates at 1 year. Continuing this work, we examined the relationship between intravascular ultrasound (IVUS)–identified external elastic lamina (EEL) disruption against the histopathology of the adventitia retrieved in the DA specimens. Of the 116 patients with TransAtlantic Inter-Society Consensus (TASC) A/B femoropopliteal lesions in our initial cohort, 108 had an IVUS examination after DA. IVUS imaging was performed by advancing the catheter to the most distal aspect of the lesion, and with data collected at 1 frame/s using a motorized pullback device set at 0.1 mm/s, raw sequential radiofrequency IVUS data were captured and transferred to a workstation for analysis. IVUS images were reconstructed utilizing IVUS lab software, and contours defining the internal elastic lamina (IEL) and EEL were identified automatically. A physician manually corrected the lumen and media-adventitial contours for each grayscale image. The IVUS definition of adventitial injury was disruption of the EEL and extension of the atherectomy cut past the EEL. Three board-certified interventional cardiologists independently reviewed all anonymized IVUS films to determine the presence or absence of EEL disruption; they had no knowledge of the histology outcomes. The IEL/EEL border was clearly identified, and the vessel structure at the site of atherectomy was comparatively examined to determine depth of disruption of vessel structures. Unanimous agreement of EEL border disturbance caused by the atherectomy device (Figure 1) was required for a “positive” EEL disruption assessment. Disagreements were classified as a negative for IVUSidentified adventitial injury. Investigator determination was then compared against histopathology findings of the atherectomy specimen as the reference gold standard. 647364 JETXXX10.1177/1526602816647364Journal of Endovascular TherapyKrishnan et al letter2016