Chengpei Xu

The three−dimensional micro− and nanostructure of the aortic medial lamellar unit measured using 3D confocal and electron microscopy imaging

Changes in arterial wall composition and function underlie all forms of vascular disease. The fundamental structural and functional unit of the aortic wall is the medial lamellar unit (MLU). While the basic composition and organization of the MLU is known, three-dimensional (3D) microstructural details are tenuous, due (in part) to lack of three-dimensional data at micro- and nano-scales. We applied novel electron and confocal microscopy techniques to obtain 3D volumetric information of aortic medial microstructure at micro- and nano-scales with all constituents present. For the rat abdominal aorta, we show that medial elastin has three primary forms: with approximately 71% of total elastin as thick, continuous lamellar sheets, 27% as thin, protruding interlamellar elastin fibers (IEFs), and 2% as thick radial struts. Elastin pores are not simply holes in lamellar sheets, but are indented and gusseted openings in lamellae. Smooth muscle cells (SMCs) weave throughout the interlamellar elastin framework, with cytoplasmic extensions abutting IEFs, resulting in approximately 20 degrees radial tilt (relative to the lumen surface) of elliptical SMC nuclei. Collagen fibers are organized as large, parallel bundles tightly enveloping SMC nuclei. Quantification of the orientation of collagen bundles, SMC nuclei, and IEFs reveal that all three primary medial constituents have predominantly circumferential orientation, correlating with reported circumferentially dominant values of physiological stress, collagen fiber recruitment, and tissue stiffness. This high resolution three-dimensional view of the aortic media reveals MLU microstructure details that suggest a highly complex and integrated mural organization that correlates with aortic mechanical properties.

Atherosclerotic enlargement of the human abdominal aorta

Aortic aneurysms usually develop in the atherosclerosis prone infrarenal abdominal aorta. To assess the role of atherosclerosis in aortic enlargement, we studied the relation between plaque formation and aortic size in 30 pressure-fixed male cadaver aortas (age 40-95 years, mean age 67 years). Morphometric analysis of transverse sections of the mid-thoracic and the mid-abdominal aortas included measurement of intimal plaque area, lumen area, plaque and media thicknesses. The area encompassed by the internal elastic lamina area (IEL area) was taken to be an index of aortic size. IEL area increased with age at both the thoracic (r=0.77, P<0.01) and abdominal (r=0.54, P<0.01) aortic levels. The aorta also enlarged with increasing plaque area at the thoracic (r=0.73, P<0.01) and abdominal (r=0.79, P<0.01) levels. Regression analysis of IEL area on age, body weight, height and plaque area revealed that the primary predictor of thoracic aortic size was age, whereas the primary predictor of abdominal aortic size was plaque area. Plaque thickness in the abdominal aorta was greater than in the thoracic aorta (P<0.01). Increased plaque area was associated with a significant decrease in media thickness in the abdominal aorta (r=-0.75, P<0.01) but not in the thoracic aorta. Aortas with relatively enlarged abdominal segments, i.e. those with a thoracic to abdominal ratio of <1.2 (n=13), were compared to those with a normal ratio (> or =1.2, n=17). Relatively large abdominal aortas had twofold greater plaque area (P<0.001), reduced medial thickness (P<0.05), fewer medial elastic lamellae (P<0.01) and greater mural tensile stress (P<0.05) than relatively normal abdominal aortas. We conclude that plaque formation in the infrarenal abdominal aorta in humans is associated with aortic enlargement and decreased media thickness. These changes may be predisposing factors for the preferential development of subsequent aneurysmal dilation in the abdominal aorta.

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.

P19 Progenitor Cells Progress to Organized Contracting Myocytes after Chemical and Electrical Stimulation: Implications for Vascular Tissue Engineering

Purpose: To test the hypothesis that a level of chemical and electrical stimulation exists that allows differentiation of progenitor cells into organized contracting myocytes. Methods: A custom-made bioreactor with the capability of delivering electrical pulses of varying field strengths, widths, and frequencies was constructed. Individual chambers of the bioreactor allowed continuous electrical stimulation of cultured cells under microscopic observation. On day 0, 1% dimethylsulfoxide (DMSO), known to differentiate cells into myocytes, was added to P19 progenitor cells. Additionally, for the next 22 days, electrical pulses of varying field strengths (0–3 V/cm), widths (2–40 ms), and frequencies (10–25 Hz) were continuously applied. On day 5, the medium containing DMSO was exchanged with regular medium, and the electrical stimulation was continued. From days 6–22, the cells were visually assessed for signs of viability, contractility, and organization. Results: P19 cells remained viable with pulsed electrical fields <3 V/cm, pulse widths <40 ms, and pulse frequencies from 10 to 25 Hz. On day 12, the first spontaneous contractions were observed. For individual colonies, local synchronization and organization occurred; multiple colonies were synchronized with externally applied electrical fields. Conclusion: P19 progenitor cells progress to organized contracting myocytes after chemical and electrical stimulation. Incorporation of such cells into existing methods of producing endothelial cells, fibroblasts, and scaffolds may allow production of improved tissue-engineered vascular grafts.

Physiological angiogenesis in electrically stimulated skeletal muscle in rabbits: Characterization of capillary sprouting by ultrastructural 3-D reconstruction study

Physiological angiogenesis occurs in electrically stimulated skeletal muscles. It is known to start as capillary sproutings, but has not yet been well characterized as ordinary angiogenesis. To characterize the sprouting process during physiological angiogenesis, we carried out an ultrastructural 3‐D reconstruction study for the extensor digitorum longus of three adult rabbits under electrical stimulation for 7 days. In addition, hemodynamic and morphological studies were carried out after stimulation for 3, 7, and 14 days. The electrical stimulation induced a twofold increase in femoral artery blood flow and tissue blood flow. This was associated with an increase in capillary density of the muscle by more than twofold at 7 and 14 days. Sproutings frequently appeared at 7 days (4.3 ± 1.4 × 103 sproutings per mm3, 13.3 ± 6.9 µm in length). All sprouting tips consisted of endothelial cytoplasmic protrusions (ECP). Besides sproutings, there were communicating networks consisting segmentally of ECP (11.6 ± 5.6 × 103 networks per mm3). Endothelial cytoplasmic protrusions began to appear at 3 days, were frequent at 7 days, and disappeared at 14 days, which corresponded well to the changes in blood flow and capillary density. We consider that in physiological angiogenesis, sproutings start as ECP, which contact other capillaries to form networks and become hollowed to form new capillary networks.

CORRELATION OF MECHANICAL BEHAVIOR AND MMP-1 PRESENCE IN HUMAN ATHEROSCLEROTIC PLAQUE

Regions of matrix metalloproteinase (MMP) activity potentially increase the susceptibility of the atherosclerotic lesion to complications associated with plaque rupture. Assessing the risk posed by this mechanism requires investigating the stress-strain environment associated with matrix metalloproteinase production in heterogeneous plaque. To this end, an experimental-computational technique was developed to perform mechanical analysis of physiologically loaded, diseased human aorta in vitro and to investigate relationships between vascular mechanics, histology, and histochemistry. Mechanical data and specimen histology were coupled through a heterogeneous finite element model, and tissue constituent material properties were determined by an optimization method. The cross-sectional distribution of MMP-1 was quantified using immunohistochemical techniques. Results show stresses and strains are strongly influenced by lesion structure and composition, and MMP-1 presence is correlated with histology and lesion mechanics. Interactions between lipid presence, mechanical stimuli, and extracellular matrix metabolism-catabolism likely affect arterial plaque remodeling, progression, and the risk of disruption and clinical symptoms.

Expression of TGF-β1 and β3 but not apoptosis factors relates to flow-induced aortic enlargement

BackgroundCell proliferation and apoptosis are both involved in arterial wall remodeling. Increase in blood flow induces arterial enlargement. The molecular basis of flow-induced remodeling in large elastic arteries is largely unknown.MethodsAn aortocaval fistula (ACF) model in rats was used to induce enlargement in the abdominal aorta. Aortic gene expression of transforming growth factors beta (TGF-β) and apoptosis-related factors was assessed at 1 and 3 days and 1, 2, 4, and 8 weeks. Expression levels were determined using a ribonuclease protection assay and western blotting. Cell proliferation and apoptosis were analyzed using BrdU incorporation and TUNEL techniques.ResultsBlood flow increased 5-fold immediately after ACF (P<0.05). Lumen diameter of the aorta was 30% and 75% larger at 2 and 8 weeks respectively than those of controls (P<0.05). mRNA levels of TGF-β1 and TGF-β3 increased after ACF, peaked at 3 days (P<0.05) and returned to normal level at 1 week and thereafter. Western blotting showed enhanced expression of TGF-β1 at 3 days and TGF-β3 at 1 and 3 days and 1 week (P<0.05). mRNA levels of Bcl-xS initially decreased at 1 day, 3 days and 1 week, followed a return to baseline level at 2 weeks. Cell proliferation was observed at all time points after ACF (P<0.001 vs. controls) with proliferation in endothelial cells more significant than smooth muscle cells. Apoptosis was not significant.ConclusionsGene expression of TGF-β1 and β3 precedes arterial enlargement. Expression of apoptosis related factors is little regulated in the early stage of the flow-induced arterial remodeling.

Inhibition of experimental abdominal aortic aneurysm in a rat model by way of tanshinone IIA

BACKGROUND The purpose of the present study was to investigate whether tanshinone IIA (Tan IIA), one of the major lipophilic components of Salvia miltiorrhiza Bunge, could inhibit the development of elastase-induced experimental abdominal aortic aneurysms (AAAs). METHODS Male Sprague-Dawley rats (n = 12/group) were randomly distributed into three groups: Tan IIA, control, and sham. The rats from the Tan IIA and control groups underwent intra-aortic elastase perfusion to induce AAAs, and the rats in the sham group were perfused with saline. Only the Tan IIA group received Tan IIA (2 mg/rat/d). The maximum luminal diameter of the abdominal aorta was measured before and 5, 12, 18, and 24 d after perfusion. The systolic blood pressure was measured twice using the tail cuff technique before administration and death. Aortic tissue samples were harvested at 24 d and evaluated using reverse transcriptase-polymerase chain reaction, Western blot, immunohistochemistry, and Millers elastin-Van Gieson staining. RESULTS The rats in the control group had significantly increased aortic sizes compared with the sham group after 24 days (P < 0.05), and the Tan IIA group had a significant reduction in aortic size (Tan IIA versus control, P < 0.05) without affecting blood pressure (P > 0.05). The overexpression of matrix metalloproteinase-2, metalloproteinase-9, monocyte chemotactic protein-1, and inducible nitric oxide synthase and the depletion of elastic fibers and vascular smooth muscle cells induced by elastase perfusion were significantly decreased by Tan IIA treatment (P < 0.05). CONCLUSIONS Tan IIA inhibited the development of elastase-induced experimental AAAs by suppressing proteolysis, inflammation, and oxidative stress and preserving vascular smooth muscle cells. It could be a new pharmacologic therapy for AAAs.

Chronic effects of pulmonary artery stenosis on hemodynamic and structural development of the lungs

Pulmonary artery (PA) stenosis is a difficult obstructive defect to manage since clinicians cannot know a priori which obstructions to treat and when. Prognosis of PA stenosis and its chronic effects on lung development are poorly understood. This study aimed to characterize the hemodynamic and structural effects of PA stenosis during development. Fourteen male Sprague-Dawley rats underwent left PA (LPA) banding at age 21 days, and 13 underwent sham operation. Hemodynamic and structural impacts were studied longitudinally at 20, 36, 52, 100, and 160 days. Chronic LPA banding resulted in a significant reduction in LPA flow (P < 0.0001) and size of both proximal LPA (P < 0.0001) and distal LPA (P < 0.01), as well as a significant increase in flow and size of the right PA (P < 0.05) throughout development. Flows and sizes adapted such that normal levels of wall shear were restored after banding. At 160 days, LPA banding resulted in a significant decrease in left lung volume and an increase in right lung volume but no significant differences in total lung volume. There was an elevation of proximal LPA pressure as well as right ventricular hypertrophy in the banded animals. The banded lung exhibited arterial disorganization, loss of vessels, and enlargement of its bronchial arteries, whereas the contralateral lung showed signs of vascular pathology. There are consequences on development of both lungs in the presence of an LPA stenosis at young age. These results suggest that early intervention may be necessary to optimize left lung growth and minimize right lung vascular pathology.

Pathophysiology of Vascular Disease

Vascular disease is the major cause of morbidity and mortality in Western civilization. Its manifestations include heart attacks, strokes, lower extremity occlusive disease, and aneurysmal disease, and its predominant underlying cause is atherosclerosis. Although atherosclerosis is a generalized disorder of the arterial tree associated with well-known risk factors—including hyperlipidemia, hypertension, cigarette smoking, and diabetes mellitus—its clinical expression tends to be focal. Not all individuals with extensive risk factors develop atherosclerotic plaques, and many patients with extensive atherosclerotic plaques have no recognized risk factors. Moreover, morbidity and mortality usually result from localized plaque deposition at certain vulnerable sites in the arterial tree rather than from diffuse disease.