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PubMed1976

Role of calcium in different layers of vascular smooth muscle in norepinephrine contraction.

The role of calcium present in adventitial and media-intimal layers of rabbit aorta in the fast (F-) and the slow (S-) components of norepinephrine (10(-5) M) contraction was evaluated in this study. An increased sensitivity and decreased maximum tension to response to norepinephrine was observed in adventitia-free aorta as compared to whole aorta. Incubation in calcium-free Ringer solution with or without EDTA decreased the F-component of norepinephrine contraction of the adventitia-free aorta, whereas the F-component of the whole aorta was not affected. The S-component of both preparations was abolished in calcium-free Ringer solution. Norepinephrine increased calcium uptake into whole and adventitia-free aortic strips. An increase in calcium uptake induced by norepinephrine during the F-component of norepinephrine contraction was also observed. 45Ca efflux studies revealed the presence of an exchangeable calcium fraction in the adventitial layer. These data suggest that F-component of norepinephrine contraction is partially dependent on the extracellular calcium distributed in the adventitial layer of rabbit aorta.

Animals↗
PubMed1979

Kinetics of alpha-adrenoreceptor blockade by phentolamine in the normal and denervated rabbit aorta and rat vas deferens.

The rates of onset and offset of alpha-adrenoreceptor blockade by phentolamine (5 X 10(-7) M) have been studied on the normal and denervated rabbit aorta and rat vas deferens. Removal of the adventitia with its accompanying sympathetic nerve component results in an approximately threefold increase in the rate of onset of alpha-adrenoreceptor blockade by phentolamine in the rabbit aorta. The rate of offset of blockade by the antagonist on the aorta is likewise faster after the adventitia has been removed (approximately threefold faster for loss of 90% of the antagonist from the region of the receptors). This effect is not likely to be attributed to the absence of sympathetic nerve terminals since surgical denervation of the rat vas deferens has no effect on the kinetics of receptor blockade in this densely innervated organ. These data suggest that the adventitia of the rabbit aorta may serve as a diffusion barrier which limits the antagonist's access to, and efflux from, the region of the receptors. The dissociation constant of phentolamine (calculated from the equilibrium dose ratio) on the alpha-adrenoreceptor is unaltered by denervation in either tissue and is identical in both tissues. It is suggested that alpha-adrenoreceptors in the rabbit aorta and rat vas deferens are of a single type and are not significantly affected, with respect to antagonist affinity, by denervation.

Animals↗
PubMedDate not supplied

Three-dimensional collagen organization of human brain arteries at different transmural pressures.

Measurements on the directional organization of collagen are necessary for relating the structure and mechanical function of blood vessels. The birefringent optical property of collagen has enabled us to assess the collagen architecture for brain arteries, which are prone to spasm and aneurysm formation. Using the universal stage and polarizing microscope, we measured the three-dimensional organization of the collagen of the main layers of the artery wall, and examined the effect of distending pressure on that organization. Adult arteries obtained from autopsy were fixed at one of three distending pressures, 30, 120 and 200 mm Hg; they were embedded in paraffin and sectioned parallel to the vessel axis at 4 microns thickness. Sections were stained with picrosirius red, a birefringent enhancement stain specific for collagen. Orientation data were obtained from tangential sections from thirteen arteries. We chose to use tangential sections that graze the curving surface of individual layers, to permit measurements that are equally sensitive to fibres in the mechanically meaningful range of directions including longitudinal, helical and circumferential. Each measurement was from a single fibre or group of fibres at a specific location; the mean direction and its variation of alignment within each artery layer were calculated. In some arteries, the adventitia and subendothelium measurements were separated into sublayers, distinguishable by the birefringent optical appearance. Main findings included a substantial component of longitudinal fibres in the adventitia and subendothelium, highly varied in coherence and mean direction, and a thin collagen layer of the adventitia, radially outside the medial muscle cells, that was highly organized circumferentially (circular standard deviation of 9 degrees). At higher pressures, the collagen fabric of all the layers was increasingly coherent and more circumferential in direction.

Aged↗
PubMed2005

Mechanisms of inducible nitric oxide synthase-mediated vascular dysfunction.

OBJECTIVE: Inducible nitric oxide synthase (iNOS) is expressed in arteries during inflammation and may contribute to vascular dysfunction. Effects of gene transfer of iNOS to carotid arteries were examined in vitro in the absence of systemic inflammation to allow examination of mechanisms by which iNOS impairs contraction and relaxation. METHODS AND RESULTS: After gene transfer of iNOS with an adenovirus (AdiNOS), constrictor responses to phenylephrine (PE) and U46619 were impaired. After AdiNOS, inhibition of soluble guanylate cyclase (sGC) with 1H-[1,2,4]oxadiazolo-[4,3,2]quinoxalin-1-one (ODQ) reduced the EC50 for PE from 4.33+/-0.78 micromol/L to 1.15+/-0.43 micromol/L (mean+/-SEM). These results imply that iNOS impairs contraction by activation of the NO/cGMP pathway. Relaxation to acetylcholine (ACh) also was impaired after AdiNOS. Sepiapterin (300 micromol/L), the precursor for tetrahydrobiopterin (BH4), improved relaxation to Ach. Because BH4 is an essential cofactor for production of NO by both iNOS and endothelial nitric oxide synthase (eNOS), these results suggest that iNOS may reduce production of NO by eNOS by limiting availability of BH4. Next, we examined effects of expression of iNOS in endothelium and adventitia. Selective expression of iNOS in endothelium, but not adventitia, impaired contraction to phenylephrine and relaxation to acetylcholine. CONCLUSIONS: We conclude that: (1) iNOS may impair contraction in part by activation of sGC; (2) iNOS impairs relaxation, at least in part, by limiting availability of BH4; and (3) expression of iNOS in endothelium may be a more important mediator of vascular dysfunction than expression of iNOS in adventitia.

Adenoviridae↗
PubMed2005

Atherosclerotic plaque progression and vulnerability to rupture: angiogenesis as a source of intraplaque hemorrhage.

Observational studies of necrotic core progression identify intraplaque hemorrhage as a critical factor in atherosclerotic plaque growth and destabilization. The rapid accumulation of erythrocyte membranes causes an abrupt change in plaque substrate characterized by increased free cholesterol within the lipid core and excessive macrophage infiltration. Neoangiogenesis is associated closely with plaque progression, and microvascular incompetence is a likely source of intraplaque hemorrhage. Intimal neovascularization is predominantly thought to arise from the adventitia, where there are a plethora of pre-existing vasa vasorum. In lesions that have early necrotic cores, the majority of vessels invading from the adventitia occur at specific sites of medial wall disruption. A breech in the medial wall likely facilitates the rapid in-growth of microvessels from the adventitia, and exposure to an atherosclerotic environment stimulates abnormal vascular development characterized by disorganized branching and immature endothelial tubes with "leaky" imperfect linings. This network of immature blood vessels is a viable source of intraplaque hemorrhage providing erythrocyte-derived phospholipids and free cholesterol. The rapid change in plaque substrate caused by the excessive accumulation of erythrocytes may promote the transition from a stable to an unstable lesion. This review discusses the potential role of intraplaque vasa vasorum in lesion instability as it relates to plaque rupture.

Animals↗
PubMedDate not supplied

Role of elastase and lysyl oxidase activity in spontaneous rupture of internal elastic lamina in rats.

Rupture of the internal elastic lamina may occur spontaneously with age in certain arteries of the rat and to various extents in different strains. This phenomenon may have some bearing on certain aspects of arterial pathology. For this study, we investigated biochemically the mechanisms of formation of interruptions in the internal elastic lamina (IIEL) by comparing aortas of Brown Norway (BN) rats, which develop numerous IIEL in the abdominal aorta, with those of Long-Evans (LE) rats, which develop none. We isolated aortic elastin from BN and LE rats and determined its amino acid composition and its susceptibility to different elastases. No differences were found between the two strains, but the quantity of elastin isolated per aorta was lower in the BN than in the LE rats. Elastase-like activity (ELA) of whole aortic extracts, measured with Suc(Ala)3NA as a substrate, was greater in the BN rats than in the LE rats of both sexes. The assay of ELA in endothelium, media, and adventitia extracted separately showed very low levels in the media compared to the endothelium and adventitia. The endothelium accounts for about one-half of the total aortic ELA, but a difference between the two strains was detected only in the adventitia. With 3H-insoluble elastins prepared from BN and LE aortas as substrates, elastinolytic activity (EA) was detected only in extracts of endothelium after prior exposure to trypsin. Extracts from BN endothelium on BN elastin were more active than were those from LE endothelium on LE elastin. The assay of lysyl oxidase activity in aortic extracts from the two strains with 3H-collagen from chick embryo calvaria as the substrate showed a lower activity in the BN than in the LE rats. Taken together, these results suggest that increased elastase activity and decreased lysyl oxidase activity may be involved in the formation of IIEL.

Animals↗
PubMed1992

Chronic rejection of rat aortic allografts. III. Synthesis of major eicosanoids by vascular wall components and effect of inhibition of the thromboxane cascade.

We have previously demonstrated that rat aortic allografts from the DA (RT1a) to the WF (RT1v) strain develop chronic arteriosclerotic changes in the vascular wall after a short spontaneously reversible acute rejection episode. These changes, which are lacking in syngeneic DA-to-DA control grafts, are virtually identical with those observed in human allografts during chronic rejection. In this study we have investigated whether eicosanoids are involved in the generation of arteriosclerotic changes. Incubation of aortic wall rings in vitro and immunochemical assays demonstrated that the arteriosclerotic allografts synthesize significantly more thromboxane B2 (TxB2) but not 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) or leukotriene B4. The increased synthesis of TxB2 in the allografts persisted for at least 5 months after transplantation. Separate incubation of the two major components of the vascular wall, after microdissection of the intima and (media plus) adventitia, demonstrated that most of the synthesis of TxB2 during chronic rejection was due to the outer layer of aorta, presumably the inflammatory cells of the adventitia. In contrast, most of the 6-keto-PGF1 alpha was synthesized by the inner layer of the aorta, presumably the endothelial cells and the smooth muscle cells of the intima. Administration of 1 mg.kg-1 x day-1 of a specific TxA2 receptor inhibitor, GR32191B, to the recipient rat reduced the proliferative response of inflammatory cells in the adventitia by 30%, as detected by in vivo tritiated-thymidine (3H-TdR) labeling and autoradiography, but did not reduce the proliferative response of smooth muscle cells in the media and intima.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Intimal thickening and medial necrosis in allograft arteriosclerosis (chronic rejection) are independently regulated.

Rat aortic allografts from the DA (RT1a) to the WF (RT1v) strain but not syngeneic DA-to-DA control grafts develop arteriosclerotic changes in the vascular wall that are virtually identical to human allografts during chronic rejection. A more prominent medial cell destruction in the rat aorta, leading ultimately to complete medial necrosis, is the major difference between rat and human allografts. If the adventitia of syngeneic grafts is exposed to starch before transplantation, these grafts also develop an inflammatory reaction in the adventitia and an extensive intimal thickening at the site of the granulomas, but the medial smooth muscle cells are preserved. In both types of transplants with an intact endothelium as determined by light microscopy, adventitial inflammatory cell proliferation was accompanied by smooth muscle cell replication in the media and thickening of the intima. We therefore propose that an adventitial proliferative response is a prerequisite for intimal thickening to occur. In the allograft but not in starch-exposed syngeneic grafts there was also a notable lymphoid activation in the adventitia, which was accompanied by medial necrosis. We suggest that the medial necrosis in the allograft is linked to a toxic effect of activated lymphoid cells on medial myocytes and is not a prerequisite for intimal proliferation. Instead, intimal proliferation and medial necrosis in the allograft seem to be independently regulated.

Animals↗
PubMed1994

Cytomegalovirus infection enhances mRNA expression of platelet-derived growth factor-BB and transforming growth factor-beta 1 in rat aortic allografts. Possible mechanism for cytomegalovirus-enhanced graft arteriosclerosis.

We have recently demonstrated that rat cytomegalovirus (RCMV) infection induces an early inflammatory response in the adventitia (perivasculitis) and in the subendothelial space (endothelialitis) as well as doubles smooth muscle cell (SMC) proliferation and intimal thickening of rat aortic allografts performed from the DA (AG-B4, RT1a) to the WF (AG-B2, RT1v) strain. In this study, the impact of RCMV infection on the structure of inflammation in the allograft adventitia and on the expression of SMC growth factors in the allograft vascular wall was investigated. The recipient rats were inoculated with 10(5) plaque-forming U of RCMV Maastricht strain or left noninfected and used as controls. The allografts were removed at 7 days and 1 and 3 months after transplantation and processed for morphometry and immunohistochemistry. RNA was isolated for reverse transcriptase polymerase chain reaction (RT-PCR). RCMV infection was associated with significantly upregulated presence (P < .05) of T helper (W3/25), T cytotoxic (OX8), and natural killer (3.2.3) cells in the allograft adventitia 7 days after transplantation but not thereafter. More monocyte/macrophages (OX42) were also present in RCMV-infected allografts, but the difference was not significant. Concomitantly, RCMV infection significantly enhanced (P < .05) the expression of major histocompatibility complex class II (OX6) and almost doubled (P = NS) the expression of interleukin-2R (CD25), intercellular adhesion molecule-1 (CD54;1A29), and lymphocyte function-associated antigen-1 alpha-chain (CD11a; WT.1) in the adventitial inflammatory infiltrate. RCMV infection was linked with an early, prominent expression of both PDGF-BB mRNA at 7 days (P < .05) and at 1 month (P < .025) and of transforming growth factor-beta 1 mRNA at 7 days (P < .025) and at 1 month (P < .025) after transplantation. A less-prominent mRNA upregulation of acidic fibroblast growth factor (P < .05) was associated with RCMV infection at 7 days and at 1 month, as well as of epidermal growth factor at 1 month after transplantation, when compared with noninfected allografts, although the mRNA expression in both groups was below the levels of nontransplanted DA aortas. RCMV infection almost doubled basic fibroblast growth factor mRNA expression (P = NS) in the allograft vascular wall at 7 days and at 1 month. RCMV infection had no additional impact on insulin-like growth factor-1 mRNA expression when compared with noninfected allografts.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Tissue-type plasminogen activator and its inhibitor in rat aorta. Effect of endotoxin.

Plasminogen activator (PA) and PA inhibitor (PAI) antigen, activity, and mRNA were analyzed in the three layers of rat aorta, and the effect of endotoxin on PA and PAI was studied. All PA activity in aorta was identified as tissue-type PA (TPA) activity; no urokinase-type PA was detected. In the tunica adventitia TPA activity, TPA antigen, and TPA mRNA were detected, whereas in the tunica media TPA antigen and TPA mRNA, but no TPA activity, were found. PAI activity was detected in the tunica media, explaining the absence of TPA activity in this layer. Removal of the endothelial cells had no effect on TPA antigen and PAI activity in intima-media preparations. Also, similar amounts of PAI-1 mRNA were found in intima-media preparations, irrespective of the presence or absence of the intima. Immunohistochemical staining showed that TPA immunoreactivity was present in all three layers of the aorta, whereas PAI-1 immunoreactivity was found in medial smooth muscle cells but not in endothelial cells. After endotoxin treatment, TPA activity was decreased in extracts of the total aorta and of the adventitia, although TPA antigen and TPA mRNA were unchanged. PAI-1 mRNA was strongly increased in the tunica adventitia and in the tunica media, as was PAI activity in the tunica media. Thus, endotoxin decreased TPA activity by increasing the synthesis of PAI-1; TPA was unaffected. Our observations in rat aorta differ from observations in mouse aorta and in rat carotid artery, and they caution against extrapolation from one tissue (or species) to another.

Animals↗
PubMed1995

Inflammation and matrix metalloproteinases in the enlarging abdominal aortic aneurysm.

The risk of rupture of an abdominal aortic aneurysm increases with aortic diameter. To obtain insight into the pathological processes associated with the vascular remodeling that accompanies aortic dilatation, we compared the histological features and the activity of matrix metalloproteinases (MMPs) in biopsies from 21 small (4.0 to 5.5 cm in diameter) and 45 larger abdominal aortic aneurysms. The histological feature most clearly associated with enlarging aneurysm diameter was a higher density of inflammatory cells in the adventitia, P = .018. This inflammation was nonspecific, principally macrophages and B lymphocytes. Fibrosis of the adventitia provided compensatory thickening of the aortic wall as the aneurysm diameter increased. A combination of zymography and immunoblotting identified gelatinase A (MMP-2) as the principal metallogelatinase in small aneurysms, whereas zymography indicated an increasing activity of gelatinase B (MMP-9) in large aneurysms. Homogenates prepared from both small and large aneurysms had similar total activity against gelatin or type IV collagen. However, the concentration of gelatinase A, determined by immunoassay, was highest for small aneurysms: median concentrations, 385, 244, and 166 ng/mg protein for small aneurysms, large aneurysms, and atherosclerotic aorta, respectively. Immunolocalization studies indicated that gelatinase A was concentrated along fibrous tissue of both the acellular media and the atherosclerotic plaque. The recruitment of inflammatory cells into the adventitia, with subsequent elaboration of metalloproteinases, including gelatinase B, may contribute to the rapid growth and rupture of larger aneurysms.

Amino Acid Sequence↗
PubMed1998

Adventitial expression of recombinant eNOS gene restores NO production in arteries without endothelium.

The current study was designed to determine the effect of recombinant endothelial nitric oxide synthase (eNOS) gene expression on endothelium-dependent relaxations to bradykinin in isolated canine basilar, coronary, or femoral arteries. Arterial rings were exposed ex vivo (30 minutes at 37 degrees C) to an adenoviral vector encoding either the eNOS gene (AdCMVeNOS) or the beta-galactosidase reporter gene (AdCMVbeta-Gal). Twenty-four hours after transduction, transgene expression was evident mainly in the adventitia. Expression of recombinant proteins was much higher in basilar arteries than in coronary or femoral arteries. Rings of control, AdCMVbeta-Gal, and AdCMVeNOS arteries with and without endothelium were suspended for isometric tension recording. Levels of cGMP were measured by radioimmunoassay. In AdCMVeNOS basilar arteries with endothelium, relaxations to low concentrations of bradykinin (3 x 10(-11) to 10(-9) mol/L) were significantly augmented. In contrast, in coronary and femoral arteries with endothelium, AdCMVeNOS transduction did not affect relaxations to bradykinin. Removal of the endothelium abolished bradykinin-induced relaxations in control and AdCMVbeta-Gal basilar arteries. However, in basilar arteries transduced with AdCMVeNOS even when the endothelium was removed, stimulation with bradykinin (3 x 10(-11) to 10(-9) mol/L) caused relaxations as well as increases in cGMP production. The relaxations to bradykinin were completely blocked by an NOS inhibitor, NG-nitro-L-arginine methyl ester. Electron microscopic analysis revealed that recombinant eNOS protein was expressed in fibroblasts of the basilar artery adventitia. These results suggest that genetically modified adventitial fibroblasts may restore production of NO in cerebral arteries without endothelium. Our findings support a novel concept in vascular biology that fibroblasts in the adventitia may play a role in the regulation of vascular tone after successful transfer and expression of recombinant eNOS gene.

Adenoviridae↗
PubMed1999

Smooth muscle-specific SM22 protein is expressed in the adventitial cells of balloon-injured rabbit carotid artery.

During the "response-to-injury" process after a mechanical insult to the porcine coronary arteries, the adventitial cells acquire the structural characteristics of myofibroblasts before being incorporated into smooth muscle (SM) layer. We assessed whether the SM-specific SM22 protein can be used as a tracer of adventitial cell-myofibroblast differentiation in the mild balloon injury of rabbit carotid artery. To achieve this goal, we used 2 monoclonal anti-SM22 antibodies (E-11 and 1-B8) and a molecular probe for the SM22alpha mRNA isoform in immunocytochemical and in situ hybridization experiments. The differentiation profile and the migratory and proliferative ability of activated adventitial cells were evaluated by a panel of antibodies to some SM and nonmuscle antigens and pulse- and end-labeling with bromo-deoxyuridine, respectively. In adventitial cells, SM22 antigenicity and SM22alpha mRNA were detectable at days 2 and 4 and, to a lesser extent, at days 7 and 21 after injury, particularly near the adventitia-media interface and mostly colocalizing with bromo-deoxyuridine-positive cells. The pulse-labeling experiments showed that the large majority of these cells penetrated the outermost layer of the tunica media without migrating to the subendothelial region. The phenotypic features of activated migrating and nonmigrating adventitial cells resembled those of vimentin-actin myofibroblast subtype and fetal-type SM cells. These findings indicate that a direct exposure of adventitia to the lumen is not required for phenotypic changes and proliferation/migration of these cells. After comparison of the SM22 expression in arterial vessels during early stages of development, we hypothesize that in the injured carotid artery the mural incorporation of adventitial cells and the spatiotemporal activation of SM22 expression are reminiscent of the vascular morphogenetic process and suggest the existence of a stem cell-like reservoir in adventitia. The early adventitial upregulation of SM22 expression in the injured vessel might be related to a multistep transition process in which nonmuscle cells are converted to myofibroblasts and, possibly, to SM cells.

Animals↗
PubMedDate not supplied

Blood flow through vasa vasorum of coronary arteries in atherosclerotic monkeys.

Morphologic studies and postmortem arteriograms provide qualitative evidence that atherosclerosis induces neovascularization in coronary arteries. In this study we obtained the first measurements of blood flow through vasa vasorum in atherosclerotic coronary arteries. We measured blood flow with microspheres to intima media and to adventitia in normal and atherosclerotic cynomolgus monkeys. Blood flow to adventitia was not altered by atherosclerosis. The blood flow to intima media, however, was increased more than fivefold by atherosclerosis: flow was 3 +/- 1 in normal animals and 16 +/- 5 in atherosclerotic monkeys (p less than 0.05). Blood flow was threefold greater in atherosclerotic than in normal coronary arteries during adenosine-induced vasodilatation, which suggests that proliferation of new vessels, not dilatation of existing vessels, accounts for the increase in flow through vasa in intima media. Histologic studies indicated that vasa vasorum to intima media in atherosclerotic coronary arteries originate from adventitial vasa. We conclude that: 1) there is a large increase in blood flow through vasa vasorum to intima media, but not to adventitia, in atherosclerotic coronary arteries; and 2) increased flow is produced primarily by proliferation of new vessels that originate from adventitial vasa.

Animals↗
PubMed2002

Dosimetric measurements in isolated human coronary arteries: comparison of commercially available iridium(192) with strontium/yttrium(90) emitters.

BACKGROUND: Intravascular brachytherapy is being applied more and more in patients with coronary artery disease for the prevention of restenosis subsequent to balloon angioplasty, in particular after stent implantation. Several radiation sources (beta- and gamma-emitters) are available in clinical routine. It was the purpose of this study to compare the radiation doses at the level of the adventitia in diseased and stented human coronary arteries for (192)Ir and (90)Sr/Y emitters in routine use. In contrast to previously published work, we performed dosimetry instead of calculating depth-dose distribution by use of the Monte Carlo system. METHODS AND RESULTS: Postmortem calcified human coronary artery segments were stented and placed in an organ bath. Commercially available gamma-emitters ((192)Ir; Cordis Checkmate) and beta-emitters ((90)Sr/Y; Novoste Beta-Cath) were used. Relative dose distributions along the adventitia were measured by a specially designed scintillation detector system. Whereas dose perturbations caused by stents and calcified plaque were negligible for the (192)Ir source, radiation from the beta source was significantly impaired (as much as 40%) at the level of the adventitia (3.0-mm vessel diameter). Dose perturbation was clearly dependent on the extent and severity of calcification, less affected by stent material. CONCLUSIONS: Dose perturbation caused by calcified plaque and metallic stents is significant for beta-sources. This dosimetric difference between beta- and gamma-emitters in diseased coronary arteries should be considered when calculating doses in intravascular brachytherapy.

Beta Particles↗
PubMed2002

In vivo evidence for a role of toll-like receptor 4 in the development of intimal lesions.

BACKGROUND: Inflammation plays an important role in atherogenesis. The toll-like receptor 4 (TLR4) is the receptor for bacterial lipopolysaccharides and also recognizes cellular fibronectin and heat shock protein 60, endogenous peptides that are produced in response to tissue injury. To explore a possible role for this receptor in arterial obstructive disease, we determined the expression of TLR4 in the atherosclerotic arterial wall, including adventitia, and studied the effect of adventitial TLR4 activation on neointima formation in a mouse model. METHODS AND RESULTS: Localization of TLR4 was studied in human atherosclerotic coronary arteries by immunohistochemistry and detected in plaque and adventitia. In the adventitia, not all TLR4-positive cells colocalized with macrophages. In primary human adventitial fibroblasts, expression of TLR4 was demonstrated by immunofluorescence, Western blot, and reverse transcriptase-polymerase chain reaction. Adding lipopolysaccharide to these fibroblasts induced activation of NF-kappaB and an increase of mRNAs of various cytokines. The effect of adventitial stimulation of TLR4 was studied in a mouse model. A peri-adventitial cuff was placed around the femoral artery. Application of lipopolysaccharide between cuff and artery augmented neointima formation induced by the cuff (intimal area+/-SEM, 9134+/-1714 versus 2353+/-1076 microm(2), P<0.01). In TLR4-defective mice, application of cuff and lipopolysaccharide resulted in a smaller neointima than in wild-type mice (intimal area, 3859+/-904 microm(2), P=0.02 versus wild type). CONCLUSIONS: A functional TLR4 is expressed in human adventitial fibroblasts and macrophages. Adventitial TLR4 activation augmented neointima formation in a mouse model. These results provide evidence for a link between the immune receptor TLR4 and intimal lesion formation.

Animals↗
PubMed2003

Diabetes-induced oxidative stress and low-grade inflammation in porcine coronary arteries.

BACKGROUND: Multiple pathways contribute to accelerated coronary atherosclerosis in diabetics, including increased oxidative stress and inflammatory burden. Accordingly, the mechanisms of abnormal formation of reactive oxygen species and the changes in inflammatory gene expression were examined in diabetic coronary arteries. METHODS AND RESULTS: In pigs with streptozotocin-induced diabetes, superoxide formation was augmented in coronary media and adventitia because of increased NAD(P)H oxidase activity (3 months) accompanied by upregulated expression of its cytosolic subunit, p22phox. Diabetes-induced oxidative stress resulted in the inflammatory response in the adventitia (increased expression of interleukin-6, tumor necrosis factor-alpha, monocyte chemotactic protein-1, vascular cell adhesion molecule-1 [VCAM-1]) and in the media (VCAM-1). To examine the mechanisms of these changes, studies with isolated coronary fibroblasts were undertaken. Advanced glycation end products (AGEs), rather than glucose itself, upregulated expression of interleukin-6, VCAM-1, and monocyte chemotactic protein-1 mRNAs. These results were paralleled by increased interleukin-6 secretion (P<0.01) and augmented leukocyte adhesion to AGE-stimulated coronary cells (P<0.001). AGEs increased expression of phosphorylated forms of mitogen-activated protein kinases in coronary cells (ERK1/2 and JNK) and resulted in redox-sensitive expression of inflammatory genes that was inhibited by several inhibitors of oxidative pathways [NAD(P)H oxidase inhibitors, N-acetylcysteine, and pyrrolidine dithiocarbamate]. CONCLUSIONS: Diabetes increased NAD(P)H oxidase activity and oxidative stress, producing inflammatory responses in porcine coronary media and adventitia. AGEs activated ERK1/2 and JNK signaling pathways and induced the expression of several inflammatory genes in coronary cells in a redox-sensitive manner. These results suggest the involvement of AGEs in the development of accelerated coronary atherosclerosis in diabetes.

Animals↗
PubMed2000

Direct in vivo evidence demonstrating neointimal migration of adventitial fibroblasts after balloon injury of rat carotid arteries.

BACKGROUND: Clinical and experimental evidence suggest that the adventitia participates in the response to endoluminal vascular injury. The current study used a direct approach to test the hypothesis that, after balloon injury of the rat carotid artery, adventitial fibroblasts migrate in a luminal direction and contribute to neointima formation. METHODS AND RESULTS: Primary syngeneic adventitial fibroblasts were stably transduced with retroviral particles coordinating expression of beta-galactosidase (LacZ) and introduced into the adventitia of right carotid arteries of rats immediately after balloon injury. At defined times after injury and fibroblast implantation, rats were euthanized, and arterial tissue was examined for detection of LacZ mRNA (reverse transcription polymerase chain reaction), DNA (polymerase chain reaction), and in situ enzymatic activity. LacZ expression was detected in the media 5 days postinjury and in both media and neointima at 7, 10, and 14 days postinjury. LacZ was undetectable in injured vessels that had not been seeded with transduced fibroblasts and was restricted to the adventitia in seeded vessels that were not injured. CONCLUSIONS: These observations provide direct demonstration of adventitial fibroblast migration into neointima of arteries after endoluminal injury.

Animals↗