Vascular smooth muscle cells can perform both contractile and synthetic functions, which are associated with and characterised by changes in morphology, proliferation and migration rates, and the expression of different marker proteins. The resulting phenotypic diversity of smooth muscle cells appears to be a function of innate genetic programmes and environmental cues, which include biochemical factors, extracellular matrix components, and physical factors such as stretch and shear stress. Because of the diversity among smooth muscle cells, blood vessels attain the flexibility that is necessary to perform efficiently under different physiological and pathological conditions. In this review, we discuss recent literature demonstrating the extent and nature of smooth muscle cell diversity in the vascular wall and address the factors that affect smooth muscle cell phenotype. (Neth Heart J 2007;15:100-8.)

Two sets of primers derived from genomic DNA libraries of Leptospira serovars icterohaemorrhagiae (strain RGA) and bim (strain 1051) enabled the amplification by PCR of target DNA fragments from leptospiral reference strains belonging to all presently described pathogenic Leptospira species. The icterohaemorr~gi~-deriv~L. santarosai and L. meyeri, whereas the bim-derived primers (B64-I/B64-11) enabled the amplification of L. kirschneri. Southern blot and DNA sequence analysis revealed inter-species DNA polymorphism within the region spanned by primers G1 and 6 2 between L. interrogans and various other Leptospira species. Using a mixture of primer sets G1/G2 and B64-I/B64-11, leptospires of serovars icterohaemorrhagiae, copenhageni, hardjo, pomona, grippotyphosa and bim were detected in serum samples collected from patients during the first 10 days after the onset of illness.

Abstract. The characterization of a novel 59-kD cytoskeletal protein is described. It is exclusively observed in smooth muscle cells by Northern blotting and immunohistochemical analysis and therefore designated "smoothelin." A human smooth muscle cDNA library was screened with the monoclonal antibody R4A, and a full-size cDNA of the protein was selected. The cDNA was sequenced and appeared to contain a 1,113-bp open reading frame. Based on the cDNA sequence, the calculated molecular weight of the polypeptide was 40 kD and it was demonstrated to contain two N-glycosylation sites. Computer assisted analysis at the protein level revealed a 56-amino acid domain with homologies of ~40% with a sequence bordering the actin-binding domains of dystrophin, utrophin, [3-spectrin and a-actinin. In situ hybridization demonstrated that human smoothelin is encoded by a single copy gene which is located on chromosome 22. Immunohistochemistry and Western blotting revealed synthesis of smoothelin in smooth muscle of species evolutionarily as far apart as human and teleost. Northern blotting indicated that sequence as well as size of the mRNA (~1,500 bases) are conserved among vertebrates. Cell fractionation studies and differential centrifugation showed that the protein cannot be extracted with Triton X-100, which indicates that it is a part of the cytoskeleton. Transfection of the human cDNA into smooth muscle cells and COS7 cells produced a protein of 59 kD, which assembled into a filamentous network. However, in rat heart-derived myoblasts association with stress fibers was most prominent. Smoothelin was not detected in primary or long term smooth muscle cell cultures. Also, transcription of smoothelin mRNA was almost instantly halted in smooth muscle tissue explants. We conclude that smoothelin is a new cytoskeletal protein that is only found in contractile smooth muscle cells and does not belong to one of the classes of structural proteins presently known.

Smoothelin is a constituent of the cytoskeleton specific for smooth muscle cells (SMCs) in a broad range of species. It has been postulated that smoothelin represents a marker of highly differentiated, contractile SMCs. Here, we present data on the presence of smoothelin in the human vascular system that support this hypothesis. For this purpose, smoothelin distribution was studied (1) during vasculogenesis of the placenta, (2) in normal adult blood vessels, and (3) in atherosclerotic lesions. Smoothelin was first observed in placental tissue at approximately week 10 to 11 of gestation. In full-term placenta, it was found in the SMCs of vessels in the large stem villi and in the chorionic plate. Furthermore, it was present in the fetal arteries of smaller stem villi, but it was not found in the veins. In adult blood vessels, a small population of aortic (approximately 10%) and large muscular artery (approximately 30% to 50%) SMCs was positive for smoothelin. In general, smoothelin and desmin were coexpressed in the same SMCs, but expression of desmin appeared to be less abundant. However, the majority of SMCs in these blood vessels were smoothelin- and desmin negative but expressed vimentin, whereas alpha-smooth muscle actin (alpha-SMA) was present in all SMCs. The SMCs in the media of small muscular arteries were positive for smoothelin and desmin (> 95%), whereas the vimentin-positive SMC type was scarce. Smoothelin was absent in capillaries, pericytic venules, and small veins but was occasionally observed in the SMCs of large veins. Thus, the distribution of smoothelin in the SMCs of the vascular system appears to be limited to blood vessels that are capable of pulsatile contraction. In atherosclerotic femoral arteries, smoothelin-positive cells were detected in the media, the atheromatous plaque, and the intimal thickening. Smoothelin-positive cells were present primarily at the luminal portion of advanced lesions. The presence of a considerable number of such smoothelin-positive cells at that location may indicate that these plaques are no longer expanding.

The progressive rise in uterine blood flow during pregnancy is accompanied by outward hypertrophic remodeling of the uterine artery (UA). This process involves changes of the arterial smooth muscle cells and extracellular matrix. Acute increases in blood flow stimulate endothelial production of nitric oxide (NO). It remains to be established whether endothelial NO synthase (eNOS) is involved in pregnancy-related arterial remodeling. We tested the hypothesis that absence of eNOS results in a reduced remodeling capacity of the UA during pregnancy leading to a decline in neonatal outcome. UA of nonpregnant and pregnant wild-type (Nos3+/+) and eNOS-deficient (Nos3-/-) mice were collected and processed for standard morphometrical analyses. In addition, cross sections of UA were processed for cytological (smoothelin, smooth muscle alpha-actin) and proliferation (Ki-67) immunostaining. We compared the pregnancy-related changes longitudinally and, together with the data on pregnancy outcome, transversally by analysis of variance with Bonferroni correction. During pregnancy, the increases in radius and medial cross sectional area of Nos3-/- UA was significantly less than those of Nos3+/+ UA. Smooth muscle cell dedifferentiation and proliferation were impaired in gravid Nos3-/- mice as deduced from the lack of change in the expression of smoothelin and smooth muscle alpha-actin, and the reduced Ki-67 expression. Until 17 days of gestation, litter size did not differ between both genotypes, but at birth the number of viable newborn pups and their weights were smaller in Nos3-/- than in Nos3+/+ mice. We conclude that absence of eNOS adversely affects UA remodeling in pregnancy, which may explain the impaired pregnancy outcome observed in these mice.

Expression of the A-type lamins was studied in the lung adenocarcinoma cell line GLC-A1. A-type lamins, consisting of lamin A and C, are two products arising from the same gene by alternative splicing. Northern blotting showed in GLC-A1 a relatively low expression level of lamin C and an even lower expression level of lamin A as compared to other adenocarcinoma cell lines. Immunofluorescence studies revealed highly irregular nuclear inclusions of lamin A, suggesting protein or gene expression abnormalities. Reverse transcriptase-polymerase chain reaction-based cDNA analysis followed by sequencing indicated the presence of an as yet unidentified alternative splicing product of the lamin A/C gene. This product differs from lamin A by the absence of the 5' part of exon 10 (90 nucleotides). Therefore we propose to designate this product lamin Adelta10. Deletion of the 30 amino acids encoded by exon 10 was predicted to result in a shift in pI of the protein from 7.4 to approximately 8.6, which was confirmed by two-dimensional immunoblotting. mRNA analysis in a variety of cell lines, normal colon tissue as well as carcinomas demonstrated the presence of lamin Adelta 10 in all samples examined, suggesting its presence in a variety of cell types.

Abstract-Long-term patency of human saphenous vein bypass grafts is low because of intimal thickening and superimposed atherosclerosis. Matrix-degrading metalloproteinases (MMPs) and changes in vascular smooth muscle cell (VSMC) phenotype are thought to be essential for the VSMC migration that contributes to intimal thickening. We examined VSMC phenotype and MMP activity in saphenous veins obtained before and after surgical manipulation. Surgical preparation of the veins significantly increased pro-MMP-1 expression by 2-fold and significantly reduced tissue inhibitor of MMPs (TIMP)-2 expression, whereas MMP-3 and TIMP-1 were unaffected. Furthermore, caseinolytic and gelatinolytic activities measured by in situ zymography were dramatically elevated by injury. The expression of desmin and smoothelin was significantly decreased by injury, whereas vimentin expression was significantly increased. In addition, these changes in phenotype and MMP activity were localized to a subpopulation of VSMCs, the circumferential medial VSMCs. Our data show that surgical preparative injury induces phenotypic modulation of a subpopulation of medial VSMCs to a synthetic phenotype and increases MMP activity. This may favor matrix degradation, VSMC migration, and the subsequent intimal thickening that leads to graft failure. Medial VSMCs exist in the normal blood vessel wall in the contractile (differentiated) phenotype. These have a "spindlelike" morphology, maintain vessel wall tone, and are rich in contractile and intermediate filament proteins (IFPs). It has been suggested that migrating and proliferating VSMCs have dedifferentiated to a synthetic phenotype, characterized by a reduction in contractile proteins and alterations in IFPs (see review by Owens 5 ). Recent studies illustrate that the amounts of the IFPs vimentin and desmin, the cytoskeleton-related protein smoothelin, and the contractile proteins ␣-smooth muscle (SM) actin, SM myosin heavy chain (SMMHC), and tropomyosin change when VSMCs shift from the contractile to the synthetic phenotype. [5][6][7] Mechanical injury to the blood vessel wall, particularly endothelial damage, is thought to trigger phenotypic modulation of medial VSMCs, shifting them toward the synthetic phenotype (see reviews by Thyberg and colleagues 8 -10 ). To enable VSMC migration, remodeling of the basement membrane and of the interstitial collagenous matrix that maintains VSMCs in a quiescent state must occur. 11 Mechanical injury of aortic explants 12 and isolated VSMCs 13 stimulates the production of extracellular matrix-degrading metalloproteinases (MMPs), which are mainly associated with VSMCs of the synthetic phenotype. 14,15 Injury of rat carotid arteries 16 and human saphenous veins 17 increases the expression of basement membrane-degrading MMP-2 and MMP-9. Furthermore, MMP inhibitors,12,[18][19][20] as well as gene transfer of the endogenous tissue inhibitors of MMPs (TIMPs), [21][22][23][24][25] have demonstrated the involvement of MMPs in injury-stimulated intimal thickening. Injury also...

In response to a chronic high plasma concentration of long-chain fatty acids (FAs), the heart is forced to increase the uptake of FA at the cost of glucose. This switch in metabolic substrate uptake is accompanied by an increased presence of the FA transporter CD36 at the cardiac plasma membrane and over time results in the development of cardiac insulin resistance and ultimately diabetic cardiomyopathy. FA can interact with peroxisome proliferator-activated receptors (PPARs), which induce upregulation of the expression of enzymes necessary for their disposal through mitochondrial β-oxidation, but also stimulate FA uptake. This then leads to a further increase in FA concentration in the cytoplasm of cardiomyocytes. These metabolic changes are supposed to play an important role in the development of cardiomyopathy. Although the onset of this pathology is an increased FA utilization by the heart, the subsequent lipid overload results in an increased production of reactive oxygen species (ROS) and accumulation of lipid intermediates such as diacylglycerols (DAG) and ceramide. These compounds have a profound impact on signaling pathways, in particular insulin signaling. Over time the metabolic changes will introduce structural changes that affect cardiac contractile characteristics. The present mini-review will focus on the lipid-induced changes that link metabolic perturbation, characteristic for type 2 diabetes, with cardiac remodeling and dysfunction.