Mitochondrial DNA deletions and depletion within paraspinal muscles.

AIMS: Although mitochondrial abnormalities have been reported within paraspinal muscles in patients with axial weakness and neuromuscular disease as well as with ageing, the basis of respiratory deficiency in paraspinal muscles is not known. This study aimed to determine the extent and basis of respiratory deficiency in paraspinal muscles from cases undergoing surgery for degenerative spinal disease and post mortem cases without a history of spinal disease, where age-related histopathological changes were previously reported. METHODS: Cervical and lumbar paraspinal muscles were obtained peri-operatively from 13 patients and from six post mortem control cases (age range 18-82 years) without a neurological disease. Sequential COX/SDH (mitochondrial respiratory chain complex IV/complex II) histochemistry was performed to identify respiratory-deficient muscle fibres (lacking complex IV with intact complex II activity). Real-time polymerase chain reaction, long-range polymerase chain reaction and sequencing were used to identify and characterize mitochondrial DNA (mtDNA) deletions and determine mtDNA copy number status. Mitochondrial respiratory chain complex subunits were detected by immunohistochemistry. RESULTS: The density of respiratory-deficient fibres increased with age. On average, 3.96% of fibres in paraspinal muscles were respiratory-deficient (range 0-10.26). Respiratory deficiency in 36.8% of paraspinal muscle fibres was due to clonally expanded mtDNA deletions. MtDNA depletion accounted for further 13.5% of respiratory deficiency. The profile of immunohistochemically detected subunits of complexes was similar in respiratory-deficient fibres with and without mtDNA deletions or mtDNA depletion. CONCLUSIONS: Paraspinal muscles appeared to be particularly susceptible to age-related mitochondrial respiratory chain defects. Clonally expanded mtDNA deletions and focal mtDNA depletion may contribute towards the development of age-related postural abnormalities.

Gene expression profile of the fibrotic response in the peritoneal cavity.

The cellular response to materials implanted in the peritoneal cavity has been utilised to produce tissue for grafting to hollow smooth muscle organs (blood vessels, bladder, uterus and vas deferens). To gain insight into the regulatory mechanisms involved in encapsulation of a foreign object, and subsequent differentiation of encapsulating cells, the present study used microarray technology and real-time RT-PCR to identify the temporal changes in gene expression associated with tissue development. Immunohistochemical analysis showed that 3-7 days post-implantation of foreign objects (cubes of boiled egg white) into rats, they were encapsulated by tissue comprised primarily of haemopoietic (CD45(+)) cells, mainly macrophages (CD68(+), CCR1(+)). By day 14, tissue capsule cells no longer expressed CD68, but were positive for myofibroblast markers alpha-smooth muscle (SM) actin and SM22. In accordance with these results, gene expression data showed that early capsule (days 3-7) development was dominated by the expression of monocyte/macrophage-specific genes (CD14, CSF-1, CSF-1R, MCP-1) and pro-inflammatory mediators such as transforming growth factor (TGF-beta). As tissue capsule development progressed (days 14-21), myofibroblast-associated and pro-fibrotic genes (associated with TGF-beta and Wnt/beta-catenin signalling pathways, including Wnt 4, TGFbetaRII, connective tissue growth factor (CTGF), SMADs-1, -2, -4 and collagen-1 subunits) were significantly up-regulated. The up-regulation of genes associated with Cardiovascular and Skeletal and Muscular System Development at later time-points suggests the capacity of cells within the tissue capsule for further differentiation to smooth muscle, and possibly other cell types. The identification of key regulatory pathways and molecules associated with the fibrotic response to implanted materials has important applications not only for optimising tissue engineering strategies, but also to control deleterious fibrotic responses.

Cytoplasmic filaments in developing and adult vertebrate smooth muscle.

An extensive study of adult and developing smooth muscle has revealed the widespread occurrence of a distinct filament with an average diameter of about 100 A (termed the 100 A filament). Unlike that of myofilaments, their appearance in longitudinal section is uniform, but in transverse section they have a round profile, occasionally exhibiting a less electron-opaque core. The 100 A filaments are almost invariably preserved under a variety of fixation procedures, whereas myofilaments, particularly the thicker filaments, are preserved inconsistently. The 100 A filaments appear to be randomly oriented throughout the cytoplasm, either singly or in small groups, although they are sometimes concentrated in the juxtanuclear region of the smooth muscle cells. The intimate association of 100 A filaments with dark bodies, in both developing and adult smooth muscle cells, may indicate that these filaments either play a role in dark body formation or, at least, constitute a part of the dark body. The 100 A filaments are conspicuous in developing smooth muscle cells and occasionally form networks or clusters; they appear to decrease in relative number as maturation proceeds, but considerable numbers are still present in adult tissue.

Fine structure of smooth muscle cells grown in tissue culture.

The fine structure of smooth muscle cells of the embryo chicken gizzard cultured in monolayer was studied by phase-contrast optics and electron microscopy. The smooth muscle cells were irregular in shape, but tended to be elongate. The nucleus usually contained prominent nucleoli and was large in relation to the cell body. When fixed with glutaraldehyde, three different types of filaments were noted in the cytoplasm: thick (150-250 A in diameter) and thin (30-80 A in diameter) myofilaments, many of which were arranged in small bundles throughout the cytoplasm and which were usually associated with dark bodies; and filaments with a diameter of 80-110 A which were randomly orientated and are not regarded as myofilaments. Some of the aggregated ribosomes were helically arranged. Mitochondria, Golgi apparatus, and dilated rough endoplasmic reticulum were prominent. In contrast to in vivo muscle cells, micropinocytotic vesicles along the cell membrane were rare and dense areas were usually confined to cell membrane infoldings. These cells are compared to in vivo embryonic smooth muscle and adult muscle after treatment with estrogen. Monolayers of cultured smooth muscle will be of particular value in relating ultrastructural features to functional observations on the same cells.

Phenotype-dependent response of cultured aortic smooth muscle to serum mitogens.

Smooth muscle cells from the aortic media of adult pigs and monkeys have been grown in primary culture by plating cells enzymatically dissociated from the intact aorta. During the first 6 d these cells are in the "contractile" phenotype. That is, they contract slowly in response to angiotensin II and their cytoplasm is filled with both thick and thin myofilaments. In this state they do not incorporate [3H]thymidine into DNA or proliferate in response to normolipemic or hyperlipemic whole blood serum (WBS). After 7 d in culture the cells undergo a spontaneous modulation of phenotype to a "synthetic" state where they cannot be stimulated to contract and their cytoplasm is filled with organelles usually associated with synthesis of secretory protein. Thick myosin-containing filaments can no longer be demonstrated. When challenged with normolipemic or hyperlipemic WBS the cells incorporate [3H]thymidine into DNA and undergo logarithmic growth. It is suggested that when smooth muscle is the contractile phenotype (as normally exists for most cells in the aortic media of adult animals) it does not divide when challenged with serum mitogens but can undergo a change of phenotype to a synthetic state in which division can be stimulated.

Different nucleosome structures on transcribing and nontranscribing ribosomal gene sequences.

Monomeric DNA lengths from Physarum nuclear chromatin occur in two subunit forms which differ from each other and from higher oligomers of nucleosomes in content of transcribed ribosomal DNA sequences. Labeled DNA restriction fragments from ribosomal RNA coding regions reanneal most rapidly with DNA from a monomeric subunit fraction. A particles, isolated from growing plasmodia and containing 144 base pairs of DNA in an extended conformation. Higher oligomers of nucleosomes are depleted in sequences from transcribing gene regions but are enriched in sequences from the nontranscribed central spacer of the ribosomal DNA palindrome. Nucleosome configuration on two 26S gene intervening sequences resembles that on adjacent coding regions.

Possible derivation of the laboratory mouse genome from multiple wild Mus species.

Laboratory strains of mice are thought to be derived from wild populations of Mus domesticus. Many instances of non-domesticus genetic information fixed in these strains have been described, however, and the amount of strain-to-strain genetic variation exceeds that found in wild domesticus populations. In order to estimate the extent of the non-domesticus contribution to laboratory mouse genomes, and to determine whether it could account for observed variation, we have used computer simulations to investigate the properties of genetically marked chromosomal segments and the distribution of residual allogenicity at various times during inbreeding. A locus or chromosomal segment is allogenic if it is unfixed within a lineage at a given time. The odds of fixation of a foreign chromosome segment are predicted to be an exponentially decreasing function of its length. The median segment length is predicted to be 17 centimorgans. Available data for markers of chromosomes 1, 9 and 12 in recombinant inbred strain sets conform to these predictions. Together, the results suggest that introgression of non-domesticus chromosomes and segregation of residual allogenicity are sufficient to account for the genetic diversity observed among inbred mouse strains and substrains.

A linkage map of mouse chromosome 12: localization of Igh and effects of sex and interference on recombination.

Inheritance of restriction fragment length polymorphisms associated with four anonymous DNA markers (D12Nyu1, 2, 3 and 4), the Fos proto-oncogene, the Mtv-9 viral integration site, and the alpha 1-antitrypsin (Aat-1) and immunoglobulin heavy chain (Igh) gene families in the mouse has been followed in a backcross experiment. A Bayesian multilocus map-building strategy yielded the map: centromere-D12Nyu2-10 cM-D12Nyu1-2 cM-D12Nyu3-15 cM-Fos-1 cM-D12Nyu4-2 cM-Mtv-9-8 cM-Aat-1-17 cM-Igh-C. A map constructed from male meiotic data was substantially shorter than one constructed from female meiotic data. Significant interference was observed for the linkage group. Two groups of markers studied in recombinant inbred strains of mice could be interpolated into the map: Es-25, D12Nyu10, D12Nyu7 and Apob form a cluster proximal to D12Nyu2, and Ly-18, Ah, and D12Nyu5 form a cluster between D12Nyu2 and D12Nyu1. These data establish an unambiguously ordered linkage group including Igh and Aat-1 that spans most of chromosome 12.

Effect of cross-transplantation on normotensive and spontaneously hypertensive rat arterial muscle membrane.

Transplantation of arteries into the anterior eye chamber of rats for 8 weeks was used to test the hypothesis that the neurohumoral environment is important in establishing the altered membrane potential (observable during electrogenic ion transport inhibition) of vascular muscle in hypertension. When caudal arteries from 12- to 16-week-old spontaneously hypertensive rats (SHR) or genetically matched Kyoto-Wistar normotensive rats (KNR) were transplanted into the opposite strain, there was no change in the transport inhibited membrane potential (Em) of the arterial muscle cells from that found in freshly excised donor arteries. However, when caudal arteries from 2-week-old animals were transplanted into the anterior eye chamber, the arteries always developed the appropriate Em for the host animal. In other words, a genetically KNR artery developed the Em of an SHR artery in an SHR host; conversely, a genetically SHR artery developed the Em of a KNR artery in the KNR host. These results provide evidence that: 1) the differences between th Em of caudal arteries from SHR and KNR are not inherent in those muscle cells; 2) the change in Em is triggered in young animals preceding development of hypertension, but not after hypertension is established; and 3) the Em alteration of the caudal artery is independent of structural changes that occur in the artery as a result of increased blood pressure (because KNR transplants were not connected in series with the host anterior eye chamber vasculature and subject to the elevated blood pressures). We conclude that the arterial muscle cells up to a certain age respond to an external factor that regulates their Em and presumably their sensitivity to vasopressor agents.

What controls smooth muscle phenotype?

Enzyme-dispersed smooth muscle cells from the adult pig aortic media in the first few days of primary culture are in the contractile phenotype and do not divide when challenged with 5% WBS. After 6--8 days the isolated cells spontaneously undergo a change in phenotype where contraction cannot be stimulated and the cells respond to mitogens in WBS by logarithmic growth. The change in phenotype is reversible if the cells are seeded sufficiently densely (5 x 10(4) to 1 x 10(5)/ml) that a confluent monolayer results after less than 1 week of proliferation, but is irreversible if the cells are seeded sparsely (1 x 10(3) to 5 x 10(3)/ml) and take more than 2 weeks of proliferation to reach confluence. When the cells are seeded so densely (10(6)/ml) that a confluent monolayer is present from day 1, the cells do not undergo a change in phenotype but remain indefinitely in the contractile state. The spontaneous modulation of phenotype of isolated smooth muscle cells can be inhibited by a confluent monolayer of contractile smooth muscle or endothelial cells in co-culture with the sparsely seeded smooth muscle such that the two cell layers are not in contact but bathed by the same nutrient medium. Smooth muscle modulation can also be inhibited by a factor extracted from pig and rabbit aortic tissue, and its effects mimicked by commercially available sodium heparin at 50 units/ml.

Effect of endothelium on beta-VLDL metabolism by cultured smooth muscle cells of differing phenotype.

The effect of endothelial cells (EC) on the binding and internalisation of beta-very low density lipoprotein (beta-VLDL) and the subsequent accumulation of lipid was investigated in cultured smooth muscle cells (SMC) of different phenotype. The following combinations were examined: (i) SMC cultured and incubated with 125I-beta-VLDL without EC: "control" cultures; (ii) SMC co-cultured with EC and incubated with 125I-beta-VLDL without EC: "separated" cultures; and (iii) SMC co-cultured with EC and incubated with 125I-beta-VLDL in the presence of EC: "co-incubated" cultures. SMC were in the contractile (CON), reversible synthetic (RS) or irreversible synthetic (IRS) phenotype and EC were either actively proliferating or confluent and quiescent. All three SMC phenotypes showed the greatest capacity to bind and internalise 125I-beta-VLDL with accumulation of lipid when "co-incubated" with confluent EC. SMC "co-incubated" with proliferating EC showed a lower capacity to bind and internalise the lipoprotein and accumulate lipid, while "control" SMC showed the lowest capacity for all phenotypes. IRS SMC bound more 125I-beta-VLDL than either RS or CON state phenotypes. In addition, IRS SMC "co-incubated" with confluent EC showed the greatest degree of binding, and IRS SMC incubated with EC-conditioned medium and EC-conditioned 125I-beta-VLDL showed a significant increase in binding above control (fresh medium and fresh 125I-beta-VLDL). The degree of binding 125I-beta-VLDL to SMC was affected by the functional state of the EC. That is, SMC "co-incubated" with confluent EC bound more lipoprotein than SMC "co-incubated" with the same number of proliferating EC. These results are consistent with observations by others who report preferential lipid accumulation in regions of denuded artery recently recovered by endothelium compared with regions lacking an endothelium. The results also indicate that the EC both modify the beta-VLDL particle and affect the biology of the SMC themselves.

Perindopril inhibits both the development of atherosclerosis in the cholesterol-fed rabbit and lipoprotein binding to smooth muscle cells in culture.

The aim of the study was to examine the effect of low doses of perindopril, approximating those used therapeutically and sub-therapeutically in human hypertensives, on the development of atherosclerosis in the cholesterol-fed rabbit. The right carotid artery of 12 week old rabbits was balloon de-endothelialized to induce the formation of a myointimal thickening. After 14 weeks rabbits were placed into 6 groups, 6 rabbits per group. Groups I, II and III were fed a 1% cholesterol diet for the following 6 week experimental period, while Groups IV, V and VI received a normolipemic diet. In addition, Groups II and V rabbits received in their drinking water 0.3 mg/kg per day perindopril, and Groups III and VI 0.01 mg/kg per day. At the end of 6 weeks' treatment, the mean arterial pressure (MAP) in Groups II and V decreased by about 12%, while that in Groups III and VI decreased by 13%. Plasma cholesterol levels of rabbits on a normolipemic diet (Groups IV, V, VI) averaged 1.3 mmol/l while those on a cholesterol-enriched diet (Groups I, II, III) averaged 10.5 mmol/l. Plasma perindoprilat concentrations and percentage of plasma angiotensin converting enzyme (ACE) inhibition in Groups II and V averaged 14 ng/ml and 92.1% respectively, while in Groups III and VI they were 5.7 ng/ml and 80.5%, respectively. The percentage luminal surface area of the thoracic aorta covered by lipid-filled plaques (as observed by en face staining with Oil-Red-O) averaged 26.3% in Group I, 4.7% in Group II and 20.0% in Group III. No lesions developed in Groups IV, V and VI. Microscopic examination of the right (manipulated) carotid arteries of Group I rabbits revealed lesions of large, lipid-filled cells radially oriented, overlying the pre-formed myointimal thickening. Both doses of perindopril in the cholesterol-fed rabbits (Groups II and III) decreased the amount of lipid-filled cells which were oriented circumferentially. More extracellular matrix was present in the lesions of Groups II and III than of Group I. No lesions were observed in the right carotid arteries of Groups IV, V and VI (normal diet) or in the unmanipulated left carotid arteries of all 6 groups. The sizes of the neointima plus lesion in Groups I, II and III were, however, not significantly different, being 42.4%, 48.5% and 46.9% of the cross-sectional area of the artery wall.(ABSTRACT TRUNCATED AT 400 WORDS)

Glycosaminoglycan synthesis by smooth muscle cells of differing phenotype and their response to endothelial cell conditioned medium.

The synthesis of glycosaminoglycans (GAG) by contractile and irreversible synthetic phenotypes of vascular smooth muscle cells (SMC), and their response to endothelial cell conditioned medium (ECCM), has been investigated. Contractile SMC, (with a high volume fraction of myofilaments) were obtained by culturing freshly isolated rabbit aortic SMC for 3 days in primary culture. Irreversible synthetic SMC (with a low volume fraction of myofilaments) were obtained by serially passaging SMC to achieve more than 5 cumulative population doublings. In fresh medium both phenotypes produced significant amounts of GAG, but irreversible synthetic cells were more than twice as active on a per cell and cell volume basis. The proportions of individual GAG also changed with change in phenotype. Hyaluronic acid (HA) was the predominant GAG (78%) synthesised by contractile SMC but was significantly reduced (47%) in the irreversible synthetic cells with a corresponding increase in sulphated GAG (SGAG). The changed levels in GAG synthesis were independent of SMC growth. Both phenotypes responded to ECCM from bovine endothelial cells (EC) and significantly increased their synthesis of GAG and by the same relative amounts (50-100%). This response was density dependent, with ECCM from low and high density cultures of EC producing maximal responses and EC of intermediate densities producing minimal increases. Furthermore, dense cultures of EC preferentially stimulated SGAG. These findings show that an increase in synthesis of SMC GAG, and especially sulphated GAG as is found in atherosclerosis, may occur either through a change in phenotype or through endothelial mediated stimulation of GAG synthesis by either phenotype.

Vascular smooth muscle phenotype and growth behaviour can be influenced by macrophages in vitro.

The effect of macrophages on rabbit vascular smooth muscle phenotype and proliferative ability was examined using ultrastructural morphometry. The volume fraction of myofilaments (Vv myo) in smooth muscle cells (SMC) from 9-week-old rabbit aorta in vivo was 39.5 +/- 1.2%. After seeding the enzymatically isolated SMC at 4 X 10(5) cells/ml in primary culture, the Vv myo was 38.9 +/- 1.2% on day 3 dropping to 29.9 +/- 2.0% by day 5. On day 6 the Vv myo was 29.2 +/- 1.8%, and the cells began to proliferate. Confluency was reached after less than 24 h proliferation and the Vv myo rose abruptly on day 7 to 36.9 +/- 1.9%. When the SMC were co-cultured with macrophages, the Vv myo fell to 31.2 +/- 0.9% on day 3 and to 25.9 +/- 0.5% on day 5 at which time cells commenced proliferation. Confluency occurred on day 6 but the SMC Vv myo did not rise throughout the rest of the culture period (27.4 +/- 1.8% and 26.9 +/- 1.3% on days 7 and 9, respectively) and the cells, unlike the controls, continued to proliferate, becoming multi-layered. Early phenotypic modulation in sparsely seeded SMC (8 X 10(4) cells/ml) co-cultured with macrophages was also found using fluorescent labelled antibodies to smooth muscle myosin. Measurement of proliferation by cell counts (and tritiated thymidine autoradiography) showed that macrophages stimulated SMC in primary culture to proliferate at a significantly greater rate than control cells grown alone in 5% whole blood serum (WBS). Proliferation of subcultured SMC co-cultured with macrophages was also stimulated.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid accumulation in arterial smooth muscle cells. Influence of phenotype.

Isolated smooth muscle cells from the adult pig and rabbit aorta in primary culture undergo a spontaneous change in phenotype from a contractile to a synthetic state over 6-8 days, losing their capacity to contract and gaining the capacity to divide. The change in smooth muscle phenotype to the synthetic state is accompanied by distinct changes in the cells' ability to metabolize LDL, with the rate of degradation of 125I-labelled LDL decreasing to about one fifth of the level in contractile state cells. This does not appear to be due to changes in the number or affinity of LDL receptors since saturable binding of LDL is unaltered. The specific activities of the lysosomal enzymes acid phosphatase and N-acetyl-beta-glucosaminidase increase with change to the synthetic state as do cytochrome c oxidase (mitochondria) and NADPH-dependent cytochrome c reductase (endoplasmic reticulum). In contrast there is a slight but not significant decrease in the specific activity of the lysosomal enzyme acid cholesteryl esterase of rabbit smooth muscle cells and a significant decrease in the activity of pig cells with change in phenotype to the synthetic state. Significantly more [3H]cholesteryl oleate is recovered in synthetic state than in contractile state cells following incubation with 20 micrograms/ml unlabelled LDL and [3H]sodium oleate. Morphologically there is no difference in the number of lipid droplets in contractile and synthetic state cells after incubation in 5% normolipemic serum, but in cells grown in 10% hyperlipemic serum for 4 days synthetic state cells become almost completely filled with lipid droplets while contractile state cells are unaffected. Lipid accumulation also occurs selectively in vivo in synthetic as compared with contractile state smooth muscle cells within intimal fibromuscular thickenings induced by de-endothelialization of the carotid artery of cholesterol-fed rabbits. We suggest that accumulation of lipid in smooth muscle cells of atherosclerotic plaques is related to reduced catabolism of LDL following smooth muscle phenotypic change from the contractile to synthetic state.

Expression of growth factor receptors in arterial smooth muscle cells. Dependency on cell phenotype and serum factors.

The effects of modulation of rabbit aortic smooth muscle cells (SMCs) from the 'contractile' phenotype on surface membrane receptors binding epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and platelet-derived growth factor-BB (PDGF-BB), as well as their responsiveness to these growth factors was investigated in cell culture. Cells predominantly of the 'contractile' phenotype expressed low numbers of high affinity EGF and bFGF receptors (EGFr: 1.09 +/- 0.18 fmol/10(6) cells; bFGFr: 0.32 +/- 0.07 fmol/10(6) cells). Upon modulation from the 'contractile' phenotype, the expression of these cell surface receptors increased greatly: 8- and 11-fold with respect to EGF and bFGF receptors. Cell surface receptors binding [125I]-PDGF-BB were largely unaltered. The elevated bFGF receptor number appeared dependent on SMC modulation from the 'contractile' phenotype and serum; the latter factor did not influence EGF receptor numbers. In both instances the increase in receptor numbers was independent of the proliferation status of the cells. Cells expressing high levels of the growth factor receptors also rapidly entered the cell cycle, proliferated, and exhibited growth factor-specific changes in shape in the presence of these growth factors. Because the effects on growth factor receptor numbers were observed in confluent cells, such alterations, are likely to play a significant role in vessel remodelling following balloon catheter angioplasty, in atherosclerotic vessels and the vascular hypertrophy associated with hypertension.

Matrix metalloproteinase can facilitate the heparanase-induced promotion of phenotype change in vascular smooth muscle cells.

Previous studies from this laboratory have shown that degradation of heparan sulphate proteoglycan by both living macrophages and macrophage lysosomal heparanase induces phenotypic change of vascular smooth muscle cells (SMC) from a high volume fraction of myofilaments (V(v)myo) to a low V(v)myo [Campbell et al. Exp Cell Res 1992; 200: 156-167]. The aim of this study was to determine whether matrix metalloproteinase (MMP) activity is also involved in the induction of SMC phenotypic change by macrophages. A specific inhibitor of MMPs (BB94) was able to block macrophage-induced SMC phenotypic change and subsequent DNA synthesis in freshly dispersed SMC seeded in primary culture at confluent density. The inhibitor did not block these SMC changes when SMC were seeded at low density without macrophages nor did it block heparanase activity directly. We also determined whether heparanase and MMP activities are upregulated together in vivo. Artery homogenates were analysed in a heparanase enzyme assay and for MMPs using zymograms. Increased heparanase activity was observed 3-14 days following balloon catheter injury of rabbit carotid arteries, and returned to control levels 6 weeks after injury. Active MMP2 was induced with heparanase after injury. MMP9 induction was also apparent 6 h after injury. Immunohistology on sections of these arteries showed the presence of MMPI1, 2, 3 and 9 with these MMPs being strongly induced in the intima 7 days after balloon catheter injury. Both heparanase and MMP activities were also present in human end-stage complex lesions from coronary arteries, carotid endarterectomies and abdominal aortic aneurysms. Because MMPs and heparanase are expressed at the same time, it is possible that MMPs facilitate heparanase activity in promotion of phenotypic modulation of SMC in vivo during neointimal thickening following injury and in atherosclerotic lesions.

ICAM-1 expression by vascular smooth muscle cells is phenotype-dependent.

Atherosclerosis is an inflammatory disease characterised by increased expression of adhesion molecules for leukocytes on both the surface of dysfunctional endothelium and on smooth muscle cells (SMC) within the lesion. It is also characterised by altered SMC phenotypic expression, indicated by a decreased volume fraction of myofilaments (V(v)myo) [1,2] and changes in gene expression [3]. The present study used an in vitro model to investigate, by immunofluorescence staining and flow cytometry, the influence of phenotype on vascular SMC expression of the adhesion molecule for leukocytes, intracellular adhesion molecule-1 (ICAM-1), and the regulatory mechanisms involved in this process. Smooth muscle cells with a high V(v)myo, freshly isolated from rat aortic media, expressed little or no ICAM-1 and this could not be induced by interleukin-1beta (IL-1beta). As SMC modulated phenotype, indicated by decreasing V(v)myo over the first 5 days of culture, there was a concomitant increase in ICAM-1 expression. At day 9 of primary culture, when SMC cultures had returned to the high V(v)myo phenotype, ICAM-1 expression was markedly lower. However, these cells retained the capacity to express ICAM-1 in response to IL-1beta. After several passages in culture, cells (with a low V(v)myo) constitutively expressed ICAM-1, with levels further up-regulated in response to IL-1beta. These changes in ICAM-1 expression were not related to proliferative state, since similar results were obtained with growth arrested SMC. Investigation of signalling pathways involved in regulating ICAM-1 expression by primary vascular SMC suggested a complex regulatory mechanism. Activation of adenyl cyclase (with forskolin) caused a significant increase in cells expressing ICAM-1. Treatment with inhibitors of protein kinase C (chelerythrine chloride), protein tyrosine kinase (genistein), or the transcription factor NF-kappaB (PDTC) had no significant effect on IL-1-induced ICAM-1 expression. However, in the presence of serum, both genistein and PDTC caused a significant increase in basal expression. The results indicate that ICAM-1 expression by SMC is phenotype-dependent, with expression evident only after cells have modulated to a low V(v)myo phenotype. They also indicate the existence of complex regulatory mechanisms, possibly involving the SMC cytoskeleton.

The effect of the aged garlic extract, 'Kyolic', on the development of experimental atherosclerosis.

The aged garlic extract 'Kyolic' lowers serum cholesterol levels in humans and experimental animals and thus is presumed to have a protective effect against atherosclerosis. However, to date no studies have examined the effect of this substance on the actual development of the disease. In the present study, the right carotid artery of 24 rabbits was de-endothelialized by balloon catheterisation in order to produce a myointimal thickening. After 2 weeks the rabbits were randomly assigned to four groups: Group I received a standard diet; Group II received the standard diet supplemented with 800 microl/kg body weight/day 'Kyolic'; Group III received a 1% cholesterol supplemented standard diet; and Group IV received a 1% cholesterol supplemented standard diet plus 'Kyolic'. After 6 weeks, the cholesterol diet caused a 6-fold increase in serum cholesterol level (Group III; 6.4 +/- 0.6 mmol/l) compared to normal diet (Group I; 1.2 +/- 0.4 mmol/l) (P < 0.05) with only a minor, non-significant reduction seen by the addition of 'Kyolic' (Group IV; 6.2 +/- 0.7 mmol/l). Group III rabbits developed fatty streak lesions covering approximately 70 +/- 8% of the surface area of the thoracic aorta, which was significantly reduced to 25 +/- 3% in the 'Kyolic'-treated Group IV. No lesions were present in Groups I and II. The hypercholesterolaemic diet caused an increase in aortic arch cholesterol (2.1 +/- 0.1 mg cholesterol/g tissue) which was significantly reduced by 'Kyolic' supplementation (1.7 +/- 0.2 mg cholesterol/g tissue) (P < 0.05). 'Kyolic' significantly inhibited the development of thickened, lipid-filled lesions in the pre-formed neointimas produced by balloon-catheter injury of the right carotid artery in cholesterol-fed rabbits (intima as percent of artery wall, Group III 42.6 +/- 6.5% versus Group IV 23.8 +/- 2.3%, P < 0.01), but had little effect in rabbits on a standard diet (Group II 18.4 +/- 5.0% versus Group I 16.7 +/- 2.0%). In vitro studies showed that 'Kyolic' has a direct effect on inhibition of smooth muscle proliferation. In conclusion, 'Kyolic' treatment reduces fatty streak development, vessel wall cholesterol accumulation and the development of fibro fatty plaques in neointimas of cholesterol-fed rabbits, thus providing protection against the onset of atherosclerosis.

Effect of estrogen on vascular smooth muscle cells is dependent upon cellular phenotype.

To investigate the growth-regulating action of estrogen on vascular smooth muscle cells (SMC), effects of beta-17-estradiol (beta-E2) on phenotypic modulation and proliferation of rabbit aortic SMC were observed in vitro. At 10(-8)M, beta-E2 significantly slowed the decrease in volume fraction of myofilaments (Vv myo) of freshly dispersed SMCs in primary culture, indicating an inhibitory effect of beta-E2 on spontaneous phenotypic modulation of SMC from a contractile to a synthetic phenotype. Freshly dispersed SMCs treated with beta-E2 also had a relatively longer quiescent phase than control cells before intense proliferation occurred. This was in contrast to SMCs in passage 2 3 (synthetic state), where beta-E2-treated cells replicated significantly faster than untreated cells. beta-E2 also markedly enhanced the serum-induced DNA synthesis of synthetic SMCs in a concentration-dependent manner within physiological range (10(-10)to 10(-8)M). These findings indicate that the growth-regulating effect of estrogen on vascular SMC is dependent on the cell's phenotypic state. It delays the cell cycle re-entry of the contractile SMCs by retarding their phenotypic modulation: however, once cells have modulated to the synthetic phenotype, it promotes their replication.