Adventitial Proliferation Research Articles

Early Adventitial Activation and Proliferation in a Mouse Model of Arteriovenous Stenosis: Opportunities for Intervention.

Background: Arteriovenous fistula (AVF) stenosis remains an important cause of AVF maturation failure, for which there are currently no effective therapies. We examined the pattern and phenotype of cellular proliferation at different timepoints in a mouse model characterized by a peri-anastomotic AVF stenosis. Methods: Standard immunohistochemical analyses for cellular proliferation and macrophage infiltration were performed at 2, 7 and 14 d on our validated mouse model of AVF stenosis to study the temporal profile, geographical location and cellular phenotype of proliferating and infiltrating cells in this model. Results: Adventitial proliferation and macrophage infiltration (into the adventitia) began at 2 d, peaked at 7 d and then declined over time. Surprisingly, there was minimal macrophage infiltration or proliferation in the neointimal region at either 7 or 14 d, although endothelial cell proliferation increased rapidly between 2 d and 7 d, and peaked at 14 d. Conclusions: Early and rapid macrophage infiltration and cellular proliferation within the adventitia could play an important role in the downstream pathways of both neointimal hyperplasia and inward or outward remodelling.

P4143IL-6 production in the vascular adventitia and adventitia-media-crosstalk in neointima formation

Abstract Background The aim of this study was to analyze the impact of the adventitial layer on vascular remodeling processes and to define the underlying cellular mechanisms. Methods and results Morphometric analysis of human coronary arteries and of murine femoral arteries at several times following vascular intervention revealed a significant correlation of neointimal and adventitial thickening (R2=0.6845, P<0.001 for human samples; R2=0.6845, P<0.001 for human samples. Immunohistochemical staining for the proliferation marker Ki-67 was performed 7, 14, and 21 days following injury of the murine femoral artery. Formation of a neointimal lesion at 21 days was preceded by high adventitial proliferation rates at 7 and 14 days (85.00±6.041 Ki67+adventitial cells vs. 5.118±0.633 Ki-67+neointimal cells at 7d, P<0.0014; 28.80±5.240 Ki-67+adv. cells vs. 19.40±2.468 Ki-67+neoint. cells at 14d, P<0.006, n=17). Complete removal of the adventitial layer prevented neointima formation, attributing pivotal importance to the adventitial layer (luminal stenosis: 71.73±3.77% vs. 7.44±1.71%, n=5, P<0.0001). Re-transplantation of the aortic adventitia of ubiquitously GFP expressing C57BL/6-Tg (CAG-EGFP)1Osb/Jmice around the medial vascular layer of the femoral artery where the native adventitia has been removed completely restored neointima formation. Importantly, only very view GFP+cells were present in the neointimal layer, indicating that a direct contribution of adventitial cells to the neointimal lesion represents an extremely rare event. To investigate a potential paracrine effect of the activated adventitial layer, we explanted adventitial transplants 14 days following injury and transplantation and incubated the respective samples in serum-free media for 24 hours. BrdU incorporation assays and scratch wound assays revealed significantly increased proliferation and migration rates of human coronary artery SMCs in response to the supernatant of adventitial transplants compared to the supernatant of control samples. Further secretome analyses of the same adventitial supernatants identified predominantly interleukin (IL)-6 to trigger SMC proliferation and migration. Accordingly, serum-free media incubated with adventitial grafts of IL-6−/− mice prevented SMC proliferation and migration. Transplantation of the adventitia of IL-6−/− mice into C57BL/6J wild type mice was not sufficient to trigger neointima formation. Plain old balloon angioplasty, bare metal stent implantation, or drug-eluting stent implantation in swine coronary arteries and analysis for Ki-67+ cell counts supported the hypothesis in the large animal model and a more clinical setting. Conclusion Acute vascular injury is followed by an expansion of cytokine-producing adventitial cells, whose paracrine function and especially whose release of IL-6 is essential for the subsequent induction of the proliferation and migration of local SMC and thus for neointima formation.

Long-term effect of PROLI/NO on cellular proliferation and phenotype after arterial injury

Vascular interventions are associated with high failure rates from restenosis secondary to negative remodeling and neointimal hyperplasia. Periadventitial delivery of nitric oxide (NO) inhibits neointimal hyperplasia, preserving lumen patency. With the development of new localized delivery vehicles, NO-based therapies remain a promising therapeutic avenue for the prevention of restenosis. While the time course of events during neointimal development has been well established, a full characterization of the impact of NO donors on the cells that comprise the arterial wall has not been performed. Thus, the aim of our study was to perform a detailed assessment of proliferation, cellularity, inflammation, and phenotypic cellular modulation in injured arteries treated with the short-lived NO donor, PROLI/NO. PROLI/NO provided durable inhibition of neointimal hyperplasia for 6 months after arterial injury. PROLI/NO inhibited proliferation and cellularity in the media and intima at all of the time points studied. However, PROLI/NO caused an increase in adventitial proliferation at 2 weeks, resulting in increased cellularity at 2 and 8 weeks compared to injury alone. PROLI/NO promoted local protein S-nitrosation and increased local tyrosine nitration, without measurable systemic effects. PROLI/NO predominantly inhibited contractile smooth muscle cells in the intima and media, and had little to no effect on vascular smooth muscle cells or myofibroblasts in the adventitia. Finally, PROLI/NO caused a delayed and decreased leukocyte infiltration response after injury. Our results show that a short-lived NO donor exerts durable effects on proliferation, phenotype modulation, and inflammation that result in long-term inhibition of neointimal hyperplasia.

Proliferation patterns in a pig model of AV fistula stenosis: can we translate biology into novel therapies?

Arteriovenous fistula (AVF) stenosis remains an important cause of AVF maturation failure for which there are currently no effective therapies. To understand the mechanisms involved, we have examined the pattern of cellular proliferation at different time points in a pig model of AVF stenosis. Immunohistochemical analysis of cellular proliferation was performed at 2, 7, 28, and 42days. The distribution of cellular proliferation within the different layers of the vessel wall was also studied. An ANOVA analysis was used to identify differences between the magnitude of cellular proliferation at different time points and within different layers of the vessel wall. Adventitial proliferation occurred at 2days and declined over time. Intimal and medial proliferation peaked at 7days and then decreased over time. There was minimal proliferation in all three layers at the 28- and 42-day time points. An important finding was the presence of active myofibroblast proliferation within "neointimal buds" at the 7-day time point. Results suggest that there could be early adventitial activation, followed by a passage of these cells into the medial and intimal layers. These suggest that the application of perivascular antiproliferative (adventitial) therapies at the time of surgery could potentially reduce AVF maturation failure.

ROLE OF ENDOTOXIN IN SYSTEMIC INFLAMMATION AND DEVELOPMENT OF ATHEROSCLEROSIS

Atherosclerotic vascular disease is an inflammatory disease. Interferon-beta (IFN-β) is an important immune modulator. However, the role of IFN-β in atherosclerotic vascular disease is still not clear. The present study is designed to determine the effects of IFN-β on atherosclerosis, abdominal aortic aneurysm (AAA) formation and proliferative vascular remodeling in apolipoprotein E (apoE) deficient mice. Six-month-old male apoE deficient mice fed a normal chow underwent ligation of the common left carotid artery, and were randomly assigned to receive either vehicle or angiotensin II (Ang II, 1.4 mg/kg daily) via a subcutaneously implanted osmotic infusion pump. The animals were further assigned to groups that were subjected to subcutaneous injection of vehicle or murine IFN-β (10 MIU/kg, daily). Ang II increased atherosclerotic area in the non-ligated carotid artery and aortic arch, induced AAA, and exacerbated ligation-induced adventitial proliferation and neointimal hyperplasia characterized by smooth muscle cell (SMC) proliferation and macrophage infiltration in the ligated carotid artery. Co-treatment with IFN-β, had no effects by itself, significantly attenuated Ang II-accelerated increase in the areas of neointima, adventitia, SMC and macrophage in the ligated carotid artery and suppressed Ang II-exacerbated atherosclerosis, but did not affect Ang II-induced AAA formation. These data indicate that IFN-β can play a prominent anti-atherosclerosis, anti-inflammation, and anti-proliferation role of vasculoprotection.

Interferon-beta attenuates angiotensin II-accelerated atherosclerosis and vascular remodeling in apolipoprotein E deficient mice

Atherosclerotic vascular disease is an inflammatory disease. Interferon-beta (IFN-β) is an important immune modulator. However, the role of IFN-β in atherosclerotic vascular disease is still not clear. The present study is designed to determine the effects of IFN-β on atherosclerosis, abdominal aortic aneurysm (AAA) formation and proliferative vascular remodeling in apolipoprotein E (apoE) deficient mice. Six-month-old male apoE deficient mice fed a normal chow underwent ligation of the common left carotid artery, and were randomly assigned to receive either vehicle or angiotensin II (Ang II, 1.4 mg/kg daily) via a subcutaneously implanted osmotic infusion pump. The animals were further assigned to groups that were subjected to subcutaneous injection of vehicle or murine IFN-β (10 MIU/kg, daily). Ang II increased atherosclerotic area in the non-ligated carotid artery and aortic arch, induced AAA, and exacerbated ligation-induced adventitial proliferation and neointimal hyperplasia characterized by smooth muscle cell (SMC) proliferation and macrophage infiltration in the ligated carotid artery. Co-treatment with IFN-β, had no effects by itself, significantly attenuated Ang II-accelerated increase in the areas of neointima, adventitia, SMC and macrophage in the ligated carotid artery and suppressed Ang II-exacerbated atherosclerosis, but did not affect Ang II-induced AAA formation. These data indicate that IFN-β can play a prominent anti-atherosclerosis, anti-inflammation, and anti-proliferation role of vasculoprotection.

Urokinase-type plasminogen activator deficiency (uPA–KO) prevented carotid artery ligation-induced vascular remodeling in mice

It has been demonstrated that urokinase-type plasminogen activator (uPA) plays an important role in vascular remodeling. This study was designed to determine whether uPA deficiency (KO) affects carotid artery ligation-induced vessel remodeling and the interaction with angiotensin II (Ang II). Ligation of the left common carotid artery in 6-month-old wild-type (C57 black/6J) mice for 4 weeks induced a concentric remodeling with vessel wall thickening, characterized by cell proliferation in neointima, media, and adventitia, and with lumen narrowing without a significant enlargement of overall vessel dimension. Intima lesions were characterized by alpha-actin positive smooth muscle cell (SMC) proliferation in a matrix background. No detectable presence of MAC-3 positive macrophages existed in the vascular wall. The ligation-induced vascular neointimal formation and adventitial proliferation, but not lumen narrowing and media expansion, were completely prevented in age-matched uPA-KO mice. Chronic infusion of Ang II (1.44 mg/kg per day) via a subcutaneously implanted osmotic minipump did not significantly affect the gross morphology of the nonligated carotid artery from both wild-type and uPA-KO mice, but it enhanced ligation-induced vascular remodeling. However, in the presence of Ang II, uPA deficiency had no effects on ligation-induced mophermetric change, but it partially and significantly reduced cell proliferation. These data indicate that uPA may play a critical role in ligation-induced vessel remodeling. Ang II may activate other mechanisms independent of uPA to exacerbate ligation-induced vascular remodeling.

Bone Marrow-Derived Cells Contribute to Pulmonary Vascular Remodeling in Hypoxia-Induced Pulmonary Hypertension

In these days, it was reported that bone marrow (BM) cells might take part in the remodeling of some systemic vascular diseases; however, it remains unknown whether the BM cells were involved in the vascular remodeling of pulmonary arteries and the progression of pulmonary hypertension (PH). The purpose of this study was to investigate whether BM-derived cells contribute to pulmonary vascular remodeling in hypoxia-induced PH. To investigate the role of BM-derived cells, we transplanted the whole BM of enhanced green fluorescent protein (GFP)-transgenic mice to the lethally irradiated syngeneic mice (n = 30). After 8 weeks, chimera mice were exposed to consistent hypoxia using a hypoxic chamber (10% O(2)) for up to 4 or 8 weeks (10 mice per group). After hemodynamics and the ratio of right ventricular (RV) weight to left ventricle (LV) weight, RV/(LV + septum [S]), were measured, histologic and immunofluorescent staining were performed. BM-transplanted mice showed a high chimerism (mean [+/- SEM], 91 +/- 2.3%). RV systolic pressure and the RV/(LV + S) ratio increased significantly with time in PH mice, indicating RV hypertrophy. Marked vascular remodeling including medial hypertrophy and adventitial proliferation was observed in the pulmonary arteries of PH mice. Strikingly, a number of GFP(+) cells were observed at the pulmonary arterial wall, including the adventitia, in hypoxia-induced PH mice, while very few cells were observed in the control mice. Metaspectrometer measurements using confocal laser scanning microscopy confirmed that this green fluorescence was produced by GFP, suggesting that these GFP(+) cells were mobilized from the BM. Most of them expressed alpha-smooth muscle actin, a smooth muscle cell, or myofibroblast phenotype, and contributed to the pulmonary vascular remodeling. A semiquantitative polymerase chain reaction of the GFP gene revealed that the BM-derived GFP-positive cells in the PH group were observed more than eightfold as often compared with the control mice. The BM-derived cells mobilize to the hypertensive pulmonary arteries and contribute to the pulmonary vascular remodeling in hypoxia-induced PH mice.

Neointimal and Not Adventitial Proliferation Plays a Role in Restenosis After Percutaneous Transluminal Coronary Angioplasty in Humans - A Histomorphometric Analysis

Neointimal and Not Adventitial Proliferation Plays a Role in Restenosis After Percutaneous Transluminal Coronary Angioplasty in Humans - A Histomorphometric Analysis

Arterial response to mild balloon injury in the normal rabbit: evidence for low proliferation rate in the adventitia.

The role of constrictive remodeling, spasm and proliferation (particularly in the adventitia) in the genesis of chronic lumen narrowing after balloon injury remains under debate. This study analyzed the time course of these components following mild injury in normal arteries. Iliac injury was induced by balloon overstretch in 32 rabbits, sacrificed at timed intervals from day 3 to 28. Angiographic response to nitrates, morphometric, immunohistochemical and biochemical analysis were performed at each time point. Quantitative angiography showed a decrease in lumen diameter and no change in response to nitrates over time. On morphometric analysis, remodeling was usually constrictive, appeared as early as day 3 and was responsible for 69+/-14% of the histologic lumen area stenosis at day 28. Constrictive remodeling was correlated negatively to intimal hyperplasia (r= 0.51, P< 0.002) and positively to the lumen area stenosis (r= 0.92, P< 0.0001). Macrophages (labeled by anti-RAM 11 antibodies) were very rare at all time points. Immunohistochemistry identified a high rate of proliferating smooth muscle cells in the media (13+/-7%) and intima (49+/-8%) at day 7, which decreased rapidly. Proliferating cells in the adventitia were rare (3+/-2% at day 7). The number of proliferating cells was time-dependent (r= 0.82, P< 0.0001) and related to cyclin A mRNA measured by reverse transcription-polymerase chain reaction (r= 0.84, P< 0.0001). In this model, luminal loss was mainly caused by constrictive remodeling rather than intimal hyperplasia. Constrictive remodeling appeared early and was not time-dependent. Macrophages, spasm and adventitial proliferation did not contribute to this constrictive remodeling.

Neointimal and not adventitial proliferation plays a role in restenosis after percutaneous transluminal coronary angioplasty in humans — a histomorphometric analysis

Neointimal and not adventitial proliferation plays a role in restenosis after percutaneous transluminal coronary angioplasty in humans — a histomorphometric analysis

Endothelial cell recoverage and intimal hyperplasia after endothelium removal with or without smooth muscle cell necrosis in the rabbit carotid artery.

Interventional-injury-induced intimal hyperplasia involves smooth muscle cell proliferation that may be limited by endothelial cell coverage. We hypothesized that endothelial cell coverage modulates intimal hyperplasia. Therefore rabbit carotid arteries were injured with (2-french Fogarty balloon) and without media necrosis (Prolene loop). After termination at 3, 7, 21, or 42 days, endothelial cell coverage was assessed with an antibody to CD31 and cross-sectional intimal hyperplasia area was measured morphometrically. At 21 and 42 days, maximal intimal hyperplasia thickness was measured at a site where endothelium was present and where endothelium was absent. Proliferating cells were identified with an antibody to the nuclear antigen Ki-67. From 3 to 42 days, endothelial cell coverage progressed from the caudal and cranial ends of the lesion towards the center and was slower in balloon- versus loop-injured arteries (p<0.001, Anova). At 21 and 42 days, intimal hyperplasia area was larger after balloon than after loop injury (21 days: 0.20 +/- 0.01 vs. 0.09 +/- 0.04 mm2, p<0.05; 42 days: 0.26 +/- 0.03 vs. 0.08 +/- 0.02 mm2, p<0.01). At 21 days, the intimal hyperplasia was maximal at the center of the lesion and diminished towards the edges in both balloon- and loop-injured arteries. Surprisingly, at 21 and 42 days, maximal intimal hyperplasia thickness was larger in CD31-positive compared to CD31-negative regions (104 +/- 8 vs. 72 +/- 12 micron, p<0.01, paired t test). At 3 and 7 days, more medial proliferation was found after balloon than after loop injury (3 days: 46.2 +/- 8.8 vs. 0.2 +/- 0.1%, p<0.01; 7 days: 18.5 +/- 6.4 vs. 1.0 +/- 0.4%, p<0.01). In the same period, abundant adventitial proliferation was found after balloon injury that was entirely absent after loop injury. We conclude, first, that endothelial cell recoverage proceeded at a lower rate over damaged than over normal media. This retarded endothelial cell recoverage may contribute to enhanced intimal hyperplasia. Second, at a late stage of vascular healing, when intimal hyperplasia had already been formed, reendothelialization seems to have been enhanced over areas with thick intimal hyperplasia. Third, dilation-induced medial damage was accompanied with massive adventitial cell proliferation.

803-3 Influence of Presence or Absence of Medial Necrosis on Endothelial Regeneration and Intimal Hyperplasia in the Rabbit Carotid Artery

Interventional injury induced intimal hyperplasia (IH) involves smooth muscle cell proliferation which may be limited by endothelial cell (ECI regeneration. We hypothesized that EC-regeneration (ECR) modulates IH. To create different ECR's we induced EC removal with 100% media necrosis (2F Fogarty balloon, 5 cm) and without media necrosis (prolene loop, 5 cm) in the rabbit carotid artery. After termination at 3, 7, 21 or 42 days, the artery was divided in segments which were alternately processed for paraffin- and frozen sections. ECR was assessed with an antibody to CD31 and expressed as percentage coverage. Proliferating cells were identified with an antibody to the nuclear antigen Ki-67 and scored as percentage positive cells. The crosssectional IH area (IHA, mm 2 ) was measured morphometrically from elastin stained sections. Wall coverage (%) with CD31 positive cells, IHA (mm 2 ) (mean ± sem, * = p &lt; 0.05, † = P &lt; 0.001, Fogarty balloon (BAL) versus loop): n ECR,3d ECR,7d ECR,21d ECR,42d IHA,7d IHA,21d IHA,42d BAL 7 3 ± 2 57 ± 14 61 ± 5 82 ± 11 0.01 ± 0.01 0.20 ± 0.01 * 0.26 ± 0.03 * loop 7 8 ± 4 81 ± 10 93 ± 4 100 ± 0 0.01 ± 0.01 0.09 ± 0.04 0.08 ± 0.02 From 3–42 days, ECR was enhanced in loop versus balloon injured arteries (P &lt; 0.001, ANOVA). At 3 and 7 days, more medial proliferation was found after balloon than after loop injury (3d: 46.2 ± 8.8% versus 0.2 ± 0.1%, 7d: 18.5 ± 6.4% versus 1.0 ± 0.4%; p &lt; 0.01, ANOVA). In the same period, abundant adventitial proliferation was found after balloon injury which was entirely absent after loop injury. Endothelial cell regeneration is slower over a damaged than over a normal media. This retarded endothelial cell regeneration may contribute to enhanced intimal hyperplasia.

Delayed effects of neutron irradiation on central nervous system microvasculature in the rat.

Pathologic examination of a series of 14 patients with malignant gliomas treated with BNCT showed well demarcated zones of radiation damage characterized by coagulation necrosis. Beam attenuation was correlated with edema, loss of parenchymal elements, demyelination, leukocytosis, and peripheral gliosis. Vascular disturbances consisted of endothelial swelling, medial and adventitial proliferation, fibrin impregnation, frequent thrombosis, and perivascular inflammation. Radiation changes appeared to be acute and delayed. The outcome of the patients in this series was not significantly different from the natural course of the disease, even though two of the patients had no residual tumor detected at the time of autopsy. The intensity of the vascular changes raised a suspicion that boron may have sequestered in vessel walls, resulting in selectively high doses of radiation to these structures (Asbury et al., 1972), or that there may have been high blood concentrations of boron at the time of treatment. The potential limiting effects of a vascular ischemic reaction in Boron Neutron Capture Therapy (BNCT) prompted the following study to investigate the delayed response of microvascular structures in a rat model currently being used for pre-clinical investigations.KeywordsCaudate NucleusBasal LaminaNeutron IrradiationBoron Neutron Capture TherapySpurr ResinThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Proliferative potential of vascular components in human glioblastoma multiforme.

Sixteen patients with glioblastoma multiforme received a 1-h intravenous infusion of 5-bromo-deoxyuridine (BrdU), 150-200 mg/m2 at the start of surgery, to label S-phase cells in tumor tissue. Labeled cells of vascular components and of tumor parenchyma were detected in excised tumor specimens by indirect immunoperoxidase staining using anti-BrdU monoclonal antibodies followed by periodic acid-Schiff staining. The BrdU labeling index (LI, defined as the percentage of labeled cells in relation to the total number of cells scored) was calculated separately for vascular components and tumor parenchyma in each specimen. The BrdU LI of vascular components of glioblastoma multiforme was remarkably higher than that of normal brain (1.1%-8.7% vs. less than 0.05%). The mean BrdU LIs of vascular components and tumor cells in eight primary glioblastomas were 4.5 +/- 0.8% (mean +/- SE) and 9.9 +/- 1.1%, respectively, while the corresponding BrdU LIs in eight recurrent tumors were 2.7 +/- 0.5% and 9.3 +/- 0.7%. The differences in the BrdU LIs of primary and recurrent tumors were not statistically significant, but the BrdU LI of vascular components was consistently much lower than that of tumor cells. BrdU labeling of vascular components was inconsistent and occurred mostly in glomerular-shaped vessels, but only about 20% of them contained labeled cells. These results suggest that unusual vascular proliferation, such as the formation of glomerular-shaped vessels and endothelial or adventitial proliferation, in glioblastoma multiforme may have been programmed to slow down or even to cease at a certain stage, and is not likely to be the result of neoplastic transformation.