Arterial enlargement in response to high flow requires early expression of matrix metalloproteinases to degrade extracellular matrix

This study investigated the effects of high flow and shear stress on the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-2 (TIMP-2) during flow-induced arterial enlargement using a model of arteriovenous fistula (AVF) creation on the carotid artery with the corresponding jugular vein in Japanese white male rabbits. Flow increased 8-fold 7 days after AVF. Endothelial cells (EC) and smooth muscle cells (SMC) proliferated with internal elastic lamina (IEL) degradation in response to high flow and shear stress. Expression of MMP-2 mRNA peaked at 2 days (1700-fold) and maintained high level expression. MMP-9 mRNA gave a 10.8-fold increase within 2 days and decreased later. Their proteins were detected in EC and SMC. Membrane type-1-MMP (MT1-MMP) mRNA increased 121-fold at 3 days and maintained high expression. TGF-beta1 was increased after AVF. Two-peak up-regulation of Egr-1 mRNA was recognized at 1 and 5 days of AVF. These results suggest that high flow and shear stress can mediate EC and SMC to express MMP-2 and MMP-9, which degrade cell basement membranes and IEL to induce arterial enlargement. The disproportional increase in MT1-MMP and TIMP-2 might contribute to MMP-2 activation. Egr-1 and TGF-beta1 might play important roles in this process.     

Intermittent short-duration exposure to low wall shear stress induces intimal thickening in arteries exposed to chronic high shear stress

We sought to determine whether intermittent short-duration exposure to low wall shear stress could induce intimal thickening in arteries chronically exposed to high shear stress. An arteriovenous fistula (AVF) was created between the left common carotid artery and the corresponding external jugular vein in 20 Japanese white male rabbits. After 4 weeks, blood flow was increased 10-fold to 182 +/- 39 ml/min and shear stress was increased to 33.4 +/- 13 dyn/cm(2). The AVF was then occluded for 1 h by finger compression with an 85% reduction in carotid artery blood flow (27 +/- 7 ml/min) and a reduction in wall shear stress to 4.9 +/- 1.7 dyn/cm(2) (P < 0.0001). Release of finger compression restored flow to the AVF and high shear stress to the carotid artery. This procedure was repeated at weekly intervals with a cumulative total of 4 h of low shear stress exposure. Arteries exposed to intermittent low shear stress developed a layer of intimal thickening which consisted of 3-4 layers of smooth muscle cells lined with thin elastic fibers and medial hyperplasia. Control arteries exposed to 8 weeks of continuous high shear had no intimal thickening. Transient exposure to low shear stress upregulated TGF-beta1, MMP-2, -14, and TIMP-2 gene expression while MMP-9 expression was downregulated. We conclude that repeated, intermittent short-duration exposure to low shear stress in the setting of high flow and high shear stress can induce arterial intimal thickening. Short-duration alterations in hemodynamic forces can induce rapid vascular cell message expression, which may effect arterial remodeling. This experiment suggests that a threshold value of 5 dyn/cm(2) may be needed in order to initiate and sustain the intimal thickening response.     

Kinetics of cellular proliferation after arterial injury. II. Inhibition of smooth muscle growth by heparin

Heparin inhibits intimal thickening after arterial injury. Whether this effect is due to inhibition of medial smooth muscle cell (SMC) migration, SMC proliferation in the intima, or synthesis and deposition of connective tissue has not been evident. In this study we have investigated these possibilities in a rat carotid balloon injury model. Heparin (0.3 mg/kg/hour) was administered intravenously by means of osmotic pumps to experimental animals, and controls received lactated Ringer's solution. Smooth muscle proliferation (thymidine index), intimal smooth muscle accumulation, and endothelial regeneration were measured at intervals between 0 and 28 days. Total smooth muscle growth as determined biochemically at 14 days was markedly inhibited by heparin if the pumps were placed 24 hours before or at the time of injury and less so if inserted 48 or 96 hours after injury. SMC thymidine indices were maximal in the media at 4 days and in the intima at 7 days for injured arteries of both heparin-treated and control rats; at each time point SMC proliferation and intimal thickening were less in heparin-treated rats. The volume of connective tissue in the intima was the same in both groups at 28 days. Medial SMC migration into the intima was diminished by heparin treatment, but endothelial regeneration was not affected. These results support the hypothesis that heparin is a specific inhibitor of SMC migration and proliferation and is most effective if started before SMC enter S-phase.     

Expression of matrix Gla protein and osteonectin mRNA by human aortic smooth muscle cells.

BACKGROUND: Recent data indicate that matrix proteins such as matrix Gla protein (MGP) and osteonectin (ON) influence not only mineralization of vasculature but smooth muscle cell (SMC) differentiation. METHODS: We examined whether MGP and ON are expressed by human aortic SMCs in vivo using Northern blotting, in situ hybridization and immunohistochemistry. RESULTS: MGP and ON mRNAs were strongly expressed in the aorta without atherosclerosis from newborn and four young subjects up to 10 years old. In the aorta from 15 adult cases, MGP and ON mRNAs were decreased as atherosclerosis developed. We determined cell type and distribution of the MGP- and ON mRNA-expressing cells by in situ hybridization and immunohistochemistry. In the aorta obtained from newborn and young subjects, SMCs in the media and thin intima expressed MGP mRNA and, to a lesser extent, ON mRNA. In the adult aorta with fibrous thickening, MGP mRNA was expressed by intimal SMCs and subpopulation of medial SMCs. Osteonectin mRNA was expressed mainly by intimal SMCs and few medial SMCs. Double immunohistochemical staining revealed that both MGP- and ON protein-expressing cells were positive for anti-alpha-smooth muscle actin antibody, aortic SMCs. CONCLUSIONS: These results suggested that MGP and ON expression by aortic SMCs might be regulated by the degree of atherosclerosis and SMC differentiation in human aorta.     

Phenotypes of smooth muscle cells in carotid arteries in Takayasu's disease

The phenotype of smooth-muscle cells (SMC) of carotid arteries in health and Takayasu's disease were studied using immunofluorescence. In contrast to unaffected carotid artery, medial SMC were represented by heterogenic cell population. Many SMC in this disease expressed fibronectin with ED-A sequence, contained cytokeratin 8 and were deprived of h-caldesmon. Similar phenotypic characteristics are typical for SMC of embryonal phenotype. SMC with this phenotype are observed in the thickened intima of the carotid arteries in Takayasu's disease. It is supposed that SMC precursors in arterial media are activated in this disease, migrate into subendothelial layer and are responsible for the thickening of the intima.     

Phenotypic features of smooth muscle cells during the evolution of experimental carotid artery intimal thickening. Biochemical and morphologic studies

Balloon catheter denudation of rat carotid artery that results in significant medial damage is followed by marked intimal smooth muscle cell (SMC) proliferation associated with limited endothelial regrowth. In this report we demonstrate that: (a) SMC of the carotid media, preceding their intimal proliferation, develop a cytoskeletal profile and morphology consistent with a de-differentiated SMC phenotype; and (b) both medial and intimal SMC subsequently revert to a cytoskeletal profile and morphology reflecting incomplete but significant re-differentiation toward normal SMC phenotype. Specifically, early after balloon injury, SMC of the media and those that have migrated into the intima contain decreased amounts of actin, desmin, and tropomyosin and increased amounts of vimentin; moreover, beta-actin becomes the dominant actin isoform, whereas alpha-actin decreases as compared with that found in normal medial SMC. Late after balloon injury, actin is still less abundant, however, desmin, tropomyosin, and vimentin return toward normal values and both medial and intimal SMC again show a predominance of alpha-actin, although the endothelium does not regenerate over the central surface of intimal thickening in this model. The SMC surface to volume ratio significantly decreases early after balloon injury, whereas it is not significantly different late after balloon injury as compared with that of SMC of the normal carotid media. We demonstrate, furthermore that: (c) adjacent luminal SMC are interconnected by gap junctions and develop focal tight junctions, a feature not reported previously to occur in smooth muscle; these cells however do not form any well defined membrane specialization with the leading edge of endothelium, supporting the view that presence of modified SMC on the luminal surface of chronically denuded vessels is not responsible for the cessation of endothelial regrowth.     

A nonantibiotic chemically modified tetracycline (CMT-3) inhibits intimal thickening

Recent research has shown that the tetracycline antibiotics are pluripotent drugs that inhibit the activity of matrix metalloproteinases (MMPs) and affect many cellular functions including proliferation, migration, and matrix remodeling. We have shown that doxycycline inhibits MMP activity and intimal thickening after injury of the rat carotid artery, however we do not know whether these effects are because of the antibiotic, anti-MMP, or other actions of doxycycline. Recently, chemically modified tetracyclines have been synthesized that lack antibiotic activity but retain anti-MMP activity (CMT-3), or lack both antibiotic and anti-MMP activity (CMT-5). In the current study we have assessed the effects of treatment with CMT-3 or CMT-5 on intimal thickening after balloon catheter injury of the rat carotid artery. Rats were treated by oral gavage with 15 mg/kg/day CMT-3 or CMT-5. CMT-3 significantly reduced smooth muscle cell (SMC) proliferation in both the medial and intimal layers of the injured rat carotid artery compared to CMT-5. Furthermore, CMT-3 inhibited SMC migration from the media to the intima by 86% at 4 days after injury. CMT-3 also decreased MMP-2 activity. Finally, we found that CMT-3 treatment resulted in a significant reduction in intimal cross-sectional area from 0.23 +/- 0.01 mm(2) in the CMT-5 control group to 0.19 +/- 0.01 mm(2). There was also a reduction in elastin and collagen accumulation within the intima. We conclude that CMT-3 attenuated intimal thickening after arterial injury by inhibiting SMC proliferation, migration and MMP activity, and accumulation of extracellular matrix. The inhibitory effects of CMT-3 were independent of the antibiotic properties, but were dependent on the anti-MMP activity of the tetracycline family.     

CD8+ T lymphocytes mediate destruction of the vascular media in a model of chronic rejection

Allograft arteriosclerosis is an important characteristic of chronic graft rejection. In allograft arteriosclerosis there is a striking loss of medial smooth muscle cells (SMCs) before the development of a concentric intimal proliferative response. In this study we evaluated the role of CD8+ T lymphocytes in this medial SMC loss. Brown Norway aortic segments were transplanted into Lewis animals for 60 days (long allo-exposure) or 20 days (short allo-exposure). After 20 days allogeneic exposure aortic segments were transplanted back into syngeneic (Brown Norway) animals for 40 days. Experimental animals were treated with mAb to CD8. Apoptosis was measured by terminal dUTP nick-end labeling at 20 days and morphometry analyzed at 60 days to evaluate medial and intimal changes. Anti-CD8 treatment significantly lowered CD8+ T cell counts in peripheral blood, reduced medial SMC apoptosis at 20 days, and increased medial SMC counts at 60 days. Both short- and long-allogeneic exposure groups confirmed these findings and demonstrated that medial SMC loss is proportional to the length of allogeneic exposure. Antibody depletion of CD8+ T cells results in reduced medial SMC apoptosis and better medial SMC preservation. This supports the hypothesis that medial SMC loss occurs by apoptotic death and is driven by CD8+ T lymphocytes.     

Smooth muscle cells of the coronary arterial tunica media express tumor necrosis factor-alpha and proliferate during acute rejection of rabbit cardiac allografts.

Graft coronary arteriosclerosis (GCA) frequently limits the long-term success of cardiac transplantation. The pathogenic mechanisms of and stimuli that provoke GCA remain uncertain. Whatever the initiating factors, deranged control of smooth muscle cells (SMC) proliferation likely contributes to the intimal hyperplasia that produces obstructive lesions. To identify mediators that may contribute to ongoing modulation of SMC functions during acute rejection and to explore the mechanisms of the pathogenesis of graft coronary arteriosclerosis, we studied the kinetics of proliferation and the expression of tumor necrosis factor-alpha (TNF-alpha), a proinflammatory and SMC growth-promoting cytokine, in coronary arterial SMCs in rabbit hearts transplanted heterotopically without immunosuppression. Hearts were harvested at 2 (n = 5), 5 (n = 5), and 8.2 +/- 0.4 (mean +/- SD, n = 5) days after transplantation, just before graft failure as judged clinically. SMC proliferation was assessed by continuous bromodeoxyuridine labeling (BrdU 10 mg/kg/d. s.q.). Whole heart cross sections were stained immunohistochemically with monoclonal antibodies that recognize TNF-alpha, BrdU, and SMCs (muscle alpha-actin). Major epicardial coronary arteries (five to nine profiles in each animal) were evaluated. Histological rejection grades by the International Society for Heart and Lung Transplantation scale at 2, 5, and 10 days were 1.6 +/- 0.9, 2.8 +/- 1.1, and 4.0 +/- 0.0, respectively. Medial SMCs in normal hearts and 2 days after transplant expressed little or no TNF-alpha and displayed negligible BrdU incorporation. At 5 days after transplantation, some medial SMCs stained for TNF-alpha and had a low BrdU labeling index (0.5 +/- 0.8%). At 8.2 days after transplant, almost all medial SMCs expressed TNF-alpha intensely and had a high labeling index (29.8 +/- 8.0%). These results demonstrate that acute rejection activates medial SMCs in coronary arteries to express TNF-alpha and that SMC-derived TNF-alpha may contribute to medial SMC proliferation in coronary arteries during acute rejection. This finding of early medial SMC replication suggests a novel and heretofore unsuspected mechanism of intimal expansion consequent to the allogeneic state. These results furnish additional insight into the possible mechanisms that link acute rejection with graft coronary arteriosclerosis. Furthermore, the close association of TNF-alpha expression with SMC replication provides not only a novel marker of SMC activation but also a potential new therapeutic target for the prevention of graft coronary disease.     

Endothelial regeneration. IX. Arterial injury followed by rapid endothelial repair induces smooth-muscle-cell proliferation but not intimal thickening.

Rat thoracic aortas were denuded of endothelial cells with a fine nylon filament, which removed a row of endothelium approximately 100 mu in width and caused platelets to adhere to the exposed subendothelium. A further group of rats was subjected to a sham operation where only the abdominal aorta was injured, and another group of rats was used as controls. Each animal was continuously labeled with 3H-thymidine (6.7 Ci/mM, 10 microCi/hr) for 7 days, at which time they were killed and the aortas perfusion-fixed with aldehydes. Sections of each aorta were processed for autoradiography. The thoracic aortas from animals subjected to the nylon filament injury showed no intimal thickening, but there was a significant increase in the thymidine index of the medial smooth muscle cells (SMCs) as compared with aortas from sham operation or control animals (1.02% +/- 0.44% versus 0.19% +/- 0.13% versus 0.15% +/- 0.12%). This experiment showed that continuous administration of 3H-thymidine permitted the detection of a small but significant increase in replication rates that could not be detected by the standard bolus administration of 3H-thymidine, and that selected loss of endothelium with minimal trauma to the vessel wall caused SMC proliferation without intimal thickening. These findings suggest that platelets may indeed provide SMC proliferation, but that migration of these cells into the intima may be controlled by different factor(s).     

Changes in the Organization of the Smooth Muscle Cells in Rat Vein Grafts

Mechanical tensile stress in vein grafts increases suddenly under the influence of arterial blood pressure. In this study, we examined the influence of increased tensile stress on the organization of the smooth muscle cells (SMCs) in the neointima and media of the rat vein grafts. An autogenous jugular vein was grafted into the abdominal aorta of the rat, and changes in the organization of the vein graft SMCs were studied by observing the distribution of SMC actin filaments and nuclei at 3 min and 1, 5, 10, and 30 days after surgery. In a normal jugular vein, the average wall circumferential tensile stress was ~ 3 kPa at an internal pressure of 3 mm Hg. The SMCs, that contained long, slender actin filamentous bundles, were oriented mainly in the circumferential direction of the vessel, and constituted a 2- to 3-cell-thick medial layer underneath the endothelium. In a vein graft, the wall circumferential tensile stress suddenly increased by ~ 140 times compared with the control level. In response to this suddenly increased stress, the SMC layer was stretched into a structure with scattered pores and disrupted SMC actin filamentous bundles within 3 min. This initial change was followed by a rapid reduction in the density of the SMC nuclei and actin filaments within 1 day and progressive SMC proliferation, that was associated with medial thickening and a change in the SMC orientation from 5 to 30 days. Further studies showed that a local inflation of normal jugular veins to 120 mm Hg for 3 min induced a similar change as found in the vein grafts, whereas the organization of the SMCs was not significantly changed in vein-vein grafts, that did not experience a change in tensile stress. These results suggested that increased tensile stress contributed to the initial damage of the SMCs and played a role in the regulation of medial SMC remodeling in vein grafts. © 1998 Biomedical Engineering Society. PAC98: 8722-q, 8745-k     

Hemodynamic forces regulate mural macrophage infiltration in experimental aortic aneurysms

Blood flow (BF) and wall shear stress (WSS) influence reactive oxygen species production and oxidative stress in abdominal aortic aneurysm (AAA) disease. To gain further insight into the mechanisms of hemodynamic influences on AAA inflammation, we examined aneurysm macrophage density, chemotaxis and survival under varying aortic flow conditions. Rat AAAs were created via porcine pancreatic elastase (PPE) infusion. In selected cohorts, AAA flow was increased via left common femoral arteriovenous fistula (AVF) creation (HF-AAA) or decreased by left common iliac ligation (LF-AAA). WSS was highest in HF-AAA (10.4 +/- 2.3 dyn/cm(2) vs. 2.4 +/- 0.4 and 0.5 +/- 0.2 for NF- and LF-AAA, respectively, P < 0.001) 7 days after PPE infusion, with reduced medial macrophage density and increased apoptosis. Adventitial macrophage density was not significantly influenced by flow. Monocyte chemoattractant protein-1 (MCP-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression correlated with observed macrophage densities in the media and adventitia. Luminal flow conditions regulate AAA inflammation in part via influences on medial macrophage density. Hemodynamic forces may modulate AAA inflammation and diameter enlargement via direct regulation of intimal macrophage adhesion, transmural migration or survival.     

Apoptosis, cell proliferation and modulation of cyclin-dependent kinase inhibitor p21cip1 in vascular remodelling during vein arterialization in the rat

Neo-intima development and atherosclerosis limit long-term vein graft use for revascularization of ischaemic tissues. Using a rat model, which is technically less challenging than smaller rodents, we provide evidence that the temporal morphological, cellular, and key molecular events during vein arterialization resemble the human vein graft adaptation. Right jugular vein was surgically connected to carotid artery and observed up to 90 days. Morphometry demonstrated gradual thickening of the medial layer and important formation of neo-intima with deposition of smooth muscle cells (SMC) in the subendothelial layer from day 7 onwards. Transmission electron microscopy showed that SMCs switch from the contractile to synthetic phenotype on day 3 and new elastic lamellae formation occurs from day 7 onwards. Apoptosis markedly increased on day 1, while α-actin immunostaining for SMC almost disappeared by day 3. On day 7, cell proliferation reached the highest level and cellular density gradually increased until day 90. The relative magnitude of cellular changes was higher in the intima vs . the media layer (100 vs . 2 times respectively). Cyclin-dependent kinase inhibitors (CDKIs) p27^Kip1 and p16^INKA remained unchanged, whereas p21^Cip1 was gradually downregulated, reaching the lowest levels by day 7 until day 90. Taken together, these data indicate for the first time that p21^Cip1 is the main CDKI protein modulated during the arterialization process the rat model of vein arterialization that may be useful to identify and validate new targets and interventions to improve the long-term patency of vein grafts.     

Type VIII collagen stimulates smooth muscle cell migration and matrix metalloproteinase synthesis after arterial injury

Type VIII collagen is a matrix protein expressed in a number of tissues undergoing active remodeling, including injured arteries during neointimal formation and in human atherosclerotic plaques; however, very little is known about its function. We have investigated whether the type VIII collagen stimulates smooth muscle cell (SMC) migration and invasion by binding to integrin receptors and up-regulating matrix metalloproteinase (MMP) production. SMCs attached to plates coated with type VIII collagen in a dose-dependent manner, with maximal attachment occurring with coating solutions containing 25 microgram/ml collagen. Type VIII collagen at 100 microgram/ml stimulated an 83-fold increase in the migration of SMCs in a chemotaxis chamber. Antibodies against beta1 integrin receptors prevented attachment and migration of SMCs. Antibodies against alpha1 or alpha2 integrins reduced attachment of SMCs to type VIII collagen by 29% and 77%, respectively. We found that SMCs grown from the rat neointima, but not medial SMCs, increased their production of MMP-2 and -9 on adherence to type VIII collagen. This suggests that there is an important difference in phenotype between intimal and medial SMCs and that intimal SMCs have distinct matrix-dependent signaling mechanisms. Our findings suggest that type VIII collagen deposited in vascular lesions functions to promote SMC attachment and chemotaxis, and signals through integrin receptors to stimulate MMP synthesis, all of which are important mechanisms used in cell migration and invasion.     

低分子量肝素抑制血管成形术后平滑肌细胞增生及再狭窄形成

观察血管成形术后平滑肌细胞增生及再狭窄形成过程，探讨低分子量肝素的抑制作用。     

Apoptosis participates in cellularity regulation during rat aortic intimal thickening.

Intimal thickening induced after endothelial denudation of rat aorta is though to be due to migration and proliferation of smooth muscle cells (SMC). When the reendothelialization is achieved, intimal thickening shows an important decrease in cellularity. Using in situ end labeling of fragmented DNA and electron microscopy, we show that this remodeling is accompanied by apoptosis of SMC. The number of apoptotic SMC becomes important 15 days after endothelial injury and reaches a maximum at 20 days; at 45 days the intimal thickening is reendothelialized and no more apoptotic SMC are detected. Apoptotic SMC show nuclear and cytoplasmic condensation as well as cytoplasmic vacuolization. Our results indicate that apoptosis is an important mechanism in the regulation of intimal thickening evolution.     

Expression of thrombomodulin in human aortic smooth muscle cells with special reference to atherosclerotic lesion types and age differences

Expression of thrombomodulin (TM) in atherosclerotic lesions of the human aorta (8 cases of diffuse intimal thickening, 4 fatty streaks, 11 atheromatous plaques, and 5 fibrous plaques) as well as in undiseased aortas of 5 infants obtained at autopsy was studied immunohistochemically using a novel polyclonal antibody against human TM. TM was expressed in intimal smooth muscle cells (SMC) besides endothelial cells and foamy macrophages in almost all patients (26/28). In addition, medial SMC in adult cases over 27 years of age expressed TM. In young adults with diffuse intimal thickening under 26 years of age, medial SMC showed no TM expression whereas intimal SMC did show it. Both intimal and medial SMC in infants showed no TM expression. An immunofluorescence method showed TM expression in cultured adult human SMC. These findings indicate that TM expression in SMC may depend on patient age as well as lesion type of atherosclerosis.     

Enhanced Intimal Proliferation upon Injury to Pre-Existing Neointima and Resistance of Neointimal Cells to Cell Death

Recent evidence supports a role for cell death and inflammation as etiologic factors in neointimal formation and restenosis after angioplasty. This study was undertaken to examine the pattern and intensity of the proliferative response, cell death, and activation of inflammatory, endothelial and smooth muscle cells (SMC) in a model of intimal reinjury. Two ballooning injuries were performed to rat aorta, the second one 14 days after the first injury. Our results demonstrate that ballooning injury to pre-existing neointima differs clearly from an injury to a normal aorta. First, ballooning injury to pre-existing neointima doubled the proliferative response of SMC and intimal thickening, but proliferation of SMC occurred only in the intima, and did not extend into the media. Second, within four hours after the first injury, the number of TUNEL-positive SMC in the media increased from 3% to 23%, but no such increase was found in the pre-existing neointima after the second injury. Third, the prompt proliferative response of intimal SMC after the second injury was linked with a significant increase in endothelial P-selectin and neointimal VCAM-1 immunoreactivity, compared to the first injury at corresponding time points, followed by high numbers of activated ED3+ macrophages and CD4+ T cells in the developing neointima. A balance in injury-induced cell death and proliferation obviously maintains stable cell numbers observed in the media, whereas in the neointima, the resistance of SMC to injury-induced cell death may contribute to a rapid lesion formation in restenosis.     

Rabbit ductus arteriosus during development: Anatomical structure and smooth muscle cell composition

The anatomical structure as well as the smooth muscle cell (SMC¹) composition of the ductus arteriosus (DA) were studied in rabbits ranging in age from 29 days of gestation to 20 days after birth. Computerassisted, three-dimensional reconstructions of hematoxylin-eosin stained serial cryosections from ductus arteriosus-aorta (DA-AO) junctures revealed that DA in animals near term is separated from the aorta by a “septumlike” structure that is continuous with the aortic wall. Two days after birth, obliteration of DA is almost complete, and a small “pocketlike” cavity appears in the pre-existing site in which DA merged into the aorta. This small cavity in the aortic arch was still evident in the large majority of animals examined even 20 days after birth, as also demonstrated by scanning electron microscopy. At this time period DA consisted of a central, fibrotic region surrounded by several layers of SMC (the ligamentum arteriosum, LA) and ended within the aortic media just above the small cavity, forming a round “scar.” Vascular SMC composition of DA during closure was examined by means of indirect and double immunofluorescence procedures, using a panel of monoclonal antibodies against some cytoskeletal and cytocontractile proteins (vimentin, desmin, smooth muscle (SM), and nonmuscle (NM) myosinisoforms). “Intimal cushions” were particularly evident from 5 hr after birth and were found to be desmin-negative, homogenously reactive for vimentin and NM myosin, and heterogeneously stained with anti-SM myosin antibody. In SMC subjacent to the “intimal cushions,” distribution of vimentin and SM myosin was homogeneous, whereas the one of desmin and NM myosin content was heterogeneous. The cytoskeletal and cytocontractile protein content displayed by SMC during the closure of DA is similar to that of “intimal thickening” found in some pathological conditions of the arterial wall in adult rabbits. Completation of DA closure (day 2) was accompanied by the disappearance of cellular heterogeneity in myosin isoform distribution in both the “intimal cushions” and the underlying media. These results give new insights into: (1) the structure of DA-AO juncture, which can be relevant to the physiology of blood circulation in the fetus, and (2) the phenotypic similarity of vascular SMC populations involved in the formation of “intimal cushions” and “intimal thickening”.     

Factors controlling smooth-muscle cell proliferation.

Injury to the arterial wall normally elicits a rapid and significant increase in smooth-muscle cell (SMC) replication with the subsequent development of intimal lesions. A variety of factors have been proposed to control SMC replication, but recent work has highlighted the role of basic fibroblast growth factor (bFGF) and platelet-derived growth factor in this process. In the carotid artery of the uninjured rat, we have shown that the SMCs express mRNA for bFGF and that bFGF can be readily extracted from these arteries. Following mechanical injury to the artery, ie, after balloon injury, we suggested that bFGF is released from damaged cells and then stimulates adjacent SMCs. In support of this concept, the infusion of a blocking antibody to bFGF was found to significantly inhibit the early SMC replication induced by use of a balloon catheter. The addition of the antibody at the time of injury, however, did not inhibit the development of intimal lesions. In contrast, studies by us and other investigators have shown that platelet-derived growth factor is not directly important for SMC replication after balloon injury, but that it plays a key role in stimulating the migration of SMCs into the intima. Intimal SMC replication was not inhibited with antibodies to either bFGF or platelet-derived growth factor. Therefore, while significant inroads have been made in understanding the initial events, we still do not fully understand all the processes involved in the proliferation of arterial intimal lesions.