John D. Mannion

Human Epicardial Adipose Tissue Is a Source of Inflammatory Mediators

Background—Inflammatory mediators that originate in vascular and extravascular tissues promote coronary lesion formation. Adipose tissue may function as an endocrine organ that contributes to an inflammatory burden in patients at risk of cardiovascular complications. In this study, we sought to compare expression of inflammatory mediators in epicardial and subcutaneous adipose stores in patients with critical CAD. Methods and Results—Paired samples of epicardial and subcutaneous adipose tissues were harvested at the outset of elective CABG surgery (n=42; age 65±10 years). Local expression of chemokine (monocyte chemotactic protein [MCP]-1) and inflammatory cytokines (interleukin [IL]-1&bgr;, IL-6, and tumor necrosis factor [TNF]-&agr;) was analyzed by TaqMan real-time reverse transcription–polymerase chain reaction (mRNA) and by ELISA (protein release over 3 hours). Significantly higher levels of IL-1&bgr;, IL-6, MCP-1, and TNF-&agr; mRNA and protein were observed in epicardial adipose stores. Proinflammatory properties of epicardial adipose tissue were noted irrespective of clinical variables (diabetes, body mass index, and chronic use of statins or ACE inhibitors/angiotensin II receptor blockers) or plasma concentrations of circulating biomarkers. In a subset of samples (n=11), global gene expression was explored by DNA microarray hybridization and confirmed the presence of a broad inflammatory reaction in epicardial adipose tissue in patients with coronary artery disease. The above findings were paralleled by the presence of inflammatory cell infiltrates in epicardial adipose stores. Conclusions—Epicardial adipose tissue is a source of several inflammatory mediators in high-risk cardiac patients. Plasma inflammatory biomarkers may not adequately reflect local tissue inflammation. Current therapies do not appear to eliminate local inflammatory signals in epicardial adipose tissue.

Adventitial myofibroblasts contribute to neointimal formation in injured porcine coronary arteries.

BACKGROUND The adventitia undergoes remodeling changes after a deep medial coronary injury. Because this process is associated with the formation of adventitial myofibroblasts, which resemble medial smooth muscle (SM) cells, we have examined myofibroblast involvement in the development of neointima. METHODS AND RESULTS In a porcine model, severe endoluminal coronary injury resulted in fibroblast proliferation and adventitial remodeling. Significant adventitial responses were associated with increased neointimal formation (P < .01). To examine the contribution of adventitial cells to the development of neointima, proliferating cells were labeled with bromodeoxyuridine (BrdU) at 12 and 24 hours after injury, and their subsequent localization was determined by immunohistochemistry (n = 24). At 2 to 3 days after severe injury, the adventitia contained numerous BrdU-labeled cells (37 +/- 4%), whereas the media demonstrated infrequent labeled cells (4 +/- 1%). Adventitial cells lacked alpha-SM actin and desmin, which distinguished them from medial SM cells. At 7 to 8 days, some labeled cells acquired characteristics of myofibroblasts expressing alpha-SM actin. They were found to translocate to the gap between dissected media and contributed to the formation of neointima (76 +/- 19%). At 18 to 35 days, labeled cells were abundant in the neointima (86 +/- 5%). They showed uniform immunostaining for alpha-SM actin but not for desmin, thereby differing from medial SM cells and blood-borne cells. CONCLUSIONS This study demonstrates translocation of adventitial fibroblasts to neointima, their phenotypic modulation to myofibroblasts, and distinct characteristics of myofibroblasts within neointima after severe endoluminal coronary injury. These findings suggest the significance of vascular fibroblasts in the process of arterial repair.

Adventitial Remodeling After Coronary Arterial Injury

BACKGROUND Intraluminal thrombus formation and medial smooth muscle (SM) cell proliferation are recognized responses of the arterial system to injury. In contrast to these well-characterized processes during vascular repair, changes involving the adventitia have been largely neglected in previous studies. Hence, the goal of this investigation was to assess the response of the adventitia to coronary arterial injury. METHODS AND RESULTS Adventitial changes in porcine coronary arteries subjected to medial injury were characterized by immunohistochemistry, histochemistry, and microscopic morphometry. The rapid development of a hypercellular response in the adventitia was evident 3 days after balloon-induced medial injury. Cell proliferation, as assessed by proliferating cell nuclear antigen immunostaining, reached the maximum level in the adventitia at 3 days, whereas at 14 and 28 days, the number of replicating cells reverted toward the baseline. The proliferating activity in the adventitia exceeded that seen in the media at all times after injury. To further define the changes in the phenotype of adventitial cells, the expression of three cytoskeletal proteins (vimentin, alpha-SM actin, and desmin) was characterized. Fibroblasts in normal adventitia expressed vimentin but no alpha-SM actin or desmin. After injury, these cells acquired characteristics of myofibroblasts expressing alpha-SM actin, which peaked at 7 and 14 days. Desmin expression was patchy in the adventitia, as opposed to its homogeneous distribution in medial SM cells. The modulation of fibroblast phenotype was transient, inasmuch as alpha-SM actin immunostaining declined at 28 days after injury, when dense, collagen-rich scar was evident within the adventitia. The above-described changes involving hypercellularity of the adventitia, myofibroblast formation, and fibrosis were associated with a significant focal adventitial thickening at 3, 7, 14, and 28 days after injury (P < .01 versus uninjured coronary arteries). CONCLUSIONS This study demonstrates the involvement of the adventitia in the vascular repair process after medial injury. The hypercellularity of the adventitial layer, proliferation of fibroblasts, and modulation of their phenotype to myofibroblasts are associated with the development of the thickened adventitia. It is postulated that these phenomena affect vascular remodeling and may provide an important insight into the mechanisms of vascular disorders.

Prevalence of Heparin-Associated Antibodies Without Thrombosis in Patients Undergoing Cardiopulmonary Bypass Surgery

BACKGROUND Patients with cardiovascular disease almost invariably receive heparin before cardiopulmonary bypass surgery, which places them at risk of developing heparin-associated antibodies with a risk of thromboembolic complications. This study was designed to determine the prevalence of heparin-induced antibodies in patients before and after cardiopulmonary bypass surgery. METHODS AND RESULTS Plasma from 111 patients was tested before surgery and 5 days after surgery for heparin-dependent platelet-reactive antibodies with a 14C-serotonin-release assay (SRA) and for antibodies to heparin/platelet factor 4 complexes with an ELISA. Heparin exposure after surgery was minimized. Heparin-dependent antibodies were detected before surgery in 5% of patients with SRA and 19% of patients with ELISA. By the fifth postoperative day, there was a marked increase in patients positive on the SRA or ELISA (13% and 51%, respectively; P < .01 for each). Patients who had received heparin therapy earlier in their hospitalization were more likely to have a positive ELISA before surgery (35%; P = .017) and a positive ELISA (68%; P = .054) or SRA (30%; P = .002) after surgery. However, there was no difference in the prevalence of thrombocytopenia or thromboembolic events between the antibody-positive and-negative groups. CONCLUSIONS Approximately one fifth of patients undergoing cardiopulmonary bypass surgery have heparin-induced platelet antibodies detectable before the procedure as a result of prior heparin exposure, and many more develop antibodies after surgery. The absence of an association between these antibodies and thromboembolic complications in this study may be, in part, attributable to careful avoidance of heparin after surgery. The high prevalence of heparin-induced antibodies in this setting suggests that these patients may be at risk of developing thrombotic complications with additional heparin exposure.

Remodeling of Autologous Saphenous Vein Grafts: The Role of Perivascular Myofibroblasts

BACKGROUND Aortocoronary saphenous vein grafts (SVGs) undergo structural changes that render them susceptible to atherosclerosis. Accordingly, the origin of neointimal hyperplasia-was examined in porcine arterialized SVGs to determine the mechanism of vein graft remodeling. METHODS AND RESULTS At 2 to 4 days after surgery, the percentage of cells lacking differentiation markers characteristic for smooth muscle (SM) cells (ie, alpha-SM actin, desmin, and SM myosin) increased within the media of SVGs interposed in the carotid arteries (P < .001). At 7 to 14 days, these cells acquired a differentiated phenotype (ie, alpha-SM-actin positive/ variable desmin/SM-myosin negative) and accumulated in the neointima. At 3 months, the neointima was positive for alpha-SM actin but mostly negative for desmin, which contrasted with medial SMCs that were invariably positive for alpha-SM actin, desmin, and SM myosin. To determine the role of nonmuscle cells in the above process, perivascular wound fibroblasts were selectively labeled and found to translocate through the media of newly placed SVGs, contributing to neointimal formation. These migrating cells differentiated to myofibroblasts exhibiting sustained alpha-SM-actin expression. The intima of human SVGs, retrieved during repeat aortocoronary bypass surgery, exhibited the profile of cytoskeletal proteins that resembled myofibroblasts seen in porcine SVGs. CONCLUSIONS Perivascular fibroblasts may infiltrate injured media of arterialized SVGs, differentiate to myofibroblasts (acquiring alpha-SM actin), and contribute to vein graft remodeling. The similarities between porcine and human SVGs regarding the repertoire of cytoskeletal proteins suggest the involvement of myofibroblasts in graft remodeling in the clinical setting.

Histochemical and fatigue characteristics of conditioned canine latissimus dorsi muscle.

To induce fatigue resistance in the latissimus dorsi muscle of the dog in preparation for possible myocardial assistance, eight adult male beagles underwent unilateral electrical stimulation of the thoracodorsal nerve at a frequency of 2 Hz (120 stimuli/min) and 10 Hz (600 stimuli/min) for a 6-week period. The conditioned muscles were compared with their unconditioned contralateral controls by fiber typing, pyrophosphate gel electrophoresis, isometric characteristics, and fatigue rates. At the end of the period of stimulation, the conditioned muscles had a greater percentage of slow-twitch, fatigue-resistant fibers on acid and alkaline stains (100 ± 0.7% and 83 ± 15.3%), respectively, than did their contralateral controls (45 ± 7.6% and 43 ± 7.0). Pyrophosphate gel electrophoresis revealed an increase in the slow myosin and a decrease in the fast myosin content in the conditioned muscles; the stimulated muscles also demonstrated a slower contraction time (87 ± 20 msec vs. 57 ± 17.9 msec), a lower initial tension (4.4 ± 1.45 kg vs. 7.2 ±2.11 kg), and a slower fatigue rate during a 30-minute fatigue test than did their contralateral controls. The muscles stimulated at 2 Hz had fatigue rates similar to those stimulated at 10 Hz, but generally had less diminution in muscle fiber diameters and less interfiber connective tissue. Thus, it is possible to make canine latissimus dorsi muscles more fatigue resistant, and, theoretically, more capable of myocardial assistance by electrical stimulation of the thoracodorsal nerve at a frequency as low as 2 Hz–the natural canine heart rate.

Origin of Extracellular Matrix Synthesis During Coronary Repair

BACKGROUND Coronary injury triggers differentiation of activated adventitial fibroblasts to myofibroblasts, which may contribute to neointimal formation and vascular remodeling. Accordingly, the purpose of this study was to examine the cellular origin of the enhanced synthesis of extracellular matrix proteins during coronary repair. METHODS AND RESULTS The time course and localization of collagen and elastin expression were examined by in situ hybridization and immunohistochemistry in porcine coronary arteries after balloon-induced injury. Procollagen-alpha 1(I) transcripts and intracellular type I procollagen protein increased in the adventitia within 2 days after injury. This was followed by a sustained synthesis of type I procollagen in neointima beginning at 7 days and the extracellular accumulation of type I collagen in both layers. The origin of synthetic cells was further examined by colocalization of type I procollagen and bromodeoxyuridine labeling to activated adventitial cells, which translocated to neointima. Neointimal cells exhibited sustained synthetic activity manifested by the presence of type I procollagen and elastin at 3 months after injury. In contrast, the media showed only minor changes in the synthesis of collagen or elastin throughout coronary repair. CONCLUSIONS Activated adventitial fibroblasts are endowed with synthetic capabilities after coronary injury. They express type I procollagen, with some of them translocating to the intima, where they continue to synthesize procollagen. The accumulation of type I collagen is evident in the adventitia and neointima, whereas elastin accumulates mainly in neointima. These findings support the involvement of adventitial fibroblasts in coronary repair and remodeling after endoluminal injury.

Effects of collateral blood vessel ligation and electrical conditioning on blood flow in dog latissimus dorsi muscle

Utilization of skeletal muscle as a myocardial substitute requires it to undergo two major modifications: mobilization to the site of action and adaptation to continuous activity. We have examined the effects of collateral blood vessel ligation, which would accompany mobilization, on blood flow in control and electrically conditioned canine latissimus dorsi (LD) muscle. Blood flows were measured at rest and during a vigorous isometric fatigue test. In 22 control muscles, electrical stimulation during the fatigue test resulted in a sevenfold increase in muscle blood flow (0.26 +/- 0.18 ml/g/min at rest, 1.69 +/- 0.84 ml/g/min during stimulation). No difference was detected in flow to distal and proximal portions of the muscle. In three muscles where collateral vessels were ligated immediately before measurement of blood flow, flow in the proximal portion of the muscle was not significantly different from control, but in the distal portion, stimulation failed to elicit an increase in flow (0.12 +/- 0.13 ml/g/min at rest, 0.16 +/- 0.07 ml/g/min during stimulation). In animals allowed a 3-week recovery period following collateral vessel ligation, stimulation-induced increases in blood flow were detected but remained lower than control. Muscles which had been conditioned by continuous electrical stimulation for 6-7 weeks at 2 or 10 Hz generated less peak isometric tension than controls (peak tension = 4.5 +/- 1.7 kg control, 2.4 +/- 0.7 kg following 2 Hz conditioning, 1.6 +/- 0.4 kg following 10 Hz conditioning). However, these muscles demonstrated an increased resistance to fatigue.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidative Stress and Lipid Retention in Vascular Grafts Comparison Between Venous and Arterial Conduits

Background—Because saphenous vein grafts (SVGs) exhibit greater cellular heterogeneity and worse clinical outcomes than arterial grafts (AGs), we examined oxidative stress and lipid retention in different vascular conduits. Methods and Results—In a porcine model of graft interposition into carotid artery, superoxide anion (·O2−) was measured at 2 weeks after surgery. SVGs demonstrated increased ·O2− production compared with AGs (SOD-inhibitable nitro blue tetrazolium reduction, P <0.01). The NAD(P)H oxidase inhibitor diphenyleneiodonium (P <0.01) abolished SVG-derived ·O2−, whereas the inhibitors of other pro-oxidant enzymes were ineffective. The change in oxidative stress was also reflected by lower activity of the endogenous antioxidant superoxide dismutase in SVGs than in AGs (P <0.001). SVG remodeling was associated with increased synthesis of sulfated glycosaminoglycans and augmented expression of a core protein, versican. These changes were accompanied by SVGs retaining significantly more 125I-labeled LDL than AGs ex vivo (P <0.001). In hyperlipemic animals, lipid accumulation and oxidized epitopes were preferentially noted in the intima of SVGs at 1 month after surgery. Conclusions—This study demonstrated significant differences in the biology of SVGs and AGs. SVGs exhibited higher oxidative stress, LDL accumulation, and the presence of oxidized epitopes. These findings suggest that proatherogenic changes in SVGs may commence early after surgical revascularization.

Wound healing around and within saphenous vein bypass grafts

BACKGROUND Myofibroblasts are a prominent cell type in wound healing. The goal of this study was to examine the extent to which myofibroblasts contribute to structural changes in saphenous vein bypass grafts. METHODS AND RESULTS Control veins and reversed saphenous vein bypass conduits of porcine carotid arteries were examined 2 to 4, 7 to 14, and 30 to 90 days after surgery with immunohistochemical markers of cellular proliferation (proliferating cell nuclear antigen), cytoskeletal protein production (alpha-smooth muscle actin and desmin), and histochemistry (Verhoeffs stain). Control veins demonstrated an extremely low level of cellular proliferation and no evidence of myofibroblasts in the adventitia, media, or intima. After bypass grafting, cellular proliferation was followed by myofibroblast formation, which occurred in the perivascular area and within the media. This was evidenced by a dense, but transient, expression of alpha-smooth muscle actin and a variable expression of desmin at 1 to 2 weeks, and with a significant increase in collagenous tissue by 1 to 3 months. Major cytoskeletal protein changes also occurred in the intima, with the appearance of alpha-smooth muscle actin positive cells at 7 to 14 days. alpha-Smooth muscle actin was still present in the neointima at 1 to 3 months, which is compatible with a persistent myofibroblast formation. CONCLUSION Myofibroblast formation occurs around and within saphenous veins after bypass grafting. This phenomenon is associated with significant remodeling of the vein grafts. The histologic changes are strikingly similar to events that occur during wound healing and may have implications for the development of neointimal hyperplasia and late vein graft disease.