Biomedical subjects

J Thyberg

Publications and source records attributed to J Thyberg.

At least 37 records · Page 2Linked to original sources

PubMed1998

Regulation of cell morphology in B lymphocytes by IL-4: evidence for induced cytoskeletal changes.

Lymphocyte activation is often accompanied by changes in cell morphology, for example, in cell adhesion or motility. IL-4 is a cytokine exerting many effects on B lymphocytes. In this study, we show that stimulation with LPS in combination with IL-4, but not LPS or IL-4 alone, results in a pronounced dendritic morphology of B cells. Using a culture system in which Abs directed to B cell surface markers are immobilized on the tissue culture plastic, we find that cell spreading can be mediated by a variety of Abs, including anti-CD44, -CD23, -LFA-1, -VLA-4, -ICAM-1, and -Ig. B cells stimulated with anti-Ig Abs plus IL-4, or anti-CD40 Abs in the presence or absence of IL-4, are also induced to spread, while IL-2, IL-5, or IL-10 in combination with LPS or alone fail to induce this. Spreading correlates with induction of tight cell aggregation. It is sensitive to cytochalasin B, indicating a requirement for intact actin cytoskeleton. CD44 is selectively detected in the detergent-insoluble fraction of cell lysates prepared from LPS plus IL-4-stimulated B cell cultures after Ab cross-linking of CD44, suggesting a membrane protein-cytoskeleton interaction. Interestingly, electron microscopy studies reveal induction of microvilli-like structures on LPS plus IL-4-stimulated blasts, suggesting that IL-4 can influence cell morphology on an ultra-structural level. In summary, our data show that stimulation with LPS plus IL-4 or ligation of CD40 is capable of inducing dramatic morphologic changes in murine B cells, which correlates with in vitro induction of strong cell adhesion.

Animals↗

PubMed1998

Partitioning of cytoplasmic organelles during mitosis with special reference to the Golgi complex.

During mitosis, not only the genetic material stored in the nucleus but also the constituents of the cytoplasm should be equally partitioned between the daughter cells. For this sake, the dividing cell goes through an extensive structural reorganization and transport along the endocytic and exocytic pathways is temporarily arrested. Early in prophase, the radiating array of cytoplasmic microtubules disassembles and the membrane systems of the secretory apparatus start to split up. In metaphase, the nuclear envelope fragments and the condensing chromosomes associate with the forming mitotic spindle. The cisternal and tubular elements of the endoplasmic reticulum and the Golgi complex break down into small vesicles, presumably as the result of an imbalance between vesicle budding and fusion. In anaphase, the two sets of chromosomes are pulled apart and a cleavage furrow forms halfway between the spindle poles. Since most organelles occur in multiple and widely dispersed copies at this stage, they will be evenly distributed between the daughter cells. During telophase and cytokinesis, the preceding fragmentation process is reversed. A nuclear envelope reappears around the chromosomes and cytoplasmic microtubules reassemble. The endoplasmic reticulum is rebuilt as a continuous system of flattened cisternae and tubules. Stacks of Golgi cisternae arise from small vesicles and are rearranged in an interconnected network. In parallel, the biosynthetic functions of the cell are normalized and intracellular membrane traffic is resumed.

Cell Compartmentation↗

PubMed1998

Tyrphostin A9 and wortmannin perturb the Golgi complex and block proliferation of vascular smooth muscle cells.

To proliferate, vascular smooth muscle cells first convert from a contractile to a synthetic phenotype. Earlier studies indicate that this process is supported by fibronectin and accelerated by platelet-derived growth factor (PDGF). Here, the mechanisms in this transition were further explored. Isolated rat aortic smooth muscle cells were treated with tyrphostin A9, a PDGF receptor tyrosine kinase inhibitor, and wortmannin, a phosphoinositide 3-kinase inhibitor. Electron microscopy did not show any effect on the reorganization of the cells during the first days in culture, i.e. the loss of actin filaments and the formation of a large secretory apparatus. Conversely, both drugs caused hypertrophy of the Golgi complex, with large and partly vacuolized cisternal stacks. Nevertheless, a juxtanuclear staining pattern for the Golgi enzyme mannosidase II, the coat protein beta-COP, and the PDGF beta-receptor was retained. Moreover, the serum-induced proliferation of the cells was blocked. These findings suggest that signaling via PDGF receptor tyrosine kinases and phosphoinositide 3-kinases is not necessary for the shift of the smooth muscle cells from a contractile to a synthetic phenotype. On the other hand, these enzymes apparently carry out important functions in the control of intracellular membrane traffic and cell division.

Androstadienes↗

PubMed1998

An animal model to study local oxidation of LDL and its biological effects in the arterial wall.

Oxidized LDL (oxLDL) is present in atherosclerotic lesions and is believed to play a key role in atherogenesis. Mainly on the basis of cell culture studies, oxLDL has been shown to produce many biological effects that influence the atherosclerotic process. To study LDL oxidation in vivo, we have established a model in which Sprague-Dawley rats are given a single injection of unmodified human LDL (> or = 4 mg/kg body weight). Within 6 hours, an accumulation of apolipoprotein B and epitopes present on oxLDL are detected in the arterial endothelium and media. The presence of oxLDL is associated with activation of the transcription factor nuclear factor-kappaB in the endothelium as well as endothelial expression of intercellular adhesion molecule-1. Injection of LDL enriched with the antioxidant probucol resulted in arterial accumulation of apolipoprotein B, but the expression of oxLDL-specific epitopes was reduced at 24 hours. Thus, this simple model has the potential to analyze the mechanisms behind and biological effects of LDL oxidation in vivo.

Adult↗

PubMed1998

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.

Angioplasty↗

PubMed1998

Role of caveolae in cholesterol transport in arterial smooth muscle cells exposed to lipoproteins in vitro and in vivo.

Arterial smooth muscle cells are able to shift between two major differentiated states with distinct morphologic and functional properties, a contractile phenotype and a synthetic phenotype. Recently, it was demonstrated that contractile smooth muscle cells have numerous caveolae and that these specialized regions of the plasma membrane, to a large extent, are lost when the cells are modified into a synthetic phenotype. At the same time, the levels of the cholesterol-binding membrane protein caveolin remained unchanged and caveolin was redistributed from the cell surface to the perinuclear cytoplasm. In the present investigation, electron microscopy was used to study how smooth muscle cells of different phenotypes react to exposure to low-density lipoprotein and other lipoproteins both in vitro and in vivo. Our findings indicate that contractile cells (present early in primary culture and in the media of normal arterial walls) do not accumulate lipids in the cytoplasm and release excess cholesterol by means of plasma membrane caveolae. Extracellularly, the expelled lipids were built into membranous configurations and piled up as myelin-like deposits. In synthetic cells (formed after a few days in primary culture and as a response to arterial injury), lipids gathered in cytoplasmic droplets and increased amounts of membranous inclusions appeared in endosomes and lysosomes. On the other hand, no signs of extracellular discharge of lipids were detected. The results suggest that contractile smooth muscle cells use caveolin and caveolae to free themselves of excess lipoprotein-derived cholesterol and so manage to maintain a balance in the influx and efflux of cholesterol. Synthetic smooth muscle cells show a Golgi-like immunostaining for caveolin but have an insufficient capacity to use this protein to transport cholesterol to the plasma membrane and out of the cell. Cholesterol will then rather be esterified and collect in lipid droplets, eventually leading to foam cell formation if the uptake of lipoprotein continues.

Animals↗

PubMed1997

Controlling amyloid beta-peptide fibril formation with protease-stable ligands.

We have previously shown that short peptides incorporating the sequence KLVFF can bind to the approximately 40amino acid residue Alzheimer amyloid beta-peptide (Abeta) and disrupt amyloid fibril formation (Tjernberg, L. O., Näslund, J., Lindqvist, F., Johansson, J., Karlström, A. R., Thyberg, J., Terenius, L., and Nordstedt, C. (1996) J. Biol. Chem. 271, 8545-8548). Here, it is shown that KLVFF binds stereospecifically to the homologous sequence in Abeta (i.e. Abeta16-20). Molecular modeling suggests that association of the two homologous sequences leads to the formation of an atypical anti-parallel beta-sheet structure stabilized primarily by interaction between the Lys, Leu, and COOH-terminal Phe. By screening combinatorial pentapeptide libraries exclusively composed of D-amino acids, several ligands with a general motif containing phenylalanine in the second position and leucine in the third position were identified. Ligands composed of D-amino acids were not only capable of binding Abeta but also prevented formation of amyloid-like fibrils. These ligands are protease-resistant and may thus be useful as experimental agents against amyloid fibril formation in vivo.

Amino Acids↗

PubMed1997

Generation of Alzheimer amyloid beta peptide through nonspecific proteolysis.

Polymerization of Alzheimer amyloid beta peptide (Abeta) into amyloid fibrils is associated with resistance to proteolysis and tissue deposition. Here, it was investigated whether Abeta might be generated as a protease-resistant core from a polymerized precursor. A 100-amino acid C-terminal fragment of the Alzheimer beta-amyloid precursor protein (C100), containing the Abeta and cytoplasmic domains, polymerized both when inserted into membranes and after purification. When subjected to digestion using the nonspecific enzyme proteinase K, the cytoplasmic domain of C100 was degraded, whereas the Abeta domain remained intact. In contrast, dissociated C100 polymers were almost completely degraded by proteinase K. Mammalian cells transfected with the human Alzheimer beta-amyloid precursor gene contained a fragment corresponding to C100, which needed similar harsh conditions to be dissolved, as did polymers formed by purified C100. Hence, it was concluded that C100 polymers are formed in mammalian cells. These results suggest that the C terminus of Abeta can be generated by nonspecific proteases, acting on a polymerized substrate, rather than a specific gamma-secretase. This offers an explanation of how the Abeta peptide can be formed in organelles containing proteases capable of cleaving most peptide bonds.

Alzheimer Disease↗

PubMed1997

Expression of phenotype- and proliferation-related genes in rat aortic smooth muscle cells in primary culture.

OBJECTIVES: After endothelial injury, smooth muscle cells (SMCs) in the arterial media are modified from a contractile to a sympathetic phenotype. This process includes a prominent structural reorganization and makes the cells able to migrate into the intima, divide, and secrete extracellular matrix components. A similar change occurs in culture and then in vitro system has been established as a useful model in which to study the control of SMC differentiation. The purpose of this study was to analyze the expression of a number of phenotype- and proliferation-related genes in vascular SMCs during the first week in primary culture. METHODS: SMCs were enzymatically isolated from rat aorta and seeded on substrates of fibronectin (an adhesive plasma protein) and laminin-collagen type IV (two major basement membrane proteins) in a serum-free medium or in uncoated dishes in a serum-containing medium. Total RNA was isolated from the cells after different times of culture and analyzed by Northern blotting for expression of specific gene transcripts. In part, expression of the corresponding proteins was also explored by Western blotting and indirect immunofluorescence microscopy. RESULTS: The results indicate that the proto-oncogenes c-fos, c-jun and c-ets-1 were already activated during the isolation of the cells and then continued to be strongly expressed for a few days. Especially in the serum-free groups, there was also early activation of the genes for the matrix metalloproteinases, stromelysin (MMP-3) and type IV collagenase (MMP-2). In parallel, an increased expression of the genes for two extracellular matrix components was observed, with an early rise in osteopontin mRNA and a later rise in collagen type I mRNA. At the end of the test period, the corresponding proteins were deposited around the cells in a fibrillar pattern. Among the matrix receptors investigated, the beta 1 integrin subunit showed a high and persistent expression, whereas the alpha 5 and alpha 1 integrin subunits showed lower and more variable mRNA level. In support of the existence of an autocrine or paracrine platelet-derived growth factor (PDGF) loop, an early rise in expression of the PDGF A-chain gene and a subsequent rise in expression of the PDGF alpha-receptor gene were noted. CONCLUSION: It is proposed that the coordinated shift in gene expression here described to take place in connection with the phenotypic modulation of vascular SMCs in primary culture is part of a predetermined genetic program that normally serves the function to engage the cells in a wound healing response.

Animals↗

PubMed1997

Cell death in human atherosclerotic plaques involves both oncosis and apoptosis.

The aim of the present study was to analyze the frequency and mechanism of cell-death in atherosclerotic plaques with a recent history (< 6 months) of rupture. Atherosclerotic plaques were obtained from patients with symptomatic ipsilateral carotid stenosis > 70% diameter reduction undergoing carotid endarterectomy. In situ tailing and nick translation of fragmented DNA, agarose gel electrophoresis of plaque DNA and electron microscopy were used to identify cell death by apoptosis (programmed cell death) and oncosis. The mean number of cells containing fragmented DNA in the plaques was 12.7 +/- 3.5% (n = 15). Focal accumulations of cells with DNA fragmentation occurred in the fibrous cap, at sites of rupture, close to lipid deposits and necrosis and was always accompanied by the presence of inflammatory cells. Electrophoretic separation of DNA isolated from part of plaques, where the presence of DNA fragmentation had previously been demonstrated by in situ DNA nick translation, resulted in multiple ladders of 180-200 base pairs characteristic of apoptosis. Electron microscopic analysis revealed presence of cells with morphological signs of degeneration in a frequency even higher than that found by in situ nick translation. Some of these cells had a characteristic apoptotic appearance with condensed chromatin and cytoplasm, but the large majority of the cells had an ultrastructure typical for cells undergoing cell death by oncosis with membrane disruption and swollen, disintegrating organelles. Thus, although apoptosis clearly takes place in atherosclerotic plaques, oncosis appears to be a much more common mechanism for cell death.

Aged↗

PubMed1997

Role of tyrosine kinases in extracellular matrix-mediated modulation of arterial smooth muscle cell phenotype.

Fibronectin (FN) promotes the modulation of freshly isolated arterial smooth muscle cells (SMCs) from a contractile to a synthetic phenotype by interacting with integrins on the cell surface. This process is characterized by a structural and functional transformation of the cells, including a reorganization of the cytoskeleton, the formation of a large secretory apparatus, and the acquisition of proliferative capacity. In this study we have investigated the role of integrin signaling through tyrosine kinases in the structural changes that occur in SMCs during primary culture on FN. A gradual increase in phosphotyrosine staining in focal adhesions and a concomitant increase in tyrosine phosphorylation of proteins including focal adhesion kinase were observed. In contrast, cells seeded on laminin formed few focal adhesions, and tyrosine phosphorylation of proteins was less than in cells cultured on FN. Treatment of cells cultured on FN with the tyrosine kinase inhibitor genistein strongly suppressed focal adhesion formation, cell spreading, and cytoskeletal reorganization. In addition, electron microscopic analysis demonstrated that the phenotypic modulation was slowed down. These results indicate that the ability of extracellular matrix components to promote a change in the phenotypic properties of SMCs depends on the assembly of focal adhesions with associated tyrosine kinase activity.

Actins↗

PubMed1997

DNA fragmentation and ultrastructural changes of degenerating cells in atherosclerotic lesions and smooth muscle cells exposed to oxidized LDL in vitro.

Degeneration of smooth muscle cells in the fibrous cap of atherosclerotic lesions is an important factor in plaque rupture. Recent studies have suggested that many plaque cells are in a process of apoptosis as determined by positive deoxyribonucleotide-transferase-mediated dUTP end labeling. In this study, we demonstrate the existence of a colocalization between deoxyribonucleotide-transferase-mediated dUTP end labeling-positive smooth muscle cells and oxidized LDL immunoreactivity in human carotid plaques. Oxidized LDL was found to induce deoxyribonucleotide-transferase-mediated dUTP end labeling positivity in cultured human smooth muscle cells, but only in the presence of tumor necrosis factor-alpha and interferon-gamma. Electron microscopic analysis of cultured smooth muscle cells exposed to oxidized LDL in the absence of cytokines demonstrated cytoplasmic swelling and disruption of the plasma membrane, suggesting cell death by oncosis. Cells exposed to both oxidized LDL and cytokines were characterized by chromatin and cytoplasmic condensation compatible with cell death by apoptosis. These findings further support the notion that oxidized lipids play a role in plaque cell death.

Arteriosclerosis↗

PubMed1997

Phenotypic modulation of smooth muscle cells after arterial injury is associated with changes in the distribution of laminin and fibronectin.

Earlier in vitro studies suggest opposing roles of laminin and fibronectin in regulation of differentiated properties of vascular smooth muscle cells. To find out if this may also be the case in vivo, we used immunoelectron microscopy to study the distribution of these proteins during formation of intimal thickening after arterial injury. In parallel, cell structure and content of smooth muscle alpha-actin was analyzed. The results indicate that the cells in the normal media are in a contractile phenotype with abundant alpha-actin filaments and an incomplete basement membrane. Within 1 week after endothelial denudation, most cells in the innermost layer of the media convert into a synthetic phenotype, as judged by loss of actin filaments, construction of a large secretory apparatus, and destruction of the basement membrane. Some of these cells migrate through fenestrae in the internal elastic lamina and invade a fibronectin-rich network deposited on its luminal surface. Within another few weeks a thick neointima forms, newly produced matrix components replace the stands of fibronectin, and a basement membrane reappears. Simultaneously, the cells resume a contractile phenotype, recognized by disappearance of secretory organelles and restoration of alpha-actin filaments. These findings support the notion that laminin and other basement membrane components promote the expression of a differentiated smooth muscle phenotype, whereas fibronectin stimulates the cells to adopt a proliferative and secretory phenotype.

Actins↗

PubMed1997

Expression of caveolae on the surface of rat arterial smooth muscle cells is dependent on the phenotypic state of the cells.

Both after vascular injury and when established in vitro, arterial smooth muscle cells pass through a characteristic change in phenotype. This process includes a prominent structural reorganization with partial loss of myofilaments and formation of a large endoplasmic reticulum and Golgi complex. As a result, the cells lose their contractility and become able instead to divide and to secrete extracellular matrix components. In the present study, the expression of plasma membrane caveolae in rat arterial smooth muscle cells was studied in primary culture and during the formation of neointimal thickenings after balloon injury. Electron microscopic analysis revealed that the number of caveolae (identified as flask-shaped invaginations of the plasma membrane) was reduced when the cells converted from a contractile to a synthetic phenotype (as defined morphologically) and then increased again when they readopted a more differentiated state. However, immunoblotting analysis did not show any changes in the cellular content of caveolin (a major protein component of caveolae) during the 1st week in culture. At the same time, immunocytochemical staining demonstrated a shift in the localization of caveolin from small spot-like structures dispersed over the cell surface to vesicular structures in the perinuclear cytoplasm. These findings indicate that the transition of smooth muscle cells from a contractile to a synthetic phenotype involves a marked decline in the number of plasma membrane caveolae. In parallel, caveolin is internalized and redistributed to Golgi-associated vesicles in the perinuclear cytoplasm. In context of the rapidly increasing awareness of the importance of caveolae both in signal transduction and intracellular cholesterol transport, it seems likely that the variations in the number of caveolae may be significant for the functional differences between smooth muscle cells in different phenotypes.

Animals↗

PubMed1997

Ca2+ channel blockers verapamil and nifedipine inhibit apoptosis induced by 25-hydroxycholesterol in human aortic smooth muscle cells.

We have characterized the death of human aortic smooth muscle cells induced by 25-hydroxycholesterol, an oxidation product of cholesterol. Chromatin condensation characteristic of apoptosis was observed by enzymatic (TUNEL) staining of chromatin, and by electron microscopy. Fourteen percent of cells treated with 5 microg/ml of 25-hydroxycholesterol for 24 h displayed chromatin degradation as determined by positive TUNEL staining. Addition of TNF alpha (10 ng/ml) and IFN gamma (20 ng/ml) increased the proportion of TUNEL positive cells to 30%, whereas the cytokines alone were without effect. After 48 h, 40% of the cells treated with 5 microg/ml of 25-hydroxycholesterol were TUNEL positive, and 21% of the cells displayed chromatin condensation. Oligonucleosomal DNA fragmentation typical of apoptosis was demonstrated by agarose gel electrophoresis. Furthermore, activation of the ICE-like protease caspase 3 (CPP32) was observed in cells treated with 25-hydroxycholesterol. Addition of the Ca2+ entry blockers verapamil or nifedipine to the culture medium inhibited apoptosis by more than 70% and reduced cytotoxicity, while removal of Ca2+ from culture medium reduced apoptosis by 42%. Within a few minutes after addition, 25-hydroxycholesterol induced intracellular Ca2+ oscillations with a frequency of approximately 0.3-0.4 min(-1). Thus it appears that Ca2+ influx through plasma membrane channels is an important signal in oxysterol-induced apoptosis. Addition of TNF alpha and IFN gamma enhanced cytotoxicity and resulted in a higher proportion of apoptotic cells, suggesting that inflammatory cytokines can increase the cytotoxicity of lipid oxidation products.

Aorta↗

PubMed1996

Arrest of beta-amyloid fibril formation by a pentapeptide ligand.

Polymerization of amyloid beta-peptide (Abeta) into amyloid fibrils is a critical step in the pathogenesis of Alzheimer's disease. Here, we show that peptides incorporating a short Abeta fragment (KLVFF; Abeta16-20) can bind full-length Abeta and prevent its assembly into amyloid fibrils. Through alanine substitution, it was demonstrated that amino acids Lys16, Leu17, and Phe20 are critical for binding to Abeta and inhibition of Abeta fibril formation. A mutant Abeta molecule, in which these residues had been substituted, had a markedly reduced capability of forming amyloid fibrils. The present data suggest that residues Abeta16-20 serve as a binding sequence duringA beta polymerization and fibril formation. Moreover, the present KLVFF peptide may serve as a lead compound for the development of peptide and non-peptide agents aimed at inhibiting Abeta amyloidogenesis in vivo.

Amino Acid Sequence↗

PubMed1996

Differentiated properties and proliferation of arterial smooth muscle cells in culture.

The smooth muscle cell is the sole cell type normally found in the media of mammalian arteries. In the adult, it is a terminally differentiated cell that expresses cytoskeletal marker proteins like smooth muscle alpha-actin and smooth muscle myosin heavy chains, and contracts in response to chemical and mechanical stimuli. However, it is able to revert to a proliferative and secretory active state equivalent to that seen during vasculogenesis in the fetus, and this is a prerequisite for the involvement of the smooth muscle cell in the formation of atherosclerotic and restenotic lesions. A similar transition from a contractile to a synthetic phenotype occurs when smooth muscle cells are established in culture. Accordingly, an in vitro system has been used extensively to study the regulation of differentiated properties and proliferation of these cells. During the first few days after seeding, the cells are reorganized structurally with a loss of myofilaments and formation of a widespread endoplasmic reticulum and a prominent Golgi complex. In parallel, they lose their contractility and instead become competent to divide in response to a large variety of mitogens, including platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF). After entering the cell cycle, they start to produce these and other mitogens on their own, and continue to replicate in the absence of exogenous stimuli for a restricted number of generations. Furthermore, they start to secrete extracellular matrix components such as collagen, elastin, and proteoglycans. The mechanisms that control this change in morphology and function of the smooth muscle cells are still poorly understood. Adhesive proteins such as fibronectin and laminin apparently have an important role in determining the basic phenotypic state of the cells and exert their effects via integrin receptors. The proliferative and secretory activities of the cells are influenced by a multitude of growth factors, cytokines, and other molecules. Although much work remains before an integrated view of this regulatory machinery can be achieved, there is no doubt that the cell culture technique has contributed substantially to our knowledge of smooth muscle differentiation and growth. At the same time, it has been crucial in exploring the role of these cells in vascular disease and developing new therapeutic strategies to cope with major causes of human death and disability.

Animals↗

PubMed1995

Phenotypic modulation of smooth muscle cells during the formation of neointimal thickenings in the rat carotid artery after balloon injury: an electron-microscopic and stereological study.

The formation of neointimal thickenings in the rat carotid artery after balloon injury was studied by a combination of electron-microscopic and stereological methods. All smooth muscle cells in the normal media had a contractile phenotype, the cytoplasm being dominated by myofilaments. Seven days after endothelial denudation, the smooth muscle cells in the innermost part of the media had assumed a synthetic phenotype by loss of myofilaments and formation of a large endoplasmic reticulum and Golgi complex. These cells moved through fine openings in the internal elastic lamina and gave rise to a growing neointima by proliferation and secretion of extracellular matrix components. Fourteen days after the operation, the neointima had almost reached its final size, and mitoses were no longer noted. Nevertheless, the cells maintained a synthetic phenotype with prominent secretory organelles, although myofilaments had started to become more abundant again. They were surrounded by an extracellular matrix made up of collagen fibrils and coalescing patches of elastin. Thirty-five days after the operation, an endothelial cell layer had reformed and covered most of the luminal vessel surface. In parallel, the smooth muscle cells in the neointima had returned to a contractile phenotype with a cytoplasm dominated by myofilaments. These findings provide a morphological basis for further analysis of the cellular and molecular interactions involved in the formation of neointimal thickenings after endothelial injury, and for the search for agents interfering with this process.

Animals↗