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Pharmacological characterization and autoradiographic localization of dopamine receptors in the portal vein.

1. Dopamine (DA) and DA receptor agonists exert a variety of effects on the cardiovascular system through interaction with specific DA receptors, including decreases in blood pressure and heart rate. 2. The decrease in blood pressure is due primarily to arterial vasodilation. This phenomenon is due to the stimulation of both postjunctional (D1-like or DA1) and prejunctional (D2-like or DA2) receptors causing respectively relaxation of arterial smooth muscle and decrease of the sympathetic vasoconstriction tone. 3. In view of the lack of detailed information on the existence of DA receptors in venous tissue, we have analysed D1-like and D2-like receptors in the rat portal vein using radioligand binding techniques associated with light microscope autoradiography. 4. No D1-like receptors were demonstrated in sections of the rat portal vein, whereas the D2-like receptor ligand, [3H]-spiroperidol, was bound to sections of the vein in a manner consistent with the labelling of D2-like sites. Anatomically, D2-like sites were located within the tunica adventitia, including the adventitia-media border, and in the endothelium. 5. These findings suggest the existence of D2-like but not D1-like receptor sites in the rat portal vein. D2-like sites of the tunica adventitia are probably prejunctional and involved in the modulation of sympathetic outflow. The functional significance of endothelial D2-like sites, if any, should be clarified in future studies.

Animals↗

Vascular smooth muscle cells: a major source of the semicarbazide-sensitive amine oxidase of the rat aorta.

Several methods have been used to study the distribution of the semicarbazide-sensitive amine oxidase (SSAO) within the wall of the rat aorta. After separation of the smooth muscle-containing layers of the tunica media from the connective tissue of the tunica adventitia, much higher specific enzyme activity (measured with 1 microM benzylamine) was found in homogenates of the media than of adventitia. Similar results were obtained for MAO-A (with 1 mM 5-HT as substrate). SSAO activity was also considerably higher in homogenates of cells (predominantly smooth muscle) isolated from medial tissue by enzymatic dissociation with collagenase and elastase compared with homogenates of cells (mostly of connective tissue origin) from the adventitia. Histochemical staining resulting from SSAO activity (with benzylamine as substrate) occurred predominantly and intensely over the tunica media in rat aortic sections, although some occasional staining of adventitial sites was also observed. Staining was prevented by the SSAO inhibitors hydroxylamine (1 microM) and semicarbazide (1 mM), but not by the MAO inhibitor, clorgyline (1 mM). These results indicate that SSAO is associated predominantly, although not exclusively, with the smooth muscle cells in the rat aorta. Our findings that beta-aminopropionitrile (BAPN) is a reversible, competitive inhibitor (Ki around 2 X 10(-4)M) of SSAO, in contrast to the irreversible inhibition of the connective tissue lysyl oxidase by BAPN reported by others, provides further evidence that these enzymes are not identical.

Amine Oxidase (Copper-Containing)↗

Effect of residual stress and heterogeneity on circumferential stress in the arterial wall.

Quantifying the stress distribution through the arterial wall is essential to studies of arterial growth and disease. Previous studies have shown that both residual stress, as measured by opening angle, and differing material properties for the media-intima and the adventitial layers affect the transmural circumferential stress (sigma theta) distribution. Because a lack of comprehensive data on a single species and artery has led to combinations from multiple sources, this study determined the sensitivity of sigma theta to published variations in both opening angle and layer thickness data. We fit material properties to previously published experimental data for pressure-diameter relations and opening angles of rabbit carotid artery, and predicted sigma theta through the arterial wall at physiologic conditions. Using a one-layer model, the ratio of sigma theta at the internal wall to the mean sigma theta decreased from 2.34 to 0.98 as the opening angle increased from 60 to 130 deg. In a two-layer model using a 95 deg opening angle, mean sigma theta in the adventitia increased (112 percent for 25 percent adventitia) and mean sigma theta in the media decreased (47 percent for 25 percent adventitia). These results suggest that both residual stress and wall layers have important effects on transmural stress distribution. Thus, experimental measurements of loading curves, opening angles, and wall composition from the same species and artery are needed to accurately predict the transmural stress distribution in the arterial wall.

Animals↗

Parameter sensitivity analysis and improvement of a two-layer arterial wall model.

The nonlinear two-layer arterial wall model introduced by von Maltzahn, et al. [11] is subjected to a rigorous parameter sensitivity and range of validity analysis. The model is based on the assumption that in large muscular conduit arteries the two mechanically significant layers are media and adventitia. Using curve-fitting techniques, the media is determined to be isotropic and the adventitia to be anisotropic. As a result of the range of validity analysis, the polynomial relationship for the energy density function of the media is changed to an exponential relationship. This leads to new coefficients for the polynomial of the adventitia. All coefficients have specific mechanical meanings. The parameter sensitivity analysis demonstrates convincingly that all model parameters are significantly important.

Arteries↗

Structural alterations of blood vessels in hypertensive rats.

Vascular changes in the mesenteric arteries were examined in three animals models for human essential hypertension. These models are: spontaneously hypertensive rats, which develop hypertension with age; Dahl model of genetic, salt-dependent hypertensive rats; and deoxycorticosterone-salt hypertensive rats. Morphometric measurements of the arterial wall components (e.g., endothelium, media) were carried out in the elastic arteries, muscular arteries, and arteriolar vessels from the mesenteric bed. The observed changes were correlated with the stages of hypertension development and the effect of antihypertension therapy, including sympathectomy. Specific emphasis was made to determine whether the changes observed were primary in nature, and related to the causes of hypertension, or they were secondary adaptive changes. A comparison of the three models showed that common changes in the intima, media, and adventitia were present in the three models. Alterations in the endothelium (e.g., enlargement of subendothelial space, necrotic changes), adventitia (collagen increase), and hypertrophy of the smooth muscle cells are secondary adaptive changes, because these changes occur subsequent to the development of hypertension, and antihypertensive therapy also prevent these changes from taking place. In contrast, hyperplasia of the smooth muscle cells is a primary change, because it occurs prior to the onset of hypertension. Functionally, alteration in the media is probably the most important change, because it can cause hyperreactivity of the arteries in response to stimulation. Damage to the endothelial cells may play a role in the maintenance of hypertension during the later phase. Alteration in adventitia is a passive change, which does not appear to have a major role in hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Acute and long-term effects of massive balloon dilation on the aortic wall and vasa vasorum.

To investigate the acute and long-term effects on the vasa vasorum after massive overdilation, canine aortic segments were dilated with Gruentzig balloon catheters to more than 100% over normal size. In the acute study, the significant lumen increase was the result of intimal and medial rupture with stretching and thinning of the adventitia. In these areas, the vasa vasorum were stretched and severed, causing adventitial hemorrhage. In the chronic study, areas of previous subtotal wall rupture with adventitial thinning were repaired by scar tissue. This repair included formation of a neomedia, hyperplasia of the adventitia, and proliferation of the vasa vasorum. No progression of luminal dilatation was seen. This study showed that in subtotal aortic wall rupture, even a severely damaged adventitia is capable of preserving the lumen from further dilatation and rupture until healing. Blood flow to the damaged vessel wall was reestablished by revascularization via capillary budding in the aortic wall.

Angioplasty, Balloon↗

Biaxial incremental homeostatic elastic moduli of coronary artery: two-layer model.

The detailed mechanical properties of various layers of the coronary artery are important for understanding the function of the vessel. The present article is focused on the determination of the incremental modulus in different layers and directions in the neighborhood of the in vivo state. The incremental modulus can be defined for any material subjected to a large deformation if small perturbations in strain lead to small perturbations of stresses in a linear fashion. This analysis was applied to the porcine coronary artery, which was treated as a two-layered structure consisting of an inner intima-media layer and an outer adventitia layer. We adopted a theory based on small-perturbation experiments at homeostatic conditions for determination of incremental moduli in circumferential, axial, and cross directions in the two layers. The experiments were based on inflation and axial stretch. We demonstrate that under homeostatic conditions the incremental moduli are layer- and direction dependent. The incremental modulus is highest in the circumferential direction. Furthermore, in the circumferential direction, the media is stiffer than the whole wall, which is stiffer than the adventitia. In the axial direction, the adventitia is stiffer than the intact wall, which is stiffer than the media. Hence, the coronary artery must be treated as a composite, nonisotropic body. The data acquire physiological relevance in relation to coronary artery health and disease.

Animals↗

Perivascular gene transfer of NADPH oxidase inhibitor suppresses angioplasty-induced neointimal proliferation of rat carotid artery.

Vascular stretch induces NADPH oxidase-derived superoxide anion (O2-), which has been implicated in hypertrophy and cell proliferation. We hypothesized that targeted delivery of an NADPH oxidase inhibitor to the adventitia would reduce stretch-induced vascular O2- and attenuate neointima formation. We designed a novel replication-deficient adenovirus containing a fibroblast-active promoter driving expression of NADPH oxidase inhibitory sequence gp91ds (Ad-PDGFbetaR-gp91ds/eGFP). 1) We characterized the specificity of this promoter using pPDGFbetaR-luciferase by showing induction of luciferase in cultured rat aortic fibroblasts but not in vascular smooth muscle cells. 2) Using RT-PCR, we observed expression of gp91ds and the reporter gene in fibroblasts after infection with Ad-PDGFbetaR-gp91ds/eGFP. 3) Using Ad-CMV-eGFP as a control, we delivered Ad-PDGFbetaR-gp91ds/eGFP to the adventitia of the rat common carotid artery (CCA). Immunohistochemistry confirmed localized delivery of the inhibitor to the adventitia. After CCAs were injured with an embolectomy catheter, we observed a significant increase in neointima-to-media area ratio in control CCAs, which was significantly attenuated in CCAs treated with the gp91ds-expressing virus. In a second group of rats, we detected a 10-fold increase in distension-stimulated O2-, which was significantly reduced in CCAs infected with gp91ds-expressing virus. These data demonstrate that localized adventitial delivery of an NADPH oxidase inhibitor is effective in reducing overall vascular O2- and neointima formation, suggesting that adventitial NADPH oxidase plays a functional role in development of neointimal hyperplasia.

Angioplasty, Balloon↗

Determination of layer-specific mechanical properties of human coronary arteries with nonatherosclerotic intimal thickening and related constitutive modeling.

At autopsy, 13 nonstenotic human left anterior descending coronary arteries [71.5 +/- 7.3 (mean +/- SD) yr old] were harvested, and related anamnesis was documented. Preconditioned prepared strips (n = 78) of segments from the midregion of the left anterior descending coronary artery from the individual layers in axial and circumferential directions were subjected to cyclic quasi-static uniaxial tension tests, and ultimate tensile stresses and stretches were documented. The ratio of outer diameter to total wall thickness was 0.189 +/- 0.014; ratios of adventitia, media, and intima thickness to total wall thickness were 0.4 +/- 0.03, 0.36 +/- 0.03, and 0.27 +/- 0.02, respectively; axial in situ stretch of 1.044 +/- 0.06 decreased with age. Stress-stretch responses for the individual tissues showed pronounced mechanical heterogeneity. The intima is the stiffest layer over the whole deformation domain, whereas the media in the longitudinal direction is the softest. All specimens exhibited small hysteresis and anisotropic and strong nonlinear behavior in both loading directions. The media and intima showed similar ultimate tensile stresses, which are on average three times smaller than ultimate tensile stresses in the adventitia (1,430 +/- 604 kPa circumferential and 1,300 +/- 692 kPa longitudinal). The ultimate tensile stretches are similar for all tissue layers. A recently proposed constitutive model was extended and used to represent the deformation behavior for each tissue type over the entire loading range. The study showed the need to model nonstenotic human coronary arteries with nonatherosclerotic intimal thickening as a composite structure composed of three solid mechanically relevant layers with different mechanical properties. The intima showed significant thickness, load-bearing capacity, and mechanical strength compared with the media and adventitia.

Aged↗

Three-dimensional mechanical properties of porcine coronary arteries: a validated two-layer model.

The normal coronary artery consists of two mechanically distinct layers: intima-media and adventitia. The objective of this study is to establish a two-layer three-dimensional (3-D) stress-strain relation of porcine coronary arteries. Experimental measurements were made by a series of biaxial tests (inflation and axial extension) of intact coronary arteries and, subsequently, their corresponding intima-media or adventitia layer. The Fung-type exponential strain energy function was used to describe the 3-D strain-stress relation for each layer and the intact wall. A genetic algorithm was used to determine the material constants in the Fung-type constitutive equation by curve fitting the experimental data. Because one layer must be sacrificed before the other layer can be tested, the material property of the missing layer was computed from the material constants of the intact vessel and the tested layer. A total of 20 porcine hearts were used: one group of 10 hearts for the left anterior descending artery and another group of 10 hearts for the right coronary artery. Each group was further divided into two subgroups of five specimens tested for the intact wall and the intima-media layer and for the intact wall and the adventitia layer. Our results show statistically significant differences in the material properties of the two layers. The mathematical model was validated by experimental stress-strain data for individual layers. The validated 3-D constitutive model will serve as a foundation for formulation of layer-specific boundary value problems in coronary physiology and cardiology.

Algorithms↗

Removal of albumin microinjected in rat lung perimicrovascular space.

We used a microinjection approach to assess hydraulic properties of lung perimicrovascular adventitia (interstitial cuff surrounding microvessels). Isolated blood-perfused rat lungs held at constant airway pressure were microscopically viewed to identify subpleural venules (20 microns diam). Venular adventitia were microinjected with 20 nl of fluorescent albumin (4 g/dl), and then adventitial fluorescence was quantified at the injection site by either photometery or imaging. Nonlinear decay of adventitial fluorescence indicated liquid flux from the injection site into normal interstitium. In some experiments, we determined that the adventitial fluorescence flowed longitudinally along the venule length and filled single lymphatics. The fluorescence decay at the injection site was best described by equations of convective but not diffusive transport. The decay time constant (time to 37% initial), which relates inversely to hydraulic conductivity, increased 10-fold above baseline on lung expansion with airway pressure from 5 to 15 cmH2O (P < 0.05). However, presence or absence of blood flow, increase in filtration pressure, and tissue edema were all without effect on the time constant. Our estimate of the lower limit of baseline adventitial hydraulic conductivity was 5 x 10(-6) ml.cm-2.s-1.cmH2O-1. We conclude that hydraulic conductivity of perimicrovascular adventitia is not augmented by edema but that it is decreased by lung expansion.

Albumins↗

Localisation of mRNA for JE/MCP-1 and its receptor CCR2 in atherosclerotic lesions of the ApoE knockout mouse.

MCP-1 has potent chemotactic activity for monocytes and is strongly implicated in the pathogenesis of atherosclerosis. In the present study, we have used in situ hybridisation to examine the gene expression of JE, the murine homologue of MCP-1, and its receptor, CCR2, during the development of atherosclerotic lesions in the ApoE knockout mouse. Interestingly, the earliest expression of JE detected during lesion development was found to be localised in mesenchymal cells in the adventitia and not in the intima. Macrophages were subsequently found to accumulate in these affected regions of the adventitia and these cells were found to express high levels of JE. At this stage, early macrophage-rich lesions with high expression of JE were also seen in the intima, but expression of mRNA for the receptor for JE (CCR2) was only found on adventitial macrophages and not in the intima. This sequence of events suggests that adventitial inflammation may be an important early event in lesion development and responsible for the subsequent accumulation of macrophages in the intima possibly by recruitment from the adventitia as well as via the vessel lumen.

Animals↗

Immunoreactivity of adventitial matrix fibrils of normal and aneurysmal abdominal aorta with antibodies against vitronectin and fibrinogen.

PURPOSE: We have reported that immunoglobulin G (IgG) harvested from specimens of aneurysmal abdominal aorta (AAA) is immunoreactive with a fibrillar component of the matrix of the aortic adventitia. In further studies we have reported the partial amino acid sequence of a 40-kDa protein, purified from the adventitia of the human aorta, which we have called aortic aneurysm antigenic protein-40 kDa (AAAP-40). AAAP-40 has homologies with bovine microfibril-associated glycoprotein-36 kDa (MAGP-36). Both AAAP-40 and MAGP-36 have homologies to fibrinogen beta (FB-b) and vitronectin (VN). The purposes of the present experiments were (1) to determine whether antibodies against VN and fibrinogen are immunoreactive with elements of the normal and/or aneurysmal aortic wall, and (2) to determine whether these antibodies are immunoreactive with soluble extracts of aortic proteins. METHODS: Paraffin-embedded tissue sections of normal and aneurysmal aorta were probed with polyclonal rabbit antihuman VN and antihuman FB-b antibodies. Immunoblots of soluble aortic proteins were evaluated with the same antibodies. Histochemical preparations with Gomori's aldehyde fuchsin and elastin-von-Gieson solutions were also performed. RESULTS: Anti-VN and FB-b antibodies reacted with matrix fibrils in the aortic adventitia in both normal and aneurysmal specimens, with a distribution that resembles the appearance of fibrils that are stained by Gomori's reaction. By comparison to normal adventitial fibrils, fibrils in the specimens from AAA appeared fragmented, coiled, and frayed. Antibodies against VN and FB-b were immunoreactive in immunoblots with a soluble aortic protein of molecular weight approximately 40 kDa, consistent with the migration of AAAP-40. CONCLUSIONS: There appear to be immunodeterminants in AAAP-40 that are recognized by polyclonal antibodies against VN and FB-b.

Aortic Aneurysm, Abdominal↗

Early remodeling of saphenous vein grafts: proliferation, migration and apoptosis of adventitial and medial cells occur simultaneously with changes in graft diameter and blood flow.

BACKGROUND: This study was designed to determine how migration and proliferation of adventitial and medial cells correlate temporally with hemodynamic characteristics including changes in diameter and blood flow in vein grafts. METHODS: Male mongrel dogs underwent end-to-side reversed saphenous vein bypass grafting across a ligated femoral artery. Hemodynamic parameters were assessed by duplex ultrasound. Proliferating cells were labeled 24-48 h after grafting with 5-bromo-2'-deoxyuridine (BrdU). Grafts were removed on postoperative days 2, 5, 7 and 14. Immunohistochemistry was performed on graft sections with antibodies to nuclear antigen Ki-67 (MIB-1), BrdU and smooth muscle cell markers. Apoptosis was identified by modified TUNEL staining. Proliferating/apoptotic cells were quantified digitally. RESULTS: Mean luminal cross-sectional area, blood flow and velocity increased from day 2 to 7. Cell proliferation was evident in adventitia and media on day 2, maximum on day 5 and decreased significantly by day 14. Apoptosis was maximum on day 5. Early proliferating cells (BrdU labeled) localized in the adventitia and media on day 2 were more prevalent in the neointima on days 5-14 suggesting inward migration. On day 2, proliferating cells stained positive only for vimentin. By days 5-14, proliferating cells stained for alpha-smooth-muscle actin, a phenotype characteristic of myofibroblasts. CONCLUSION: These results indicate that cell proliferation and apoptosis occur simultaneously within the adventitia and media of the vein during the first week following grafting, when changes in diameter and blood flow are greatest. In addition, proliferating adventitial cells subsequently migrate inwards to contribute to the formation of neointima.

Animals↗

External collar inhibits balloon-induced intimal hyperplasia in rabbits.

Intimal hyperplasia is a common complication following vascular interventions. To understand the underlying pathophysiology, the focus has mainly been on the intima and media. The adventitia has been less investigated, although adventitial hyperplasia is seen together with intimal hyperplasia. If the adventitial response is an important part of the process, the adventitia might be a target to inhibit intimal hyperplasia. In the present study we investigated whether an external collar attenuating the adventitial thickness could inhibit a balloon-induced intimal hyperplasia. The common carotid artery was injured in rabbits (n = 6) with a 3-french balloon catheter. The mid portion of the injured artery was encircled with a silicone collar (diameter = 2.0 mm). After 14 days the balloon-induced neointima was reduced by 54 +/- 6.3% underneath the collar. The adventitial and medial thickenings were also attenuated (36 +/- 8.7 and 44 +/- 4.3%, respectively). This study demonstrates that intimal hyperplasia following balloon injury can be inhibited with an external collar. This supports the idea of the adventitia as a potential target to inhibit intimal hyperplasia.

Actins↗

Beta-galactosidase-tagged adventitial myofibroblasts tracked to the neointima in healing rat vein grafts.

OBJECTIVE: Myofibroblasts are present transiently in normal healing wounds. However, they have been found to persist in the stroma of neoplasms, fibrotic conditions and other pathological settings. In rat vein grafts, we have observed the prolonged presence of myofibroblasts. Our aim was to determine the origin of myofibroblasts in vein grafts. METHODS: Epigastric vein to femoral artery grafts were microsurgically placed in male Lewis rats and harvested. Neointimal development, cellular death and proliferation, and cell phenotypes were analyzed using immunohistochemistry and light and electron microscopy. To follow cellular movement in the vessel wall, vein grafts were transfected with replication-defective adenovirus containing the gene encoding beta-galactosidase (n = 50), and harvested at 1, 2, 3, 4, 5, 6, 7, 14 and 28 days. Grafts were analyzed after X-gal staining. RESULTS: Myofibroblasts were detected in the outer adventitia at 4 days, in the media at 1 week and in the developing neointima at 2 weeks. Cells tagged using adenoviral beta-galactosidase demonstrated adventitia to neointima cell migration. CONCLUSIONS: Although there may be other sources of myofibroblasts in this model, the adventitia has been shown to be an origin of myofibroblasts which subsequently migrate through the vessel wall to the neointima during graft remodeling and contribute to neointimal formation.

Adenoviridae↗

Collagen biomechanics in cerebral arteries and bifurcations assessed by polarizing microscopy.

Collagen is the main matrix protein of the artery wall. We have used the known correlation between collagen birefringence and its mechanical properties to assess the wall structural integrity in brain arteries and their bifurcation regions, which are the sites of formation of saccular aneurysms. Segments of 28 brain arteries, including bifurcations, were pressure fixed and sectioned in one of three orthogonal planes. Measurements were taken by polarizing microscopy of the birefringence of collagen fibers at the apex of bifurcations and in the main layers of the artery wall - adventitia, media and intima. Dimensional data were obtained of the layers in order to estimate wall properties. Along the apex of the flow divider we measured a narrow band of collagen (birefringence 30% higher than the adjacent adventitia) providing strength and stiffness in that region. There is a thin cell-free outer layer of the tunica media (mean thickness 11 microm) comprised of densely packed coaligned collagen with high birefringence. From the fiber birefringence and directional alignment of the individual layers we calculated that the adventitia contributes about one third of circumferential and almost all of longitudinal strength of intracranial arteries.

Adult↗

3H-spiroperidol binding sites in the rabbit superior mesenteric artery. A histoautoradiographic study.

The distribution of 3H-spiroperidol binding sites within rabbit superior mesenteric artery was studied in normal as well as 6-hydroxydopamine (6-OHDA) sympathectomized animals using a histoautoradiographic technique. The labelled drug was located in the three layers of the artery (adventitia, media and intima), with the greater density in the media. In the adventitia the radiolabelled drug was located at the level of fibrous and connective cells. In the media 3H-spiroperidol was bound by smooth muscle cells and is found in the cellular membrane of the same cells. 6-OHDA administration causes an increase in the number of 3H-spiroperidol binding sites in the adventitia and as well as a 20-25% increase in the media. The possible existence of a direct dopaminergic innervation of the superior mesenteric artery is discussed.

Animals↗