Ascorbate affects proliferation of guinea-pig vascular smooth muscle cells by direct and extracellular matrix-mediated effects

Proliferation of smooth muscle cells and deposition of extracellular matrix proteins are important events in the formation of atherosclerotic plaques. We have investigated the direct and matrix-mediated effects of ascorbate on the proliferation rate of vascular smooth muscle cells (VSMC) isolated from the guinea-pig aorta. In the presence of ascorbate, cells showed a bi-phasic growth pattern. At 125 microM ascorbate, -3H--thymidine incorporation was stimulated 25%. However, higher concentrations of ascorbate gradually decreased cell-incorporated radioactivity up to 50% at 2 mM ascorbate. These effects of ascorbate on DNA synthesis in VSMC were paralleled by the changes in cell number and were not due to ascorbate cytotoxicity. Alpha-tocopherol (0.1 mM), individually and in combinations with 1 mm ascorbate, also inhibited DNA synthesis in VSMC. Ascorbate also influenced proliferation of smooth muscle cells through matrix-mediated effect. New VSMC culture plated on extracellular matrices deposited by smooth muscle cells in the presence of 0.1-1 mM ascorbate had up to 50% lower proliferation rate than on matrices from ascorbate-deficient cells, as assessed by [3H]-thymidine incorporation. This effect was independent from alpha-tocopherol and specific inhibitors of collagen synthesis: L-azetidine-2-carboxylic acid and pyridine-2,4-dicarboxylic acid. An ascorbate-dependent matrix effect was specific for smooth muscle cells grown on VSMC and human skin fibroblast-originated matrices, but not for human vascular endothelial cells. The possible involvement of ascorbate in the regulation of smooth muscle cells proliferation by its antioxidant/pro-oxidant effects and regulation of extracellular matrix composition are discussed.     

Transitions in cell organization and in expression of contractile and extracellular matrix proteins during development of chicken aortic smooth muscle: evidence for a complex spatial and temporal differentiation program

Whereas the understanding of the mechanisms underlying skeletal and cardiac muscle development has been increased dramatically in recent years, the understanding of smooth muscle development is still in its infancy. This paper summarizes studies on the ontogeny of chicken smooth muscle cells in the wall of the aorta and aortic arch-derived arteries. Employing immunocytochemistry with antibodies against smooth muscle contractile and extracellular matrix proteins we trace smooth muscle cell patterning from early development throughout adulthood. Comparing late stage embryos to young and adult chickens we demonstrate, for all the stages analyzed, that the cells in the media of aortic arch-derived arteries and of the thoracic aorta are organized in alternating lamellae. The lamellar cells, but not the interlamellar cells, express smooth muscle specific contractile proteins and are surrounded by basement membrane proteins. This smooth muscle cell organization of lamellar and interlamellar cells is fully acquired by embryonic day 11 (ED 11). We further show that, during earlier stages of embryogenesis (ED3 through ED7), cells expressing smooth muscle proteins appear only in the peri-endothelial region of the aortic and aortic arch wall and are organized as a narrow band of cells that does not demonstrate the lamellar-interlamellar pattern. On ED9, infrequent cells organized in lamellar-interlamellar organization can be detected and their frequency increases by ED10. In addition to changes in cell organization, we show that there is a characteristic sequence of contractile and extracellular matrix protein expression during development of the aortic wall. At ED3 the peri-endothelial band of differentiated smooth muscle cells is already positive for smooth muscle alpha actin (alphaSM-actin) and fibronectin. By the next embryonic day the peri-endothelial cell layer is also positive for smooth muscle myosin light chain kinase (SM-MLCK). Subsequently, by ED5 this peri-endothelial band of differentiated smooth muscle cells is positive for alphaSM-actin, SM-MLCK, SM-calponin, fibronectin, and collagen type IV. However, laminin and desmin (characteristic basement membrane and contractile proteins of smooth muscle) are first seen only at the onset of the lamellar-interlamellar cell organization (ED9 to ED10). We conclude that the development of chicken aortic smooth muscle involves transitions in cell organization and in expression of smooth muscle proteins until the adult-like phenotype is achieved by mid-embryogenesis. This detailed analysis of the ontogeny of chick aortic smooth muscle should provide a sound basis for future studies on the regulatory mechanisms underlying vascular smooth muscle development.     

Phenotypic modulation of smooth muscle cells during formation of neointimal thickenings following vascular injury

Smooth muscle cells build up the media of mammalian arteries and constitute one of the principal cell types in atherosclerotic and restenotic lesions. Accordingly, they show a high degree of plasticity and are able to shift from a differentiated, contractile phenotype to a less differentiated, synthetic phenotype, and then back again. This modulation occurs as a response to vascular injury and includes a prominent structural reorganization with loss of myofilaments and formation of an extensive endoplasmic reticulum and a large Golgi complex. At the same time, the expression of cytoskeletal proteins and other gene products is altered. As a result, the cells lose their contractility and become able to migrate from the media to the intima, proliferate, and secrete extracellular matrix components, thereby contributing to the formation of intimal thickenings. The mechanisms behind this change in morphology and function of the smooth muscle cells are still incompletely understood. A crucial role has been ascribed to basement membrane proteins such as laminin and collagen type IV and adhesive proteins such as fibronectin. A significant role is also played by mitogenic proteins such as platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF). An improved knowledge of the regulation of smooth muscle differentiated properties represents an important part in the search for new methods of prevention and treatment of vascular disease.     

A review of the actions and control of intracellular pH in vascular smooth muscle

OBJECTIVE: This review is an account of the physiological issues involved in the effects of pH on vascular smooth muscle tone. The following criteria were considered when reviewing the literature: (i) the type of smooth muscle, i.e. either tonic or phasic, (ii) the source of the smooth muscle i.e. pulmonary, systemic, large artery, resistance artery, vein or cell line, (iii) the effects of changing intracellular or extracellular pH alone, (iv) the acute or chronic effects of altered pH (v) the influence of extracellular pH on intracellular pH and (vi) the influence of altered intracellular pH on basal or agonist induced tone. Studies of the effects of pH on the individual intracellular components of vascular tone, specifically sarcoplasmic reticulum and contractile proteins function are considered. Finally, the pH sensitivity of molecular components that contribute to smooth muscle cell tone are reviewed. CONCLUSIONS: There appear to be distinct differences in the response of large arteries and resistance arteries to altered intracellular pH which may be based on the different properties of the smooth muscle within the wall of each blood vessel. Similarly, systemic and pulmonary vessels may respond differently, but no systematic study exists to allow a more definitive conclusion. Factors controlling intracellular pH such as intracellular buffering power and sarcolemmal pH regulating mechanisms may differ across the vascular bed and may contribute to some of the differences observed in response to altered extracellular pH. Finally, few studies have examined the pH sensitivity the intracellular processes involved in basal tone and pharmaco-mechanical coupling in vascular smooth muscle. More information concerning these latter aspects of smooth muscle function is required to progress the understanding of the modulator action on pH on vascular tone.     

Trophoblast cell-mediated modifications to uterine spiral arteries during early gestation in the macaque

A specialized subset of invasive embryonic cytotrophoblast cells gains access to maternal uterine arteries early in the gestation of higher primates. These cells continue to migrate extensively within the lumina of spiral arteries, converting them to the highly modified uteroplacental arteries of pregnancy. Although trophoblast cell-mediated modifications are considered critical to the progress of normal pregnancy, few studies have addressed the cellular interactions between maternal arteries and embryonic cells in situ. Macaque placentas and endometrial tissues were collected from 12 animals from day 14 of gestation (blastocyst implantation begins on day 9) to day 49. Standard indirect immunoperoxidase methods were used to identify matrix metalloproteinases (MMP-1, MMP-3, MMP-9), cathepsin B, cathepsin D, platelet-endothelial cell adhesion molecule, cytokeratins, smooth muscle actin, CD68, and factor VIII-related antigen. Cytotrophoblast cells were located deep within spiral arteries in each of the specimens examined. In some examples tightly packed clusters of cytotrophoblast occluded the lumina of invaded arteries. Initial penetration of arterial tunica intima was revealed by discontinuities in the staining pattern for factor VIII and cytotrophoblast intrusion was indicated by cytokeratin staining of the trophoblast cells. Continued cytotrophoblast intrusion into the tunica media was apparent by gaps in the smooth muscle. MMP-1, MMP-3, and MMP-9 were localized within intraluminal and intramural cytotrophoblast. By contrast, neither cathepsin B nor cathepsin D were present, although both were seen in uterine macrophages and stromal cells. Upon reaching the surrounding uterine stroma the cytotrophoblast cells ceased migration. As cytotrophoblast accumulated in the arterial wall the vascular lumen expanded. Evidence of cell death was rarely encountered in associated maternal or embryonic tissues. We conclude that intra-arterial cytotrophoblast cells express several proteinases with substrate specificities sufficient to permit independent remodeling of the extracellular matrix comprising uterine artery walls. The remodeling of the arteries, which involves extensive displacement of maternal endothelium and smooth muscle, in addition to degradation and synthesis of extracellular matrix, is accomplished with little evidence of cell death or loss of the integrity of the arteries. This process provides an interesting example of cooperation between different types of interacting tissues from genetically distinct individuals.     

Expression and function of a recombinant PDGF B gene in porcine arteries

Platelet-derived growth factor (PDGF) B is a mitogen and chemoattractant for smooth muscle cells in vitro, and expression of a recombinant PDGF B gene in porcine arteries stimulates intimal thickening. To define the mechanisms by which PDGF B gene expression induces intimal thickening in vivo, we examined its effects on smooth muscle cell proliferation and migration, extracellular matrix synthesis, and inflammatory cell infiltration in intimal lesions of pig arteries after direct gene transfer of a recombinant PDGF B gene. PDGF B gene expression was associated with rapid formation of an intima, including 3- to 10-fold increases in intimal thickness and intima-to-media area ratio 4 to 21 days after gene transfer compared with control transfected arteries. Intimal smooth muscle cell proliferation was detected at 2 days, peaked at 7 days (P < .01), and declined by 14 days, although the total number of intimal nuclei progressively increased to 21 days (P < .01). Calculations of expected-to-observed ratios of intimal cells, based on BrdC proliferation indexes, demonstrated that the increases in intimal cell number on days 2 through 7 could not be accounted for by proliferation alone, suggesting that recombinant PDGF BB acts to stimulate cell proliferation and migration of smooth muscle cells into the intima. Extracellular matrix deposition and procollagen synthesis were observed after 7 days (P < .01) and were associated with a decline in cell density in the intima, suggesting that extracellular matrix synthesis may contribute to progressive intimal thickening in response to PDGF B gene expression. There was minimal accumulation of inflammatory cells, including macrophages and CD3(+) lymphocytes, in transfected arteries. These data suggest that PDGF B gene expression promotes intimal expansion by both proliferation and migration of smooth muscle cells followed by synthesis of extracellular matrix and therefore acts through several mechanisms to play a role in the pathogenesis of intimal lesions in vivo.     

Vascular endothelial cells and renin-angiotensin system

The vascular renin-angiotensin system (RAS) is regulated independently from circulating RAS and plays a role in the local regulation of vascular tone, the modulation of sympathetic activity and vascular remodeling. Endothelial cells are a major source of angiotensin converting enzyme (ACE), which produces angiotensin II and degrades bradykinin, in normal arteries. Mechanical stress such as transmural pressure, stretch stress and shear stress appear to contribute to the regulation of endothelial ACE activity. In contrast, vessels with intimal proliferation such as atheromatous plaque and neointima following balloon injury show expression of ACE in smooth muscle cells and macrophages in the intimal lesions. Activation of ACE in intimal SMC may relate to a phenotypic change of SMC from the contracting type of the synthetic type. Activation of ACE in macrophages is also related to the transformation of macrophages from monocytes. Concerning the role of the activated RAS, elevated blood pressure and vascular tonus by angiotensin II are candidates of vascular injury and plaque rupture. Angiotensin II stimulates migration and proliferation of smooth muscle cells and production of extracellular matrix. Furthermore, angiotensin II increases oxidized-LDL which may be related to the forming of macrophages. These evidence suggest that activation of vascular RAS following endothelial dysfunction/injury play an important role in the pathogenesis of vascular remodeling and atherosclerosis.     

Commissioning for health

Whether or not the pericytes exist in the intra-acinar pulmonary arteries and their normal structure and morphological changes during development of the structural remodeling of pulmonary vessels were observed using a pulmonary hypertension model in rats induced by monocrotaline injection. The results showed that the pericytes in the peripheral pulmonary vessels proliferated and transformed into smooth muscle cells during development of pulmonary hypertension, and at the same time, the pericytes could synthesize and secrete extracellular matrix including collagen, suggesting that the pericytes play an important role in the development of pulmonary hypertension and structural remodeling of the pulmonary vessels.     

The genetics of black larva, an autosomal recessive lethal mutation located on chromosome 3 in the mosquito Anopheles albimanus

Lung tissue from 14 normal residents of high altitude regions, 10 patients with chronic bronchitis and emphysema, and 1 patient with Pickwickian syndrome was studied with regard to the occurrence of pulmonary vascular changes. In addition to the well-known pulmonary arterial alterations, lesions in small pulmonary veins were found in the great majority of the cases. These changes, consisting of medial hypertrophy and arterialization and of bundles of smooth muscle cells within the venous intima, have not been described before in man. These findings suggest that alveolar hypoxia acts not only on small pulmonary arteries and arterioles but also on veins of small caliber, probably by inducing venoconstriction.     

Mast cell collagenase correlates with regression of pulmonary vascular remodeling in the rat

Pulmonary vascular remodeling, produced by cell hypertrophy and extracellular matrix protein synthesis in response to hemodynamic stress, regresses after reduction of blood pressure, possibly by proteolysis of structural proteins. To test this postulate, we assessed the breakdown of extracellular matrix proteins and expression of collagenase and elastase in pulmonary arteries of rats exposed to hypoxia (10% O2 for 10 d) followed by normoxia. During hypoxia, contents of collagen and elastin increased in pulmonary arteries and latent rat interstitial collagenase was expressed without increased collagenolytic activity or mRNA levels. At 3 days after normoxia, collagen and elastin contents decreased coincident with the new appearance of activated collagenase and transient increases in collagenolytic and elastolytic activities. The amount of immunoreactive collagenase, localized predominately in connective tissue-type mast cells, was increased in the adventitia and media of hypertensive vessels. We conclude that mast cells containing latent collagenase are recruited into the outer walls of pulmonary arteries during remodeling. It is possible that mast cell-derived collagenase contributes to collagen breakdown in pulmonary arteries during early recovery from hypoxia and plays a role in restoration of vascular architecture.     

Pathophysiology of restenosis following percutaneous transluminal coronary angioplasty

Symptomatic restenosis occurs in approximately 30-40% of patients after percutaneous transluminal coronary angioplasty (PTCA). Despite intensive research, the primary pathophysiological mediators have not been defined, and pharmacological therapy has not been effective in preventing restenosis. Restenosis is a multifactorial and sequential process, which is initiated by mechanical injury of the vessel wall, and involves neointima formation caused by the local proliferation of smooth muscle cells and production of an extracellular matrix, followed by vascular remodelling. Numerous mediators are involved in these processes, e.g., protooncogenes, growth factors, cytokines and nitric oxide. This review discusses the pathobiological mechanisms underlying coronary restenosis, and outlines the prospects for future therapy.     

Proliferation and extracellular matrix production by human infragenicular smooth muscle cells in response to interleukin-1 beta

PURPOSE: Atherosclerotic peripheral vascular disease commonly involves the infragenicular arterial tree. Our study evaluated the effect of interleukin (IL)-1 beta on the proliferation of vascular smooth muscle cells (VSMCs) derived from atherosclerotic infragenicular arteries of human subjects who underwent below-knee amputation, as well as the role of IL-1 beta in VSMCs' production of extracellular matrix components, substances that are important in the transformation of VSMCs from the contractile to the synthetic phenotype. This transformation to the synthetic phenotype is an important step in the formation of the atherosclerotic lesion. METHODS: Cultures were identified as being of smooth muscle origin through staining with the cytoskeletal marker, alpha-smooth muscle actin. Proliferation assays were performed by seeding confluent cultures of passages 4 to 7 into six-well plates at 10,000 cells per well. After serum starvation, samples were incubated with IL-1 beta (1 ng/ml). Cell number was determined on a daily basis. To study extracellular matrix production, cells were propagated in tissue culture chamber slides in the absence or presence of growth media containing IL-1 beta. After fixation with 100% methanol, each sample was stained with a primary antibody specific for an extracellular matrix component. After staining with the fluorescein-tagged secondary antibody, each sample was examined using immunofluorescent microscopic examination. RESULTS: The results of our proliferation assays showed that IL-1 beta caused a significant increase in the proliferation of VSMCs at 24, 48, 72, and 96 hours (p < or = 0.003 when comparing IL-1 beta-treated samples with control specimens at each time period using unpaired t test). The number of IL-1 beta-treated cells at 96 hours was double the number present in the control samples (16,033 +/- 238 vs 8102 +/- 824). When compared with control samples, IL-1 beta was found to affect the production of extracellular matrix proteins by infragenicular VSMCs. IL-1 beta caused an increase in the production of fibronectin, a decrease in the production of laminin, and no change in the production of collagen type IV. CONCLUSIONS: These results suggest that interleukin-1 beta acts as a potent stimulant of the proliferation of human infragenicular VSMCs. IL-1 beta also acts to augment the production of fibronectin by these cells. Fibronectin has been implicated in the phenotypic transformation of VSMCs from the contractile to the synthetic state. Therefore, IL-1 beta may serve as an important regulatory factor in the development of atherosclerosis by stimulating the proliferation of VSMCs and their transformation to the synthetic state, two important steps in the formation of the atherosclerotic lesion.     

Urokinase but not tissue plasminogen activator mediates arterial neointima formation in mice

To define the role of the plasminogen activators (PAs) tissue PA (t-PA) and urokinase PA (u-PA) in vascular wound healing, neointima formation and reendothelialization were evaluated after electric or mechanical arterial injury in mice with a single or combined deficiency of t-PA (t-PA-/-) and/or u-PA (u-PA-/-). In both models, neointima formation and neointimal cell accumulation were reduced in u-PA-/- and in t-PA-/-/u-PA-/- arteries but not in t-PA-/- arteries. The electric injury model was used to characterize the underlying cellular mechanisms. Topographic analysis of vascular wound healing in electrically injured wild-type and t-PA-/- arteries revealed a similar degree of migration of smooth muscle cells from the noninjured borders into the necrotic center. In contrast, in u-PA-/- and t-PA-/-/u-PA-/- arteries, smooth muscle cells accumulated at the uninjured borders but failed to migrate into the necrotic center. Cultured u-PA-/- but not t-PA-/- smooth muscle cells also failed to migrate in vitro after scrape wounding. Proliferation of smooth muscle cells was not affected by PA deficiency. Reendothelialization after electric injury was similar in all genotypes. In situ analysis revealed markedly elevated u-PA zymographic activity, mRNA, and immunoreactivity in smooth muscle cells, endothelial cells, and leukocytes within 1 week after injury, eg, when cells migrated into the wound. Thus, u-PA plays a significant role in vascular wound healing and arterial neointima formation after injury, most likely by affecting cellular migration.     

The effect of fibroblasts, vascular smooth muscle cells, and pericytes on sprout formation of endothelial cells in a fibrin gel angiogenesis system

We have recently shown that during angiogenesis in situ, sprouting and newly formed capillaries appear to be composed of two cell types, endothelial cells and nonendothelial, pericyte-like cells. The effect of pericytes on the process of neovessel formation is largely unknown. To study the influence of nonendothelial cell types on endothelial tubule formation, we have performed coculture experiments in a fibrin-clot angiogenesis system. When seeded below a critical density on the surface of fibrin gels, endothelial cells (from macro- or microvascular origin) did not show spontaneous formation of sprouts. However, in superconfluent cell cultures or after stimulation of endothelial cells with basic fibroblast growth factor (bFGF), endothelial cells frequently acquired an elongated shape. By stimulation of endothelial cells with both bFGF and vascular endothelial growth factor (VEGF), development of short capillary-like structures was induced. When endothelial cells were cocultivated with a cell type of high fibrinolytic potential, i.e., fibroblasts, development of capillary-like formations could not be detected. Cocultivation of endothelial cells with vascular smooth muscle cells or with retinal pericytes also did not increase the number of capillary-like formations in fibrin gels. In contrast, vascular smooth muscle cells on their own could be demonstrated to give rise to branched capillary-like networks in fibrin, which easily could be mistaken for true capillaries. Our results indicate that periendothelial cells contribute to angiogenesis not only by fibrinolysis and proteolytic permeation of the extracellular matrix. Rather, the interactions of endothelial cells and pericyte-like cells, as frequently observed during neovessel formation in situ, appear to be more specific and may require factors hitherto unknown.     

Activation of mitogen-activated protein kinases (ERK/JNK) and AP-1 transcription factor in rat carotid arteries after balloon injury

Smooth muscle cell proliferation is a key event in neointimal formation after balloon angioplasty. The molecular signals that mediate this process have yet to be identified. Mitogen-activated protein (MAP) kinases are thought to play a pivotal role in transmitting transmembrane signals required for cell proliferation in vitro. The present studies were designed to investigate whether the signal transduction pathways of MAP kinases were involved in the development of restenosis in the injured arteries. Rat carotid arteries were isolated at various time points after balloon injury, and activities of MAP kinases, including extracellular signal-regulated kinases (ERK), and stress activated protein kinases (SAPK)/c-Jun N-terminal protein kinases (JNK), were determined in protein extracts of the vasculature using protein kinase assay and Western blot analysis. After balloon angioplasty, ERK2 and JNK1 activities in the vessel wall increased rapidly, reached a high level in 5 minutes and maintained for 1 hour. A sustained increase in ERK2 kinase activity was observed over the next 7 days in the arterial wall and 14 days in neointima after injury. In contrast, opposite and uninjured arteries did not show significant changes in these kinase activities. Concomitantly, Western blot analysis confirmed that the ERK2 kinase in the injured vessels was indeed activated or phosphorylated, showing a slowly migrating species of a 42-kDa protein containing phosphorylated tyrosine. Kinase activation is followed by an increase in c-fos and c-jun gene expression and enhanced activator protein 1 (AP-1) DNA-binding activity. Thus, balloon injury rapidly activates the MAP kinases in rat carotid arteries. These kinase activations may be crucial in mediating smooth muscle cell proliferation in response to vascular angioplasty.     

Molecular control of vascular smooth muscle cell differentiation

Changes in the differentiated state of the vascular smooth muscle cell (SMC) including enhanced growth responsiveness, altered lipid metabolism, and increased matrix production are known to play a key role in development of atherosclerotic disease. As such, there has been extensive interest in understanding the molecular mechanisms and factors that regulate differentiation of vascular SMC, and how this regulation might be disrupted in vascular disease. Key questions include determination of mechanisms that control the coordinate expression of genes required for the differentiated function of the smooth muscle cell, and determination as to how these regulatory processes are influenced by local environmental cues known to be important to control of smooth muscle differentiation. Of particular interest, a number of common cis regulatory elements including highly conserved CArG [CC(A/T)6GG] motifs or CArG-like motifs and a TGF beta control element have been identified in the promoters of virtually all smooth muscle differentiation marker genes characterized to date including smooth muscle alpha-actin, smooth muscle myosin heavy chain, telokin, and SM22 alpha and shown to be required for expression of these genes both in vivo and in vitro. In addition, studies have identified a number of trans factors that interact with these cis elements, and shown how the expression or activity of these factors is modified by local environmental cues such as contractile agonists that are known to influence differentiation of smooth muscle.     

Acromegaly caused by pulmonary carcinoid tumours

Vascular input impedance and associated hydraulic power was measured in rabbit isolated lungs. The study was focused on changes in impedance and in pulsatile hydraulic power during relaxation and contraction of vascular smooth muscle. Pulsatile power was found to be at a minimum when smooth muscle tone was such that the pulmonary arterial pressure was in the physiological range, and increased both when the vessels were relaxed and further constricted. Input impedance was found to be determined mainly by the large, proximal ('extra-alveolar') arteries.     

Immunoelectron microscopic localization of plasminogen activator inhibitor type 1 (PAI-1) in smooth muscle cells from morphologically normal and atherosclerotic human arteries

Vascular wall fibrinolytic system proteins are believed to play a pivotal role in atherogenesis. Tissue-type plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) influence persistence of luminal thrombi and proteolysis of extracellular matrix, respectively. The major physiologic inhibitor of t-PA and u-PA is plasminogen activator inhibitor type 1 (PAI-1). All three of these fibrinolytic system proteins have been detected in vascular endothelial cells, smooth muscle cells, and macrophages by light microscopic immunohistochemistry. This study was undertaken to delineate, by immunoelectron microscopy, the loci of PAI-1 in smooth muscle cells from intact morphologically normal and atherosclerotic human arteries as well as in isolated and cultured smooth muscle cells from arteries. In intact vessels, PAI-1 immunoreactivity was associated with contractile filaments in cells in both normal and atherosclerotic tissues. Lipid-laden smooth muscle cells in atherosclerotic vessels were mainly of the synthetic phenotype and displayed lesser amounts of PAI-1 associated with rough endoplasmic reticulum and contractile filaments. Isolated smooth muscle cells exhibited either a contractile or synthetic phenotype. In the cells with a contractile phenotype, PAI-1 was associated with the contractile elements, whereas in the cells with a synthetic phenotype, the PAI-1 was associated predominantly with elements of the endoplasmic reticulum. Because PAI-1 is associated predominantly with contractile filaments in smooth muscle cells, the net amount of immunodetectable PAI-1 appears to be greater in contractile compared with synthetic phenotype cells.     

Effects of changes in blood flow rate on cell death and cell proliferation in carotid arteries of immature rabbits

Spontaneous and experimental changes in arterial blood flow rates affect tissue accumulation in developing arteries. To examine whether cell proliferation and/or cell death are affected by alterations in blood flow, we ligated the left external carotid artery of 3-week-old rabbits, which reduces left common carotid blood flow by 71%. In control arteries and after 2 days of flow reduction, agarose gel electrophoresis of DNA extracted from all carotid arteries resolved multiple low molecular weight bands characteristic of apoptosis; however, DNA fragmentation in arteries carrying reduced blood flow was 2.5-fold higher than that of control arteries. The effect of reduced blood flow on cell death subsequently waned but remained significant at 7 days. Cell death in carotid arteries was also detected by in vivo uptake of propidium iodide, a DNA-binding fluorescent dye that labels the nuclei of nonviable cells. Both smooth muscle and endothelial cells exhibited large and statistically significant increases in labeling index in the flow-reduced artery. Propidium iodide-labeled cells were cleared from the vessel wall within 1 to 4 hours of labeling, and nuclear staining displayed condensation (clumping) of chromatin in all labeled cells at later time points. This time course and nuclear morphology and the rapid clearance of labeled cells are consistent with death via apoptosis. Many propidium iodide-positive cells did not display chromatin condensation immediately after labeling; however, this was also true of cultured endothelial cells that were driven into apoptosis with sphingomyelinase treatment and then double-labeled with propidium iodide and the apoptosis marker annexin V. We infer that propidium iodide can label apoptotic vascular cells before these cells display chromatin condensation that is detectable with fluorescence labeling of DNA. Replication rates of smooth muscle and endothelial cells, determined by 5-bromo-2'-deoxyuridine uptake, were inhibited by >75% with decreased blood flow. The inhibition of proliferation was unabated after 7 days of reduced flow. These findings indicate that the coordinated regulation of cell death and cell proliferation, in response to changes in arterial blood flow rates, contributes to arterial remodeling during development.     

Functional and structural adaptation of the yak pulmonary circulation to residence at high altitude

The high-altitude (HA) native yak (Bos grunniens) has successfully adapted to chronic hypoxia (CH) despite being in the same genus as domestic cows, which are known for their great hypoxic pulmonary vasoconstrictor responses (HPVRs), muscular pulmonary arteries, and development of severe pulmonary hypertension on exposure to CH. To determine possible mechanisms by which the pulmonary circulation may adapt to CH, yak pulmonary vascular reactivity to both vasoconstrictor and vasodilator stimuli and yak pulmonary artery structure were assessed. Hypoxia caused a small but significant HPVR, and norepinephrine infusion caused a greater rise in pulmonary arterial pressure (Ppa) than did hypoxia. Acetylcholine, an endothelium-dependent vasodilator, had no effect on Ppa but lowered pulmonary resistance (Rp) by causing an increase in cardiac output. Sodium nitroprusside, an endothelium-independent vasodilator, decreased both Ppa and Rp significantly. Yak small pulmonary arteries had a 4.1 +/- 0.1% medial thickness, with vessels < or = 100 microns devoid of smooth muscle. Yak pulmonary artery endothelial cells were much longer, wider, and rounder in appearance than those of domestic cows. Thus the yak has successfully adapted to HA conditions by maintaining both a blunted HPVR and thin-walled pulmonary vessels. Differences in both endothelial cell morphology and response to acetylcholine between the yak and those reported in the domestic cow suggest the adaptation to HA may include changes not only in the amount of pulmonary vascular smooth muscle but in endothelial cell function and structure as well.