Eiketsu Sho

Flow Loading Induces Macrophage Antioxidative Gene Expression in Experimental Aneurysms

Objective—Reactive oxygen species may act as proinflammatory mediators in abdominal aortic aneurysm (AAA) disease. Flow loading increases antioxidative enzyme expression and limits reactive oxygen species production in vascular smooth muscle cells in vitro, limits experimental AAA enlargement in rodent models, and is indirectly associated with reduced clinical AAA risk. We attempted to determine the mechanism or mechanisms by which flow loading limits AAA enlargement. Methods and Results—Rodent AAAs were flow loaded via femoral arteriovenous fistula creation. Aortic wall shear stress and relative wall strain were significantly higher in flow-loaded rodents. Flow loading reduced AAA diameter by 26% despite evidence of flow-mediated aortic enlargement proximal to the aneurysmal segment. Messenger RNA from AAA tissue was harvested for cDNA labeling and hybridization to a 384-clone DNA microarray. Twenty-nine genes were differentially expressed (relative intensity/relative intensity of control ratio >1.5 and <0.67) in flow-loaded compared with normal flow AAA tissue, including heme oxygenase 1 (HO-1). Increased HO-1 expression was confirmed via reverse transcriptase–polymerase chain reaction. Immunohistochemistry localized HO-1 expression to infiltrative macrophages. &agr;-Tocopherol was found to be as effective as flow loading in limiting AAA enlargement. Flow loading and &agr;-tocopherol therapy reduced AAA reactive oxygen species production. Conclusions—Flow loading may attenuate AAA enlargement via wall shear or strain-related reductions in oxidative stress.

Wall shear stress and strain modulate experimental aneurysm cellularity.

OBJECTIVE Clinical evidence indicates that hemodynamic conditions influence abdominal aortic aneurysm (AAA) disease. We modified blood flow to evaluate the effects of wall shear stress (WSS) and relative wall strain (RWS) on aneurysm structure and cellularity. METHODS Rodent AAAs were created with porcine pancreatic elastase infusion. In group 1 AAA WSS was increased with left femoral arteriovenous fistula creation, whereas in group 2 AAA WSS was decreased with left iliac artery ligation. Aortic flow, wall motion, and blood pressure were recorded in both groups. AAA diameter, endothelial and smooth muscle cellularity (CD31 and alpha-smooth muscle actin immunostaining), markers for cell proliferation (5-bromodeoxyuridine), endothelial and smooth muscle cell growth factor production (vascular endothelial growth factor-D and platelet-derived growth factor-beta, respectively), and apoptosis (deoxyuridine triphosphate nick end-labeled [TUNEL] stain) were compared between groups when the animals were killed. RESULTS Arteriovenous fistula creation increased WSS (high-flow AAA) by 300% and RWS by 150%. Iliac ligation reduced WSS (low-flow AAA) by 60%. Neither procedure significantly altered systolic, diastolic, or mean aortic pressure. When the animals were killed 7 days after elastase infusion, low-flow AAAs were significantly larger than high-flow AAAs. High-flow AAAs also contained more endothelial cells and smooth muscle cells, and evidence of increased growth factor production, cell proliferation, and decreased apoptosis. No difference in type or severity of AAA inflammatory cell infiltrate was noted between groups. CONCLUSIONS High flow conditions stimulate endothelial cell and smooth muscle cell proliferation in experimental aneurysms. Enhanced cellularity may stabilize aortic integrity, limiting aneurysm growth. Increased lower extremity activity may prevent or retard AAA disease through salutary effects on aortic remodeling mediated by endothelial cells and smooth muscle cells.

High flow drives vascular endothelial cell proliferation during flow-induced arterial remodeling associated with the expression of vascular endothelial growth factor.

Endothelial cell activation and proliferation are the essential steps in flow-induced arterial remodeling. We investigated endothelial cell turnover in the early stages of high-flow in the rabbit common carotid arteries using an arteriovenous fistula (AVF) model by kinetic investigation of cell proliferation and cell molecular analysis. BrdU was administrated to label endothelial cells (ECs) in DNA synthetic phase (S-phase) of the cell mitotic cycle. Pulse labeling revealed that ECs entered S-phase at 1.5 days of AVF (0.93 +/- 0.19%). Endothelial cell labeling index (EC-LI) peaked at 2 days of AVF (8.90 +/- 0.87%) with a high index of endothelial cell mitosis (EC-MI, 1.67 +/- 0.47%). Endothelial cell density increased remarkably at 3 days of AVF with a significant decrease in EC-LI (54%) and EC-MI (60%). Study of kinetics of EC proliferation revealed that endothelial cells took 16-24 h to finish one cycle of cell mitosis. Tracking investigation of pulse BrdU-labeled endothelial cells at 1.5 days showed that more than 66% of endothelial cells were BrdU-labeled 1.5 days after labeling. VEGF, integrin alphanubeta3, PECAM-1, and VE-cadherin were upregulated significantly preceding endothelial cell proliferation and kept at high levels during endothelial cell proliferation. These data suggest that endothelial cell proliferation is the initial step in flow-induced arterial remodeling. Hemodynamic forces may drive endothelial cell downstream migration. Expression of VEGF and cell junction molecules contribute to flow-induced arterial remodeling.

Hyperglycemia limits experimental aortic aneurysm progression.

OBJECTIVE Diabetes mellitus (DM) is associated with reduced progression of abdominal aortic aneurysm (AAA) disease. Mechanisms responsible for this negative association remain unknown. We created AAAs in hyperglycemic mice to examine the influence of serum glucose concentration on experimental aneurysm progression. METHODS Aortic aneurysms were induced in hyperglycemic (DM) and normoglycemic models by using intra-aortic porcine pancreatic elastase (PPE) infusion in C57BL/6 mice or by systemic infusion of angiotensin II (ANG) in apolipoprotein E-deficient (ApoE(-/-)) mice, respectively. In an additional DM cohort, insulin therapy was initiated after aneurysm induction. Aneurysmal aortic enlargement progression was monitored with serial transabdominal ultrasound measurements. At sacrifice, AAA cellularity and proteolytic activity were evaluated by immunohistochemistry and substrate zymography, respectively. Influences of serum glucose levels on macrophage migration were examined in separate models of thioglycollate-induced murine peritonitis. RESULTS At 14 days after PPE infusion, AAA enlargement in hyperglycemic mice (serum glucose ≥ 300 mg/dL) was less than that in euglycemic mice (PPE-DM: 54% ± 19% vs PPE: 84% ± 24%, P < .0001). PPE-DM mice also demonstrated reduced aortic mural macrophage infiltration (145 ± 87 vs 253 ± 119 cells/cross-sectional area, P = .0325), elastolysis (% residual elastin: 20% ± 7% vs 12% ± 6%, P = .0209), and neovascularization (12 ± 8 vs 20 ± 6 vessels/high powered field, P = .0229) compared with PPE mice. Hyperglycemia limited AAA enlargement after ANG infusion in ApoE(-/-) mice (ANG-DM: 38% ± 12% vs ANG: 61% ± 37% at day 28). Peritoneal macrophage production was reduced in response to thioglycollate stimulation in hyperglycemic mice, with limited augmentation noted in response to vascular endothelial growth factor administration. Insulin therapy reduced serum glucose levels and was associated with AAA enlargement rates intermediate between euglycemic and hyperglycemic mice (PPE: 1.21 ± 0.14 mm vs PPE-DM: 1.00 ± 0.04 mm vs PPE-DM + insulin: 1.14 ± 0.05 mm). CONCLUSIONS Hyperglycemia reduces progression of experimental AAA disease; lowering of serum glucose levels with insulin treatment diminishes this protective effect. Identifying mechanisms of hyperglycemic aneurysm inhibition may accelerate development of novel clinical therapies for AAA disease.

Analysis of In Situ and Ex Vivo Vascular Endothelial Growth Factor Receptor Expression During Experimental Aortic Aneurysm Progression

Objective—Mural inflammation and neovascularization are characteristic pathological features of abdominal aortic aneurysm (AAA) disease. Vascular endothelial growth factor receptor (VEGFR) expression may also mediate AAA growth and rupture. We examined VEGFR expression as a function of AAA disease progression in the Apolipoprotein E–deficient (Apo E−/−) murine AAA model. Methods and Results—Apo E−/− mice maintained on a high-fat diet underwent continuous infusion with angiotensin II at 1000 ng/kg/min (Ang II) or vehicle (Control) via subcutaneous osmotic pump. Serial transabdominal ultrasound measurements of abdominal aortic diameter were recorded (n=16 mice, 3 to 4 time points per mouse) for up to 28 days. Near-infrared receptor fluorescent (NIRF) imaging was performed on Ang II mice (n=9) and Controls (n=5) with scVEGF/Cy, a single-chain VEGF homo-dimer labeled with Cy5.5 fluorescent tracer (7 to 18 &mgr;g/mouse IV). NIRF with inactivated single chain VEGF/Cy tracer (scVEGF/In, 18 &mgr;g/mouse IV) was performed on 2 additional Ang II mice to control for nonreceptor-mediated tracer binding and uptake. After image acquisition and sacrifice, aortae were harvested for analysis. An additional AAA mouse cohort received either an oral angiogenesis inhibitor or suitable negative or positive controls to clarify the significance of angiogenesis in experimental aneurysm progression. Aneurysms developed in the suprarenal aortic segment of all Ang II mice. Significantly greater fluorescent signal was obtained from aneurysmal aorta as compared to remote, uninvolved aortic segments in Ang II scVEGF/Cy mice or AAA in scVEGF/In mice or suprarenal aortic segments in Control mice. Signal intensity increased in a diameter-dependent fashion in aneurysmal segments. Immunostaining confirmed mural VEGFR-2 expression in medial smooth muscle cells. Treatment with an angiogenesis inhibitor attenuated AAA formation while decreasing mural macrophage infiltration and CD-31+ cell density. Conclusion—Mural VEGFR expression, as determined by scVEGF/Cy fluorescent imaging and VEGFR-2 immunostaining, increases in experimental AAAs in a diameter-dependent fashion. Angiogenesis inhibition limits AAA progression. Clinical VEGFR expression imaging strategies, if feasible, may improve real-time monitoring of AAA disease progression and response to suppressive strategies.

Hemodynamic Regulation of CD34+ Cell Localization and Differentiation in Experimental Aneurysms

Objectives—Bone marrow-derived vascular progenitor cells (CD34+) are present in human and animal models of abdominal aortic aneurysm (AAA) disease. These preterminally differentiated cells may modulate disease resistance. We examined the influence of variable hemodynamic conditions on progenitor cell localization and differentiation in experimental AAAs. Methods and Results—Murine AAAs were created via porcine pancreatic elastase (PPE) infusion. AAA blood flow was increased by aortocaval fistula (ACF) formation (HF-AAA), decreased via left iliac ligation (LF-AAA), or left unchanged (NF-AAA). ACF creation increased flow by 1700%, whereas iliac ligation decreased flow 79% compared with baseline (0.6±0.1 mL/min). Wall shear stress (WSS) increased or decreased accordingly, and remained elevated (9.2±2.0 dynes/cm2) in HF-AAA 14 days after PPE infusion. CD34+ cells were identified throughout the aortic wall in all flow conditions. Seven days after PPE infusion, HF-AAAs had more CD34+ cells than LF-AAA (187±10 versus 155±7 CD34+ cells/cross sectional, P<0.05), more medial smooth muscle cells, fewer infiltrative macrophages, and a smaller diameter than LF-AAA. LF-AAAs also contained more adventitial capillaries (CD34+ capillaries 181±12 versus 89±32/cross-sectional area in HF-AAA, P<0.05). The total progenitor cell/capillary index (CD34+ capillary plus CD31+ capillary/cross sectional area) was higher in LF-AAA (282±31 versus 129±47, P<0.05). Vascular endothelial (VEGF) and platelet-derived growth factor (PDGF) expression varied directly with capillary density between groups. Increased granulocyte-macrophage colony-stimulating factor (GM-CSF) expression was also present in LF-AAAs. Conclusions—Hemodynamic conditions influence CD34+ cell localization and differentiation in experimental AAA. Adventitial capillary angiogenesis may augment inflammation and disease progression. Modulating cell lineage differentiation of mature progenitor cells may represent a novel therapeutic strategy to maintain medial cellularity and extracellular matrix integrity in AAA disease.

Enhanced Abdominal Aortic Aneurysm Formation in Thrombin-Activatable Procarboxypeptidase B–Deficient Mice

Objective—To determine whether procarboxypeptidase B (pCPB)−/− mice are susceptible to accelerated abdominal aortic aneurysm (AAA) development secondary to unregulated OPN-mediated mural inflammation in the absence of CPB inhibition. Methods and Results—Thrombin/thrombomodulin cleaves thrombin-activatable pCPB or thrombin-activatable fibrinolysis inhibitor, activating CPB, which inhibits the generation of plasmin and inactivates proinflammatory mediators (complement C5a and thrombin-cleaved osteopontin [OPN]). Apolipoprotein E−/−OPN−/− mice are protected from experimental AAA formation. Murine AAAs were created via intra-aortic porcine pancreatic elastase (PPE) infusion. Increased mortality secondary to AAA rupture was observed in pCPB−/− mice at the standard PPE dose. At reduced doses of PPE, pCPB−/− mice developed larger AAAs than wild-type controls (1.01±0.27 versus 0.68±0.05 mm; P=0.02 [mean±SD]). C5−/− and OPN−/− mice were not protected against AAA development. Treatment with tranexamic acid inhibited plasmin generation and abrogated enhanced AAA progression in pCPB−/− mice. Conclusion—This study establishes the role of CPB in experimental AAA disease, indicating that CPB has a broad anti-inflammatory role in vivo. Enhanced AAA formation in the PPE model is the result of increased plasmin generation, not unregulated C5a- or OPN-mediated mural inflammation.

Weaving Hypothesis of Cardiomyocyte Sarcomeres : Discovery of Periodic Broadening and Narrowing of Intercalated Disk during Volume-Load Change

To investigate how cardiomyocytes change their length, echocardiographic and morphological studies were performed on rabbit hearts that were subjected to volume overload, overload removal, and repeated cycles of overload and overload removal. These conditions were created by arterio-venous fistula between the carotid artery and jugular vein, closure of the fistula, and cycles of repeatedly forming and closing fistula, respectively. After overload, hearts dilated and myocytes elongated. Intercalated disks repeatedly broadened and narrowed with a 2-day cycle, which continued for 8 weeks in many animals. The cycle consisted of shifts between five modes characterized by two interdigitation elongation-and-shortenings as follows: (I) flat with short ( approximately 1/4 to approximately 1/3 sarcomere long) interdigitations; (II) flat with long (one sarcomere long) interdigitations; (III) grooved with short interdigitations; (IV) grooved with long interdigitations; (V) flat with short interdigitations intermingled by sporadic long interdigitations; and return to (I). After overload removal, hearts contracted and myocytes shortened with similar 2-day broadening and narrowing cycle of intercalated disks, in which the five modes were reversed. Repeated overload and overload removal resulted in the repetition of myocyte elongation and shortening. We hypothesize that a single elongation-and-shortening event creates or disposes one sarcomere layer, and the two consecutive elongation-and-shortenings occur complementarily to each other so that the disks return to their original state after each cycle. Our hypothesis predicts that intercalated disks weave and unravel one sarcomere per myocyte per day.