Light- and electron-microscopic study of the rat esophagus following intraluminal argon laser irradiation

36 rat esophagi were irradiated by argon laser via an applicator with circumferential light distribution. They were perfused with glutaraldehyde and studied by light and transmission electron microscopy immediately, 2 days and 14 days after irradiation. Immediately after irradiation the laser center showed destruction of the keratinized stratified squamous epithelium. The collagenous fibers of the connective tissue were altered; fibrocytes and fibroblasts were severely damaged, and the microvascular lumina were occluded. The smooth muscle tissue and skeletal muscle tissue showed myofilament defects and initial karyonecrosis. There was decreasing damage of both fiber types up to 4 mm from the laser center. After 2 days the morphology of the laser center was not different from that seen immediately after irradiation. At a distance of 2 mm a partly differentiated new epithelium emerged below the necrotic epithelium. An inflammatory reaction was found in the connective tissue. After 14 days the esophageal wall was replaced and the lumen was occluded by young granulation tissue in the former laser center. Peripherally the esophageal wall appeared almost normal. As the rat esophagus serves as a model for esophagotracheal fistulae in newborn children, our findings indicate that the argon laser should be capable of occluding these fistulae likewise.     

Arterial wall remodeling under sustained axial twisting in rats

Blood vessels often experience torsion along their axes and it is essential to understand their biological responses and wall remodeling under torsion. To this end, a rat model was developed to investigate the arterial wall remodeling under sustained axial twisting in vivo. Rat carotid arteries were twisted at 180° along the longitudinal axis through a surgical procedure and maintained for different durations up to 4 weeks. The wall remodeling in these twisted arteries was examined using histology, immunohistochemistry and fluorescent microscopy. Our data showed that arteries remodeled under twisting in a time-dependent manner during the 4 weeks post-surgery. Cell proliferation, MMP-2 and MMP-9 expressions, medial wall thickness and lumen diameter increased while collagen to elastin ratio decreased. The size and number of internal elastic lamina fenestrae increased with elongated shapes, while the endothelial cells elongated and aligned towards the blood flow direction gradually. These results demonstrated that sustained axial twisting results in artery remodeling in vivo. The rat carotid artery twisting model is an effective in vivo model for studying arterial wall remodeling under long-term torsion. These results enrich our understanding of vascular biology and arterial wall remodeling under mechanical stresses.     

CO2-laser radiation damage of the arterial wall

This study describes the effects of CO2 laser radiation on the histology of the normal rabbit arterial wall, using models that simulate laser angioplasty and anastomosis. Rabbit arteries were exposed to laser treatments similar to those used clinically; 40, 0.5 sec pulses of 40-60 mW, CO2 continuous wavelength laser, or a 1/2-circumferential laser anastomosis with a 60-80 mW continuous pulse. Aneurysms developed in 8 of 22 femoral, 1 of 22 carotid, and no controls at 12 week. There were small breaks in the internal elastic lamina with atrophy, loss of muscularis, "packing" of the elastica, thinning of the muscularis at the damage site, and enlargement of the arterial diameter. Aneurysms developed in one femoral and no carotid anastomosed artery. Laser anastomoses demonstrated more muscle damage and loss, with extensive scarring and a wider area of elastic loss than the controls. The intima was reestablished with focal reduplication of the internal elastic lamina. There were no histologic differences between the arteries which developed aneurysms and those which did not in either series. These results suggest that low power laser damage of the arterial wall consists mainly of destruction of the muscularis propria, with minimal damage to the elastica.     

Expression of T-cadherin in the rat carotid artery wall after balloon injury and in different rat organs

T-cadherin is an unusual glycosilphosphatidylinositol (GPI)-anchored member of the cadherin family of cell adhesion proteins. In contrast to classical cadherins, tissue distribution of T-cadherin so far remained unknown. We examined tissue distribution of T-cadherin in rats using Western blotting and immunohistochemical method. Our results show that T-cadherin is expressed in all types of muscles (cardiac, striated, and smooth muscles), in brain neurons, and spinal cord, in the vessel endothelium, at the apical pole of intestinal villar epithelium, in the basal layer of skin, and eosophagal epithelium. Blood-derived and lymphoid cells as well as connective tissue were T-cadherin-negative. The highest level of T-cadherin expression was revealed in the cardiovascular system. Although T-cadherin was detected in smooth muscle cells, its role in the intimal thickening and restenosis is not known. We examined T-cadherin expression within 1-28 days after balloon injury of rat left carotid arteries. T-cadherin expression was valued immunohistochemically with semiquantitative method. In uninjured arteries, T-cadherin was expressed in endothelial (vWF-positive) cells, and smooth muscle (alpha-actin-positive) cells (SMCs). After denudation of arterial wall, T-cadherin was present both in the media and neointima. We revealed dynamics of T-cadherin expression in the media of injured artery: an essential increase being registered at the stage of cell migration and proliferation in the media and neointima (1-7 days), followed by its decrease to the baseline level (10-28 days). The high upregulation of T-cadherin expression in the media and neointima during migration and proliferation of vascular cells after vessel injury enables us to suggest the involvement of T-cadherin in vessel remodeling after balloon catheter injury.     

Arterial repair after microvascular anastomosis. Scanning and transmission electron microscopy study

In order to study the morphological aspects of endothelial regeneration and vascular wall reaction after microvascular anastomosis, rat femoral arteries were sectioned and successively sutured (end-to-end anastomosis) with microsurgical techniques. Control arteries and anastomosed vessels (recovered after 1, 4, 7, 14, 21, 30, 60, 120, 180 and 360 days) were studied by means of scanning (SEM) and transmission electron microscopy (TEM). The reendothelialization phenomena started after 7 days and were mainly evident at 21 days. Areas of subendothelial connective tissue with fibrin deposition remained exposed to the blood stream up to 21-30 days. Thrombus formations or post-anastomotic stenosis have been occasionally observed. Regenerating endothelium showed evident morphological differences from the control. These changes mainly consisted of shortened cell length, absence of pinocytotic vesicles, presence of cytoplasmic prolongations, and microvillous proliferations. The arterial wall showed subintimal thickening. The anastomotic site appeared completely covered by new endothelium after 30-60 days. Subintimal vascular wall changes (thickening of the media) as well as slight alterations of endothelial cells (shortened length, reduced number of pinocytotic vesicles) were evident in 60-day vessels. Lumen reduction, due to the protruding of endothelial-covered sutures, was occasionally observed in 60- to 120-day arteries. Endothelial cell morphology normalized after 60-120 days. However, thickening of the media and occasional lumen reduction were observed also after 180-360 days. Although the endothelial regeneration phenomena were clearly evident after 2 weeks, nevertheless the reestablishment of arterial wall took longer time.(ABSTRACT TRUNCATED AT 250 WORDS)     

The origin of neointimal cells in the rat carotid artery after balloon angioplasty

At present the issue of a possible role of circulating stem cells and precursors in pathological vascular wall remodeling after angioplasty remains unsolved. Therefore the origin of neointimal cells was examined in the rat carotid artery after balloon angioplasty using morphological and immunocytochemical approaches. It is shown that at the early stages (1-7 days) after vessel injury acute inflammatory response arises in the arterial wall recruiting neutrophils, monocytes, macrophages as well as large amounts of low-differentiated blood-derived cells. At the late stages (10-28 days), at the area of injured intima, a new hyperplastic intima (neointima) is formed, which consists of cells carrying specific smooth muscle markers--alpha-actin and smoothelin. The study on cell proliferative behaviour in the injured vessel wall by bromodeoxyuridine showed that in the process of neointima formation blood-born rather than resident cells are involved. Probably, early smooth muscle and endothelial precursor cells penetrate into injured area with blood stream, where they proliferative and differentiate into mature cells.     

Essential role of PKC-zeta in normal and angiotensin II-accelerated neointimal growth after vascular injury

The contribution of atypical protein kinase C (PKC)-zeta to ANG II-accelerated restenosis after endoluminal vascular injury was investigated by using the rat carotid balloon injury model. Exposure of injured arteries to ANG II resulted in an extensive neointimal thickening (1.9 times) compared with vehicle at day 14. Treatment with PKC-zeta antisense, but not scrambled, oligonucleotides reduced neointimal formation observed in the presence or absence of ANG II. Examination of early events (2 days) after injury showed an increase in cellularity in the perivascular area of the artery wall that was transferred to the adventitia and media after exposure to ANG II, events blocked by PKC-zeta antisense, but not scrambled, oligonucleotides. A positive correlation between medial cellularity at day 2 and extent of neointimal growth at day 14 was established. Immunohistochemical analysis showed that upregulation of inflammatory markers after injury, as well as infiltration of ED1(+) monocytes/macrophages from the perivascular area to the adventitia, was accelerated by ANG II. However, ANG II-stimulated medial increase in cellularity was proliferation independent, and these cells were monocyte chemoattractant protein-1(+)/vimentin(+) but ED1(-)/VCAM(-). PKC-zeta is degraded after injury, and inhibition of its neosynthesis in medial vascular smooth muscle cells or in infiltrating cells with PKC-zeta antisense attenuated medial cellularity and expression of inflammation mediators without reversing smooth muscle cell dedifferentiation. Together, these data indicate that PKC-zeta plays a critical role in normal and ANG II-accelerated neointimal growth through a mechanism involving upregulation of inflammatory mediators, leading to cell infiltration in the media of the vascular wall.     

As human pial arteries (internal diameter 200-1000 microm) get smaller, their wall thickness and capacity to develop tension relative to their diameter increase.

Pial arteries play a key role in the regulation of human cerebral blood flow. However, many of the features and mechanisms that regulate the tone and diameters of these vessels cannot be studied in situ. One approach is to study in vitro segments of arteries obtained during neurosurgical procedures. The ratios of arterial media thickness to lumen diameter and of the capacity to develop wall force to lumen diameter have important functional consequences and are known to change in disease. Experiments were carried out on pial arteries from normotensive humans to determine the way in which these parameters vary with vessel size. Vessel dimensions--media thickness and lumen diameter were derived from fixed sections using quantitative morphometry. Wall force was measured using a resistance artery myograph. The ratio of media thickness to lumen diameter and of maximum tension developed to lumen diameter both increased as vessel diameter decreased. These ratios do not change over the age range of 15-75 years. These findings show that although in vivo intralumenal pressure falls as human pial arteries become smaller, their media thickness and capacity to develop tone increase.     

Comparison of left anterior descending coronary artery hemodynamics before and after angioplasty

Coronary artery disease (CAD) is characterized by the progression of atherosclerosis, a complex pathological process involving the initiation, deposition, development, and breakdown of the plaque. The blood flow mechanics in arteries play a critical role in the targeted locations and progression of atherosclerotic plaque. In coronary arteries with motion during the cardiac contraction and relaxation, the hemodynamic flow field is substantially different from the other arterial sites with predilection of atherosclerosis. In this study, our efforts focused on the effects of arterial motion and local geometry on the hemodynamics of a left anterior descending (LAD) coronary artery before and after clinical intervention to treat the disease. Three-dimensional (3D) arterial segments were reconstructed at 10 phases of the cardiac cycle for both pre- and postintervention based on the fusion of intravascular ultrasound (IVUS) and biplane angiographic images. An arbitrary Lagrangian-Eulerian formulation was used for the computational fluid dynamic analysis. The measured arterial translation was observed to be larger during systole after intervention and more out-of-plane motion was observed before intervention, indicating substantial alterations in the cardiac contraction after angioplasty. The time averaged axial wall shear stress ranged from -0.2 to 9.5 Pa before intervention compared to -0.02 to 3.53 Pa after intervention. Substantial oscillatory shear stress was present in the preintervention flow dynamics compared to that in the postintervention case.     

Characterization of the in vivo wall shear stress environment of human fetus umbilical arteries and veins

The endothelial cells of the umbilical vessels are frequently used in mechanobiology experiments. They are known to respond to wall shear stress (WSS) of blood flow, which influences vascular growth and remodeling. The in vivo environment of umbilical vascular WSS, however, is not well characterized. In this study, we performed detailed characterization of the umbilical vascular WSS environments using clinical ultrasound scans combined with computational simulations. Doppler ultrasound scans of 28 normal human fetuses from 32nd to 33rd gestational weeks were investigated. Vascular cross-sectional areas were quantified through 3D reconstruction of the vascular geometry from 3D B-mode ultrasound images, and flow velocities were quantified through pulse wave Doppler. WSS in umbilical vein was computed with Poiseuille’s equation, whereas WSS in umbilical artery was obtained via computational fluid dynamics simulations of the helical arterial geometry. Results showed that blood flow velocity for umbilical artery and vein did not correlate with vascular sizes, suggesting that velocity had a very weak trend with or remained constant over vascular sizes. Average WSS for umbilical arteries and vein was 2.81 and 0.52 Pa, respectively. Umbilical vein WSS showed a significant negative correlation with the vessel diameter, but umbilical artery did not show any correlation. We hypothesize that this may be due to differential regulation of vascular sizes based on WSS sensing. Due to the helical geometry of umbilical arteries, bending of the umbilical cord did not significantly alter the vascular resistance or WSS, unlike that in the umbilical veins. We hypothesize that the helical shape of umbilical arteries may be an adaptation feature to render a higher constancy of WSS and flow in the arteries despite umbilical cord bending.     

Study of the evolution of the shear stress on the restenosis after coronary angioplasty

In this article, we analyze the influence of fluid dynamics variables on the development of obstructive coronary artery disease in the medium term after percutaneous coronary intervention with stent implantation. We have analyzed a group of seven patients and the study is focused on the mid-right coronary artery. In these patients we have studied the relationship between wall shear stress and arterial wall thickness both immediately after stent implantation and six months later. The realistic three-dimensional (3D) reconstruction of the arteries is performed with the data obtained with intravascular ultrasound (IVUS) and angiography. The commercial code Fluent is used to solve the Navier-Stokes equations. Special attention is paid to the shear stress on the wall arteries and the corresponding thickness. The results show that there is a negative correlation for most of the cases between the wall shear stress and increase in wall thickness. A model is proposed to study the instability at the wall, and qualitative agreement is found.     

Measurement in vitro of pulsatile arterial diameter using a helium-neon laser

A noncontacting in vitro measurement of pulsatile arterial diameter using a scanning optical micrometer is described. The major component of this system is a He-Ne laser whose beam scans the pulsating artery to be measured. The laser micrometer was integrated into a pulsatile perfusion apparatus that imposed various hemodynamic conditions on excised canine vessels. The laser system reliably tracked the pulsating arterial diameter at a particular longitudinal site as well as at various increments in the presence of an experimentally created stenosis. The He-Ne laser measured the radial motion of canine arteries and various vascular substitutes anastomosed in an end-to-end fashion. From these novel measurements, calculations were made of arterial compliance and bending stress, two biomechanical parameters that are implicated as potential causes of anastomotic intimal hyperplasia and graft failure. Although this device is inherently limited to in vitro use, it is a potentially useful instrument for vascular physiology and biophysics.     

Modelling intravascular delivery from drug-eluting stents with biodurable coating: investigation of anisotropic vascular drug diffusivity and arterial drug distribution

In-stent restenosis occurs in coronary arteries after implantation of drug-eluting stents with non-uniform restenosis thickness distribution in the artery cross section. Knowledge of the spatio-temporal drug uptake in the arterial wall is useful for investigating restenosis growth but may often be very expensive/difficult to acquire experimentally. In this study, local delivery of a hydrophobic drug from a drug-eluting stent implanted in a coronary artery is mathematically modelled to investigate the drug release and spatio-temporal drug distribution in the arterial wall. The model integrates drug diffusion in the coating and drug diffusion with reversible binding in the arterial wall. The model is solved by the finite volume method for both high and low drug loadings relative to its solubility in the stent coating with varied isotropic–anisotropic vascular drug diffusivities. Drug release profiles in the coating are observed to depend not only on the coating drug diffusivity but also on the properties of the surrounding arterial wall. Time dependencies of the spatially averaged free- and bound-drug levels in the arterial wall on the coating and vascular drug diffusivities are discussed. Anisotropic vascular drug diffusivities result in slightly different average drug levels in the arterial wall but with very different spatial distributions. Higher circumferential vascular diffusivity results in more uniform drug loading in the upper layers and is potentially beneficial in reducing in-stent restenosis. An analytical expression is derived which can be used to determine regions in the arterial with higher free-drug concentration than bound-drug concentration.     

Close relation of arterial ICC-like cells to the contractile phenotype of vascular smooth muscle cell

This work aimed to establish the lineage of cells similar to the interstitial cells of Cajal (ICC), the arterial ICC-like (AIL) cells, which have recently been described in resistance arteries, and to study their location in the artery wall. Segments of guinea-pig mesenteric arteries and single AIL cells freshly isolated from them were used. Confocal imaging of immunostained cells or segments and electron microscopy of artery segments were used to test for the presence and cellular localization of selected markers, and to localize AIL cells in intact artery segments. AIL cells were negative for PGP9.5, a neural marker, and for von Willebrand factor (vWF), an endothelial cell marker. They were positive for smooth muscle alpha-actin and smooth muscle myosin heavy chain (SM-MHC), but expressed only a small amount of smoothelin, a marker of contractile smooth muscle cells (SMC), and of myosin light chain kinase (MLCK), a critical enzyme in the regulation of smooth muscle contraction. Cell isolation in the presence of latrunculin B, an actin polymerization inhibitor, did not cause the disappearance of AIL cells from cell suspension. The fluorescence of basal lamina protein collagen IV was comparable between the AIL cells and the vascular SMCs and the fluorescence of laminin was higher in AIL cells compared to vascular SMCs. Moreover, cells with thin processes were found in the tunica media of small resistance arteries using transmission electron microscopy. The results suggest that AIL cells are immature or phenotypically modulated vascular SMCs constitutively present in resistance arteries.     

Vasoactive mediators and pulmonary hypertension after cigarette smoke exposure in the guinea pig.

The pathogenesis of pulmonary hypertension in patients with chronic obstructive pulmonary disease is not understood. We have previously shown increased levels of mediators that control vasoconstriction (endothelin-1), vascular cell proliferation (endothelin-1 and vascular endothelial growth factor), and vasodilation (endothelial nitric oxide synthase) in the intrapulmonary arteries of animals exposed to cigarette smoke. To determine whether these mediators could be implicated in the structural remodeling of the arterial vasculature and increased pulmonary arterial pressure caused by chronic cigarette smoke exposure, guinea pigs were exposed to daily cigarette smoke for 6 mo. Pulmonary arterial pressures were measured. Intrapulmonary artery structure was analyzed by morphometry, artery mediator protein expression by immunohistochemistry, and artery mediator gene expression by laser capture microdissection and real-time RT-PCR. We found that the smoke-exposed animals developed increases in pulmonary arterial pressure and increased muscularization of the small pulmonary arteries. Gene expression and protein levels of all three mediators were increased, and pulmonary arterial pressure correlated both with the levels of mediator production and with the degree of arterial muscularization. We conclude that chronic smoke exposure produces increased vasoactive mediator expression in the small intrapulmonary arteries and that these mediators are associated with vascular remodeling as well as increased pulmonary arterial pressure. These findings support the idea that hypertension in chronic obstructive pulmonary disease is a result of direct cigarette smoke-mediated effects on the vasculature and suggest that interference with endothelin and VEGF production and activity or augmentation of nitric oxide levels may be beneficial.     

Biomechanical and morphometric properties of the arterial wall referenced to the zero-stress state in experimental diabetes

Morphometric and passive biomechanical properties were studied in isolated segments of the thoracic and abdominal aorta, left common carotid artery, left femoral artery and the left pulmonary artery in 20 non-diabetic and 28 streptozotocin (STZ)-induced diabetic rats. The diabetic and non-diabetic rats were divided into groups living 1, 4, 8, and 12 weeks after the induction of diabetes (n = 7 for each diabetic group) or sham injection (n = 5 for each group). The mechanical test was performed as a distension experiment where the proximal end of the arterial segment was connected via a tube to the container used for applying pressures to the segment and the distal end was left free. The vessel diameter and length were obtained from digitized images of the arterial segments at pre-selected pressures and at no-load and zero-stress states. Circumferential and longitudinal stresses (force per area) and strains (deformation) were computed from the length, diameter and pressure data and from the zero-stress state data. The zero-stress state was obtained by cutting vessel rings radially causing the rings to open up into a sector. Diabetes was associated with pronounced morphometric changes, e.g., wall thickness. With respect to the biomechanical data, the opening angle increased and reached a plateau in 4 weeks after which it decreased again (p < 0.05). The opening angle was smallest in the thoracic aorta and largest in the pulmonary artery. Furthermore, it was found that the circumferential stiffness of the arteries studied increased with the duration of diabetes. In the longitudinal direction significant differences were found 8 weeks after injection of STZ in all arteries except the pulmonary artery. In the 12 weeks group, the femoral artery was stiffest in the circumferential direction whereas the thoracic aorta was stiffest in the longitudinal direction. The accumulated serum glucose level correlated with the arterial wall thickness and elastic modulus (correlation coefficient between 0.56 and 0.81).     

17beta-estradiol reduces tumor necrosis factor-alpha-mediated LDL accumulation in the artery wall

Estrogens have direct effects on the vascular wall that may prevent the development of atherosclerosis. In particular, estrogens, such as 17beta-estradiol (estradiol), are known to have potent antioxidant activity. Tumor necrosis factor-alpha (TNF) is found in human atheroma and produces oxygen-derived free radicals. These oxygen-derived free radicals may modify low density lipoproteins (LDL) and increase LDL binding in the artery wall. We asked: 1) does TNF increase LDL accumulation in the artery wall and 2) can the TNF-mediated increase in LDL accumulation be prevented by the antioxidant activity of estradiol? Carotid arteries from ovariectomized 3-month-old rats were removed and perfused with fluorescently labeled LDL and arterial LDL flux was measured using quantitative fluorescence microscopy. In six arteries, addition of TNF (10 ng/ml) to the perfusate resulted in a 2.3-fold increase in the rate of LDL accumulation (1.50 +/- 0.37 ng/min per cm2 vs. 3.38 +/- 0.48 ng/min per cm2; P < 0.01). Estradiol (65 pg/ml) and alpha-tocopherol (6 mg/L) both attenuated TNF-mediated LDL accumulation (P < 0.05), indicating that TNF may exert its effects on LDL accumulation through cellular production of oxygen-derived free radicals. These results support an antioxidant role for estradiol in the protection against LDL accumulation in the artery wall and subsequent progression of atherosclerosis.