Smooth Muscle Cell Alpha-actin Research Articles

Comparative Anatomy of the Male Guinea‐Pig and Human Lower Urinary Tract: Histomorphology and Three‐Dimensional Reconstruction

The guinea-pig is often used for experimental studies in urology. However, the anatomy of the lower urinary tract of the guinea-pig is poorly described in the literature. The structure and function of the lower urinary tract, i.e. continence, micturition and sexual function, are closely related to the gross anatomy of the pelvis and the fine structure of the musculature. We investigated the anatomy and histomorphology of the lower urinary tract by serial sections in male guinea-pigs and compared it to that in humans. Immunohistochemical stainings for alpha-smooth muscle cell actin were used to differentiate between smooth and striated muscles. By using whole pelvic preparations, including all internal organs preserved in their in situ location for three-dimensional reconstruction, we developed three-dimensional models, which elucidate the spatial relationship of all muscular structures and can help to deduce functional aspects of lower urinary tract function. In the guinea-pig, most of the muscles found in humans can be demonstrated in comparable location and extension. However, the structure of the prostate and the existence of the so-called coagulation glands define a significant difference in the morphology of the prostatic urethra.

Antifibrotic effect of Stephania tetrandra on experimental liver fibrosis induced by bile duct ligation and scission in rats.

We examined the antifibrotic effect of a methanol extract from Stephania tetrandra (ST) on experimental liver fibrosis. Liver fibrosis was induced by bile duct ligation and scission (BDL/S) in rats. In BDL/S rats, activity levels of aspartate transaminase (AST), alanine transaminse (ALT), alkaline phosphatase (ALP), concentration of total bilirubin in serum, and hydroxyproline content of the liver were significantly increased. The ST treatment (either 100 mg/kg/day or 200 mg/kg/day, p.o. for 4 weeks) in BDL/S rats reduced the serum AST, ALT and ALP activity levels significantly (p< 0.01). Similarly, when compared to the control group, the concentration of hydroxyproline in the livers of the BDL/S rats treated with 100mg or 200mg ST treated rats decreased by 40% and 33% respectively, when compared to the BDL/S control group (p<0.01). The morphological characteristics of fibrotic liver that were observed in the BDL/S control group, improved in the ST treated BDL/S group. In the fibrotic liver of BDL/S rats treated with ST, a marked reduction in the numbers of alpha smooth muscle cell actin positive stellate cells was observed. These results indicate that doses of either 100 or 200 mg/kg/day of methanol extract from S. tetrandra, had an antifibrotic effect in rats with liver fibrosis induced by bile duct ligation and scission.

P75 NTR Mediates Neurotrophin-Induced Apoptosis of Vascular Smooth Muscle Cells

The development of atherosclerotic lesions results from aberrant cell migration, proliferation, and extracellular matrix production. In advanced lesions, however, cellular apoptosis, leading to lesion remodeling, predominates. During lesion formation, the neurotrophins and the neurotrophin receptor tyrosine kinases, trks B and C, are induced and mediate smooth muscle cell migration. Here we demonstrate that a second neurotrophin receptor, p75(NTR), is expressed by established human atherosclerotic lesions and late lesions that develop after balloon injury of the rat thoracic aorta. The p75(NTR), a member of the tumor necrosis factor/FAS receptor family, can modulate trk receptor function as well as initiate cell death when expressed in cells of the nervous system that lack kinase-active trk receptors. p75(NTR) expression colocalizes to neointimal cells, which express smooth muscle cell alpha-actin and are expressed by cultured human endarterectomy-derived cells (HEDC). Areas of the plaque expressing p75(NTR) demonstrate increased TUNEL positivity, and HEDC undergo apoptosis in response to the neurotrophins. Finally, neurotrophins also induced apoptosis of a smooth muscle cell line genetically manipulated to express p75(NTR), but lacking trk receptor expression. These studies identify the regulated expression of neurotrophins and p75(NTR) as an inducer of smooth muscle cell apoptosis in atherosclerotic lesions.

Photodynamic Therapy Induces Apoptosis in Intimal Hyperplastic Arteries

Photodynamic therapy (PDT) generates free radicals through the absorption of light by photosensitizers. PDT shows promise in the treatment of intimal hyperplasia, which contributes to restenosis, by completely eradicating cells in the vessel wall. This study investigates the mechanisms of PDT-induced cell death. PDT, using the photosensitizer chloroaluminum-sulfonated phthalocyanine (1 mg/kg) and laser light (lambda = 675 nm) 100 J/cm(2) was administered to rat carotid arteries after balloon injury-induced intimal hyperplasia. Apoptosis was determined by cell morphology with light microscopy and transmission electron microscopy, DNA cleavage by terminal dUTP nick-end labeling staining, and nucleosomal fragmentation (ladder pattern) by DNA agarose gel electrophoresis. Four hours after PDT, apoptosis was observed in vascular cells, as evidenced by terminal dUTP nick-end labeling staining and transmission electron microscopy. Within 24 hours no cells were present in the neointima and media. Immunofluorescence using an alpha-smooth muscle cell actin antibody confirmed the disappearance of all neointimal and medial cells within 24 hours. No inflammatory cell infiltrate was observed during this time frame. Apoptosis was sharply confined to the PDT treatment field. These data demonstrate that vascular PDT induces apoptosis as a mechanism of rapid, complete, and precise cell eradication in the artery wall. These findings and the lack of inflammatory reaction provide the basis for understanding and developing PDT for a successful clinical application in the treatment of hyperplastic conditions such as restenosis.

Cardiac alpha-actin in smooth muscle cells: detection in umbilical cord vessels and in atherosclerotic lesions.

Phenotypic modulation of smooth muscle cells (SMC) is a key event during the development of atherosclerotic and restenotic lesions. During this process, the composition of the cytoskeleton is substantially altered, with changes predominantly in actin expression reflecting a shift from smooth muscle alpha-actin to the non-muscle beta-isoform. We now demonstrate that yet another actin isoform, cardiac alpha-actin, is synthesized, de novo, in SMC of various atherosclerotic lesions. Using a highly specific monoclonal antibody against cardiac alpha-actin, we analyzed and compared the accumulation of this actin isoform in diverse SMC by immunofluorescence microscopy and immunoblotting. As expected, cardiac alpha-actin was present in human myocardium but not in healthy SMC of adult aorta, coronary arteries, trabeculae of the spleen, colon, stomach or skeletal muscle. Interestingly, the presence of cardiac alpha-actin was detected in umbilical cord vessels, human myometrium, in atherosclerotic coronary lesions and atherosclerotic lesions from peripheral vascular disease. The distribution of cardiac alpha-actin often paralleled that of cytokeratins 8 and 18, intermediate filament proteins typically found in dedifferentiated SMC. Taken together, the data presented here illustrate the expression of cardiac alpha-actin to be limited to either fetal vessels or those vessels or tissue having suffered damage or atrophy, outside its 'native' environment in the heart. The demonstration of cardiac alpha-actin in SMC of umbilical cord vessels and in atherosclerotic lesions but not in apparently healthy vessels supports the notion that SMC in atherosclerotic lesions exhibit a dedifferentiated phenotype.

A rat small bowel transplant model of chronic rejection: histopathologic characteristics.

The major impediment to long-term success in solid organ transplantation is the development of chronic rejection (CR). The vascular lesion of CR, transplant vascular sclerosis (TVS) is characterized by neointimal smooth muscle cell proliferation, and is driven by both immune- and nonimmune-mediated mechanisms. Although the features of chronic heart and kidney allograft rejection have been well characterized, the more immunogenic small bowel allograft has not received similar study. F344 small bowel (SB) was transplanted heterotopically into Lewis recipients that were treated with low-dose Cyclosporine A for 15 days. Lewis recipients of F344 or Lewis SB grafts without immunosuppression, served as controls. Grafts were assessed histologically when recipients showed clinical signs of rejection or at predetermined time points. The immunological components involved in the chronic rejection process were evaluated by immunohistochemical staining. All SB allografts (100%) developed histologic evidence of CR Cyclosporine A. TVS was seen in 36 of the 46 (78%) of these allografts. The median time to develop TVS was 45 days. Immunohistochemical staining of chronically rejected grafts showed infiltration predominantly by CD4+ cells and macrophages, uniform up-regulation of class II MHC molecule expression, moderate to intense ICAM-1 staining in grafts harvested at postoperative day 45, and uniform neointimal cell staining for smooth muscle cell alpha-actin in the TVS lesions. This F344 to Lewis SB transplant model is a useful model that reproduces significant features of CR. The highly immunogenic nature of the SB allografts allows this model to serve as a stringent test for protocols designed to prevent CR.

Endothelin converting enzyme is located on alpha-actin filaments in smooth muscle cells.

Endothelin converting enzyme is the key enzyme in the generation of endothelin-1 from big-endothelin-1. The mature endothelin-1 is a potent vasoconstrictor which also promotes mitogenesis and proliferation of smooth muscle cells. The objectives were to demonstrate in smooth muscle cells the presence of a phosphoramidon-sensitive endothelin converting enzyme activity, reveal the subcellular localization of the enzyme protein and determine the effects of the metalloproteinase inhibitor, phosphoramidon, the lysosomotrophic drug, chloroquine, and colchicine on the cycling pathway of the enzyme. Subcellular localization of endothelin converting enzyme on human smooth muscle cells and the rat cell line, A7r5, was by immunofluorescence and confocal microscopy or by biotinylation of cell cultures and immunoblotting, after treatment of cell cultures with cytochalasin D, colchicine, chloroquine and phosphoramidon. Converting enzyme activity was determined by high performance liquid chromatographic assay. We detected phosphoramidon-sensitive endothelin converting enzyme activity in smooth muscle cells. In addition to its plasma membrane location, for the first time we revealed a striking co-localization of endothelin converting enzyme and alpha-actin filaments in smooth muscle cells. Colchicine treatment results in a perinuclear accumulation of endothelin converting enzyme. An increased level of endothelin converting enzyme protein was shown to be present in smooth muscle cells which had been grown in the presence of phosphoramidon or chloroquine. The 120 kDa endothelin converting enzyme co-localizes with alpha-actin in smooth muscle cells and resembles that found in endothelial cells in that it is present on both the plasma membrane and intracellularly.

Identification of Two Amino Acids within the EIIIA (ED-A) Segment of Fibronectin Constituting the Epitope for Two Function-blocking Monoclonal Antibodies

Alternative splicing of the fibronectin gene transcript gives rise to a group of adhesive glycoproteins showing restricted spatial and temporal expression during embryonic development, tumor growth, and tissue repair. Alternative splicing occurs in three segments termed EIIIB, EIIIA, and V. The EIIIA (or ED-A) segment of fibronectin is expressed prominently but transiently in healing wounds coincident with fibroblast expression of an activation marker, smooth muscle cell alpha-actin. A monoclonal antibody (IST-9) to the EIIIA segment blocks transforming growth factor-beta-mediated smooth muscle cell alpha-actin expression by fibroblasts in culture. A second monoclonal antibody (DH1) blocks chondrocyte condensation in chicken embryos. We find that IST-9 and DH1 react with human, rat, and chicken but not with mouse or frog EIIIA, suggesting that His44 may be important for antibody binding. A series of deletion mutants of rat EIIIA, constructed as glutathione S-transferase fusion proteins, do not react with either IST-9, DH1, or a third monoclonal antibody (3E2). Mutations of pairs of amino acids to alanine have little effect, except for either (Val34Thr35) or (Tyr36Ser37), which are located in a beta strand upstream from His44. For these double mutants, the binding to all three monoclonal antibodies is markedly reduced. By contrast, single mutants at Thr35, Tyr36, or Ser37 retain full activity, suggesting that the epitope for these antibodies is determined in part by conformation. Alanine-scanning mutagenesis of rat EIIIA demonstrates the importance of Ile43 and His44 for binding. Mutation of frog EIIIA (normally Val43Lys44) to rat (Ile43His44) is sufficient to restore fully IST-9 binding and much of the activity of DH1 and 3E2. Our findings demonstrate that the function-blocking antibodies, IST-9 and DH1, bind to the Ile43 and His44 residues in a conformationally dependent fashion, implicating the loop region encompassing both residues as critical for mediating EIIIA function.

Design of a muscle cell-specific expression vector utilising human vascular smooth muscle alpha-actin regulatory elements.

The facility to direct tissue-specific expression of therapeutic gene constructs is desirable for many gene therapy applications. We describe the creation of a muscle-selective expression vector which supports transcription in vascular smooth muscle, cardiac muscle and skeletal muscle, while it is essentially silent in other cell types such as endothelial cells, hepatocytes and fibroblasts. Specific transcriptional regulatory elements have been identified in the human vascular smooth muscle cell (VSMC) alpha-actin gene, and used to create an expression vector which directs the expression of genes in cis to muscle cells. The vector contains an enhancer element we have identified in the 5' flanking region of the human VSMC alpha-actin gene involved in mediating VSMC expression. Heterologous pairing experiments have shown that the enhancer does not interact with the basal transcription complex recruited at the minimal SV40 early promoter. Such a vector has direct application in the modulation of VSMC proliferation associated with intimal hyperplasia/restenosis.

Targeted overexpression of IGF-I evokes distinct patterns of organ remodeling in smooth muscle cell tissue beds of transgenic mice.

Smooth muscle cells (SMC) of the vascular wall, bladder, myometrium, and gastrointestinal and respiratory tracts retain the ability to proliferate postnatally, which enables adaptive responses to injury, hormonal, or mechanical stimulation. SMC growth is regulated by a number of mesenchymal growth factors, including insulin-like growth factor I (IGF-I). To explore the function of IGF-I on SMC in vivo, the mouse SMC alpha-actin promoter fragment SMP8 (-1074 bp, 63 bp of 5'UT and 2.5 kb of intron 1) was cloned upstream of rat IGF-I cDNA, and the fusion gene microinjected to fertilized eggs of the FVB-N mouse strain. Mating of hemizygous mice with controls produced about 50% transgenic offspring, with equal sex distribution. Transgenic IGF-I mRNA expression was confined to SMC-containing tissues, with the following hierarchy: bladder > stomach > aorta = uterus > intestine. There was no transgene expression in skeletal muscle, heart, or liver. Radioimmunoassayable IGF-I content was increased by 3.5- to 4-fold in aorta, and by almost 10-fold in bladder of transgenic mice at 5 and 10 wk, with no change in plasma IGF-I levels. Wet weight of bladder, stomach, intestine, uterus, and aorta was selectively increased, with no change in total body or carcass weight of transgenic animals. In situ hybridization showed that transgene expression was exquisitely targeted to the smooth muscle layers of the arteries, veins, bladder, ureter, stomach, intestine, and uterus. Paracrine overproduction of IGF-I resulted in hyperplasia of the muscular layers of these tissues, manifesting in remarkably different phenotypes in the various SMC beds. Whereas the muscular layer of the bladder and stomach exhibited a concentric thickening, the SMC of the intestine and uterus grew in a longitudinal fashion, resulting in a marked lengthening of the small bowel and of the uterine horns. This report describes the first successful targeting of expression of any functional protein capable of modifying the phenotype of SMC in transgenic mice. IGF-I stimulates SMC hyperplasia, leading to distinct patterns of organ remodeling in the different tissue environments.

Immunocytochemical determination of cell type and proliferation rate in human vein graft stenoses.

Vascular reconstructions are prone to fail as a result of the development of stenotic lesions, which have historically been attributed to myointimal hyperplasia. In animal models, these lesions are associated with marked proliferative smooth muscle cell (SMC) response to vascular injury. However, recent studies using sensitive immunocytochemical techniques in human lesions have generally failed to detect significant cellular proliferation. To clarify the role of cellular proliferation in humans, we characterized the cellular composition and proliferative index of 14 early infrainguinal vein graft stenoses. All infrainguinal vein grafts at our institution are prospectively enrolled in a duplex surveillance protocol, the details of which have been previously reported. Among 98 grafts placed within the last year, 11 patients were identified with 14 progressive, focal, high-grade lesions that met previously established threshold criteria for prophylactic revision to prevent graft thrombosis. Lesions were first detected from 1 week to 7 months after surgery and were removed and replaced with segmental interposition grafts (1.5 to 10 months). Freshly excised lesions were placed in Methyl Carnoy's fixative, paraffin embedded, and serially sectioned. The cellular composition of each lesion was determined with cell-specific immunochemical reagents: alpha SMC actin, von Willebrand factor (endothelial cell), CD 68 (macrophage), and CD 45RB (monocyte). Actively proliferating cells were identified using antibody to proliferating cell nuclear antigen (PCNA). The identity of PCNA-positive cells was determined by double-label immunocytochemical staining, and the proliferative index (PCNA-positive cells/total cells x 100) was calculated by computer-assisted counts of representative gridded cross-sections of each lesion. All excised lesions demonstrated marked thickening with severe luminal encroachment and were highly cellular, with a predominance of alpha SMC actin+. Endothelial cells on the blood flow surface were present to a variable degree, and seven lesions exhibited striking numbers of macrophages and monocytes. The latter cell types were most abundant near microvessels in the deep neointima and adventitia. Active cellular proliferation was identified primarily in SMCs, with a mean PCNA index of 1.34%. However, significant PCNA reactivity was not limited to SMCs, but was also identified in macrophages and monocytes, particularly in lesions greater than 3 months old. Previous immunocytochemical studies of human coronary restenosis atherectomy specimens have generally detected low rates of cellular proliferation (0.5%), but these lesions may not truly represent myointimal hyperplasia, rather a mixture of atherosclerosis, thrombosis, and "restenosis." In contrast, the present study of early human vein graft lesions detected by duplex surveillance indicates that significant cellular proliferation occurs, although rates are lower than those obtained in animals such as the rat carotid injury model. In addition, although SMCs are the predominant proliferating cell type in human vein grafts, our identification of proliferating monocytes and macrophages raises the question of the contribution of an inflammatory component to the development of human lesions. The present study represents the first report of PCNA determination in a series of human infrainguinal vein grafting procedures.

Healing Characteristics of Intraarterial Stent Grafts in an Injured Artery Model

Previous investigations reveal in the absence of endothelial cell (EC) injury, intraarterial polytetrafluoroethylene (PTFE) stent graft (SG) exhibit greater EC repaving than PTFE interposition grafts (CG). The investigation evaluated EC repaving of SG compared to CG after balloon injury. Twenty adult dogs underwent SG (n = 10) or CG (n = 10) placement in the infrarenal aorta after balloon injury with harvest at 1 and 6 weeks. Endothelial repaving, intima-to-media height ratios (IMHR), and inflammatory stains were performed. Endothelial repaving was greater in 6-week SG compared to CG (51% +/- 5.0 versus 10% +/- 5.0, p < or = 0.05). IMHR was less in 6-week SG compared to CG at the proximal (1.22 +/- 0.16 versus 1.82 +/- 0.16, p < or = 0.05) and distal anastomosis (0.81 +/- 0.25 versus 1.33 +/- 0.25, p < or = 0.05). Smooth muscle cell (SMC) alpha-actin was greater in 1-week SG compared to CG at the distal anastomosis (121.5 +/- 7.2 versus 94.0 +/- 7.2, p < or = 0.05). Proliferating cell nuclear antigen (PCNA) was less in 6-week SG compared to CG at the proximal (5.6 +/- 1.4 versus 9.4 +/- 1.1, p < or = 0.05) and distal anastomosis (3.8 +/- 0.6 versus 11.2 +/- 1.1, p < or = 0.05). Macrophage CD-44 was less in 6-week SG compared to CG at the proximal (10.4 +/- 1.6 versus 32.6 +/- 3.6, p < or = 0.05) and distal anastomosis (8.6 +/- 0.9 versus 35.6 +/- 3.6, p < or = 0.05). Intraarterial SG placed after balloon injury exhibited significantly greater endothelialization and less intimal hyperplasia when compared to CG.

Smooth muscle cell migration and proliferation is enhanced in abdominal aortic aneurysms.

The aetiology of abdominal aortic aneurysms (AAA) is as yet undetermined. Smooth muscle cells (SMC) have been implicated in the pathogenesis of AAA as a result of their ability to produce elastin degrading proteases. The present study was undertaken to examine AAA SMC and aortic occlusive disease (AOD) SMC in terms of their respective migration and proliferation in vitro, in order to identify intrinsic differences between these cells. Five AAA specimens, four AOD and five inferior mesenteric artery (IMA) specimens were established in culture. The cultures were examined for the extent and the rate of SMC outgrowth and proliferation. Cells were counted following trypsinization using a haemocytometer. For the AAA explants, the cellular outgrowths were first seen at 6.7 days, after culture initiation, while the corresponding outgrowth in the AOD group required 8.8 days (P < 0.05) and the IMA group 11.4 days (P < 0.05). AAA cells reached confluency at a mean of 22.4 days while AOD SMC required 28.6 days (P < 0.05) and IMA 31 days (P < 0.05). In the first passage, the time for AAA SMC doubling was 5.3 days compared to 6.2 days for AOD (P < 0.05) and 8.1 days for the IMA group (P < 0.05). Greater than 98% of the cells, in both groups, stained positive to SMC alpha-actin. From these data it is clear that there are intrinsic differences in cellular kinetics between SMC from the two disease states, supporting the hypothesis that AAA are not the result of atherosclerosis.

The influence of collapse of the lung parenchyma on the morphology of pulmonary blood vessels in the rat.

During a search for resident subendothelial smooth muscle cells in pulmonary vessels of the rat we found that in expanded lungs the muscular pads in the veins, considered by some authors as sphincters, were hardly visible whereas in collapsed lungs they were very conspicuous. In a separate study intended to quantify the degree of collapse or expansion the left lung was examined in 5 rats with a collapsed and in 5 rats with an expanded lung: the expansion was produced by filling the airways by gravity with Methacarn fixative. The degree of expansion was determined by morphometry measuring the volume density of the tissue fraction of the pulmonary parenchyma in the microscopic sections: in the expanded lung the mean value was 8.5% (range 6.7-12.6%), in the collapsed lung 20.1% (range 18.7-22.3%), a highly significant difference (p < 0.000). Serial sections generally 60-100, 6-microns-thick, were stained by PAS, Sirius red hematoxylin and Verhoeff's elastic stains. Immunohistochemical staining was done with monoclonal antibody against alpha smooth muscle cell actin and desmin. Graphic reconstructions of representative vessels were performed. It was shown that the muscular media of the veins was interrupted and that the muscular pads corresponded to the contracted smooth muscle cell segments alternating with the noncontracted segments devoid of muscle. In the expanded lungs muscular pads were flattened and often hardly detectable. This indicates that the structures considered as sphincters are postmortem contraction rings in collapsed lungs.

Immunohistochemical and molecular characterization of the differential response of the rat mesenteric microvasculature to angiotensin-II infusion.

The present study focuses on the differential response of three branch levels of the mesenteric resistance arterial vasculature of 450-gram Sprague-Dawley rats infused continuously with angiotensin II (A-II) for 4, 7 and 14 days at a rate of 435 ng/kg/min, with an associated period of hypertension. The three branch levels (types I, II and III) were characterized by light microscopy and immunostaining using monoclonal antibodies for proliferating cell nuclear antigen, ED-1 (specific for rat monocytes/macrophages) and alpha smooth muscle cell (SMC) actin. Cross-sectional areas of the vascular walls were determined morphometrically. In situ hybridizations were performed on paraffin sections using both sense and antisense 35S-labeled cRNA probes generated from rat SMC osteopontin and elastin cDNAs. In the type-I (penetrating) arteries from A-II-infused animals, there was massive fibrinoid necrosis, a marked fibroproliferative perivascular response, intense monocyte/macrophage infiltration, striking SMC osteopontin and elastin gene expression; SMC, fibroblast and monocyte/macrophage DNA synthesis; and significant increase in the cross-sectional areas of the vascular walls. In the same animals, DNA synthesis also occurred in the larger mesenteric arteries of types II and III where it is associated with significant enlargement of the walls by SMC hypertrophy but without overt morphologic damage. It is suggested that the monocyte/macrophage infiltration and fibroproliferative response of type-I arteries may be related to A-II-induced osteopontin gene expression. Angiotensin infusion in the rat may represent a reproducible model of microvascular injury that can be utilized to elucidate the cellular and molecular biology of a variety of disease states such as hypertension and diabetes mellitus.

Low-dose radioactive endovascular stents prevent smooth muscle cell proliferation and neointimal hyperplasia in rabbits.

Restenosis induced by smooth muscle cell (SMC) migration and proliferation and neointimal thickening limits the clinical success of balloon angioplasty and stent implantation. In this study, the long-term effect of endovascular irradiation via low-dose radioactive stents on neointima formation was compared with conventional stent implantation in a rabbit model. Palmaz-Schatz stents were made radioactive in a cyclotron. The stents had a very low activity (maximum, 35 microCi), and thus, manipulation did not require extensive radiation protection. One, 4, 12, and 52 weeks after the implantation of nonradioactive stents and radioactive stents in rabbit iliac arteries, neointimal thickening was analyzed by quantitative histomorphometry. Immunostaining for endothelial cell von Willebrand factor, macrophages, SMC alpha-actin, collagen type I, and proliferating cell nuclear antigen (PCNA) was performed to determine radiation-induced changes in the arterial wall. SMC proliferation was quantified by computer-assisted cell counting of PCNA-immunoreactive cells. Neointima formation was markedly suppressed by the implantation of radioactive stents in a dose-dependent fashion at all observed time points. At peak proliferative activity of SMCs 1 week after nonradioactive stent implantation, 30 +/- 2% of SMCs in the neointima were proliferating, compared with 0.5 +/- 0.1% of SMCs after implantation of stents with an initial activity of 35 microCi (P < .001). The neointima covering radioactive stents was characterized by decreased smooth muscle cellularity and increased extracellular matrix formation. Further, we observed a delayed endothelialization depending on the radiation dose. No difference in vascular thrombosis was found after nonradioactive and radioactive stent implantation. The results of this study clearly indicate that low-dose radioactive endovascular stents potently inhibit SMC proliferation and neointimal hyperplasia in rabbits.

Definitive proof of endothelialization of a dacron arterial prosthesis in a human being

A 10 mm woven Dacron axillofemoral bypass graft was removed from a 65-year-old patient during redo surgery after an implant period of 26 months, because of a large seroma that surrounded the entire length of the graft. Tissue blocks were taken from representative areas along the entire length of the graft surface and evaluated by light microscopy with hematoxylin and eosin and Masson trichrome staining, scanning electron microscopy, transmission electron microscopy, and immunocytochemical staining. Paraffin-embedded sections were stained with smooth muscle cell alpha-actin, which demonstrated smooth muscle cells in the pseudointima, and Ham 56 stain to identify macrophages. Endothelial factor VIII/von Willebrand factor and Ulex europaeus agglutinin identified human endothelial cells on the flow surface, in areas far removed from the anastomoses to the native vessels. This is the first definitive proof in a human of endothelialization of a synthetic arterial graft beyond the pannus ingrowth zone.

The Molecular Basis of Increased Glomerulosclerosis after Blockade of the Renin Angiotensin System in Growth Hormone Transgenic Mice

Angiotensin converting enzyme inhibitor (ACEi) therapy delays the onset of renal failure in diabetic nephropathy and inhibits or delays the onset of proteinuria in several animal models. We examined this question using a transgenic model of chronic glomerulosclerosis caused by an excess production of growth hormone (GH) in which there is progressive glomerular scarring leading to uremia. In addition, since GH mice do not have systemic hypertension or an elevated glomerular filtration rate, we could address the question of whether ACEi or angiotensin II receptor antagonists (AII RA) had an effect on the development of glomerulosclerosis under these conditions. Since excess matrix accumulates in glomerulosclerosis because of alterations in the balance between its synthesis and degradation, we examined the effect of ACEi and AII RA on these parameters. Systemic blood pressure was unaffected by ACEi treatment, but the glomerular filtration rate decreased 85%. ACEi-treated mice had increased mesangial deposition of type I collagen and decreased 105 kD complex collagenase activity. In addition, ACEi-treated GH mice had increased glomerular alpha 1 type I collagen, alpha 1 type IV collagen, and alpha-smooth muscle cell actin mRNAs. No changes were noted in beta actin, or 72 kD metalloproteinase mRNAs. The result of these changes was a net increase in sclerosis. Surprisingly, GH mice treated with ACEi or AngII RA developed marked renal arteriolar lesions. In some forms of glomerulosclerosis, the lesions develop independently of angiotensin II. Pharmacological inhibition of angiotensin II, in this circumstance, may aggravate the lesions through disregulation of the levels and the balance between glomerular matrix synthesis and degradation.

Ultrastructural and immunocytochemical study on normal human palmar aponeuroses.

Human palmar aponeurosis can be affected by a fibrotic process whose aetiopathology is unknown. As the organization of that normal tissue has not been completely investigated, the aim of the present study was to define the ultrastructure of the aponeurosis in order to better understand its biology and behaviour in pathology. Bioptic samples from normal subjects of different ages were analysed by optical and electron microscopy and by immunocytochemistry. The aponeurotic branches consisted of thick, almost parallel collagen bundles containing columns of prominent cells, characterized by long cytoplasmic projections. Cells did not change in number and distribution with age and appeared longer and slighter in the old than in the young subjects. They exhibited plasma membrane almost completely decorated by pinocytic vesicles, intracytoplasmic bundles of thin filaments with zonal thickenings close to the cell membrane, and well-developed subcellular structures. Cells expressed smooth muscle cell alpha-actin, as revealed by immunostaining. The external surface of the plasma membrane was underlined by a discontinuous basement membrane-like structure and by a thick coat of interwoven filaments, highly positive to hyaluronan-recognizing antibodies. Immunocytochemical analyses revealed that collagen fibrils were positive for collagen types I, III, and VI and that elastin fiber composition was rather complex. Independently of the age, normal palmar aponeurotic cells show peculiar morphological features and peculiar cell-matrix interactions, very likely mediated by hyaluronan. These findings indicate that normal aponeurotic cells cannot be regarded as typical tenocytes and suggest the need for a better definition of their phenotype in order to understand their behaviour in pathological processes.

Liposuction-derived human fat used for vascular graft sodding contains endothelial cells and not mesothelial cells as the major cell type.

Endothelial cell transplantation has been suggested as a method to improve the patency of prosthetic grafts used for vascular reconstruction. A major technical concern of all cell transplantation studies has been the purity of cells in the primary isolate used for subsequent transplantation. Accordingly we have evaluated the cellular constituents of liposuction-derived human fat with immunocytochemistry and scanning electron microscopy. Samples of liposuction-derived human fat were processed for immunohistochemistry and subsequently stained for the presence of von Willebrand factor (vWF), alpha-smooth muscle cell actin, cytokeratin (peptide 18), and the endothelial cell-specific marker EN4. We also performed histochemistry studies on the cells derived from this fat after collagenase dispersion of the liposuction far. Immunohistochemistry revealed that 86.1% of the cells in intact, liposuction-derived fat express vWF, whereas 5.7% of the cells exhibited alpha-smooth muscle cell actin, and 1.0% expressed the mesothelial cell-related antigen, cytokeratin peptide 18. Expression of EN4 was found in 89.6% of the cells counted in intact far. After digestion of fat with collagenase and centrifugal separation of adipocytes from vascular and stromal cells, the expression of vWF, alpha-smooth muscle cell actin, and cytokeratin was 77.5%, 5.8%, and 2.1%, respectively. EN4 expression was observed in 74.6% of the isolated cells. Thus most cells present in liposuction-derived fat, even before tissue digestion and cell isolation, were characterized as endothelium. Although other cells common to mesodermally derived tissue were identified (e.g., adipocytes, smooth muscle cells, and mesothelium), they represented a minor fraction of the total cells present. On isolation, the number of cells expressing vWF- and EN4-specific antigens was less than that observed in intact fat. This finding suggests that a portion of cells reacting with antibodies in situ lose vWF and EN4 staining during the isolation procedure. Unlike omentum, liposuction-derived fat predominantly contains adipocytes and endothelial cells. On digestion of liposuction-derived fat and separation of cells, vascular endothelial cells represent the major cellular component.