Regulation and characteristics of vascular smooth muscle cell phenotypic diversity.

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.).

Novel smooth muscle markers reveal abnormalities of the intestinal musculature in severe colorectal motility disorders.

Histopathological studies of gastrointestinal motility disorders have mainly focused on enteric nerves and interstitial cells of Cajal, but rarely considered the enteric musculature. Here we used both classical and novel smooth muscle markers and transmission electron microscopy (TEM) to investigate muscular alterations in severe colorectal motility disorders. Full-thickness specimens from Hirschsprung's disease, idiopathic megacolon, slow-transit constipation and controls were stained with haematoxylin/eosin (HE) and Masson's trichrome (MT), incubated with antibodies against smooth muscle alpha-actin (alpha-SMA), smooth muscle myosin heavy chain (SMMHC), smoothelin (SM) and histone deacetylase 8 (HDAC8) and processed for TEM. Control specimens exhibited homogeneous immunoreactivity for all antibodies. Diseased specimens showed normal smooth muscle morphology by HE and MT. While anti-alpha-SMA staining was generally normal, immunoreactivity for SMMHC, HDAC8 and/or SM was either absent or focally lacking in Hirschsprung's disease (80%), idiopathic megacolon (75%) and slow-transit constipation (70%). Ultrastructurally, clusters of myocytes with noticeably decreased myofilaments were observed in all diseases. SMMHC and the novel smooth muscle markers SM and HDAC8 often display striking abnormalities linked to the smooth muscle contractile apparatus unnoticed by both routine stainings and alpha-SMA, suggesting specific defects of smooth muscle cells involved in the pathogenesis of gastrointestinal motility disorders.

Expression of the smoothelin gene is mediated by alternative promoters.

OBJECTIVE: Two major isoforms of smoothelin have been reported, a 59-kDa smoothelin-A in visceral smooth muscle cells and a 110-kDa smoothelin-B in vascular smooth muscle cells. The present study was undertaken to investigate the expression of these smoothelin isoforms in different smooth muscle tissues and to determine how they are generated. METHODS: Western blotting with a new, well-defined, smoothelin antibody was used to confirm the existence of two major smoothelin isoforms. Northern blotting, RT-PCR, primer extension and 5'RACE were applied to analyse the expression of these isoforms in human and mouse. Promoter reporter assays were carried out to establish the existence of a dual promoter system governing the expression pattern of the gene. RESULTS: Antibody C6G confirmed the existence of two smoothelin proteins. Northern blotting showed that in vascular tissues a larger smoothelin transcript is generated than in visceral tissue. The cDNA of this larger smoothelin-B was cloned. Computer analysis of the open reading frame suggests an alpha-helical structure of 130 amino acids at the amino terminus of smoothelin-B. The smoothelin gene was cloned and sequenced. It comprises about 25 kb and contains 21 exons. The translational start of smoothelin-B is located in exon 2, whereas transcription and translation of the previously described smoothelin-A starts inside exon 10. Smoothelin-A and -B were demonstrated to be generated by two physically separated promoters. Splice variants within the calponin homology domain at the 3' end of the gene were found for both isoforms. CONCLUSIONS: Two major smoothelin isoforms are generated from a single gene by a dual promoter system in a tissue specific manner. Further variation in the smoothelin proteins is achieved by alternative splicing in the calponin homology domain.

Smoothelin expression during chicken embryogenesis: detection of an embryonic isoform.

Two isoforms of a novel smooth muscle cell (SMC) -specific cytoskeletal protein, smoothelin, have been described. In the adult chick, the 55-kDa smoothelin-A is expressed in visceral SMC, whereas the 120-kDa smoothelin-B is the major product in vascular SMC. Chicken was chosen to study smoothelin expression during embryogenesis and neonatally. Smoothelin-B was found in vascular SMC from stage 20 onward. In visceral SMC, smoothelin-B was present from stage 29 until hatching. Perinatally, a strong up-regulation of smoothelin synthesis was observed in visceral tissues, coinciding with a switch to the A-isoform. Transient smoothelin synthesis was observed in the somites and the developing heart. Western blotting revealed in these tissues a 62-kDa smoothelin isoform, designated smoothelin-C. Expression of the smoothelin isoforms seems to be strictly controlled with respect to cell type and developmental stage and may be related to the mode of contraction of the different cells.

Injury induces dedifferentiation of smooth muscle cells and increased matrix-degrading metalloproteinase activity in human saphenous vein.

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.

Expression of transient receptor potential mRNA isoforms and Ca(2+) influx in differentiating human stem cells and platelets.

Store-regulated Ca(2+) entry (SOCE) is an important mechanism of elevating cytosolic [Ca(2+)]i in platelets, though the Ca(2+) influx channels involved are still unclear. We screened human platelets and their precursor cells (human stem cells and megakaryocytes) for the presence of candidate influx channels, i.e., isoforms of the Trp family of proteins. Primary stem cells were cultured with thrombopoietin to allow differentiation into megakaryocytes. The undifferentiated stem cells (CD34(+)) showed mRNA expression of only a spliced variant Trp1A. Immature (CD61(+)/CD42b(low)) and mature (CD61(+)/CD42b(high)) megakaryocytes as well as platelets expressed in addition unspliced Trp1 as well as Trp4 (less abundant) and Trp6 isoforms. This unspliced isoform appeared to be specific for cells of the megakaryocyte/platelet lineage, since immature (CD14(+)/CD61(-)/CD42b(-)) and mature monocytes expressed only the Trp1A isoform. This conclusion was confirmed by the presence of Trp1A, 3, 4 and 6 transcripts in the immature megakaryocytic Dami cell line, and of Trp1, 1A, 4 and 6 transcripts in the more mature CHRF-288 cell line. The up-regulation of Trp1, 4 and 6 in the lineage from primary stem cells to mature megakaryocytes and platelets was accompanied by increased influx of extracellular Ca(2+) after pretreatment of the cells with thapsigargin or thrombin. Expression of new Trp isoforms in the differentiated cells is thus accompanied by increased SOCE.

Structural changes of atrial myocardium during chronic atrial fibrillation.

Of all known arrhythmia's, atrial fibrillation (AF) is the most often met in the clinical setting and it is associated with an increase in mortality risk. Several risk factors for AF have been described and several mechanisms of induction and maintenance have been proposed. Studies in patients with AF have shown that structural changes occur in the atria, but the relationship between the structural remodelling and the chronicity of the arrhythmia are not well understood. The changes mainly concern adaptive (dedifferentiation of cardiomyocytes) and maladaptive (degeneration of cells with replacement fibrosis) features. In order to characterise the time course of the structural remodelling the need for animal models which adequately mimic chronic atrial fibrillation in humans is felt essential. In this review, the structural changes that are observed during prolonged sustained AF in patients and animal models, are described. Furthermore, the time course and potential mechanisms of structural remodelling are discussed and methods for elucidation of the underlying molecular mechanisms are presented.

Dynamics of the nuclear lamina as monitored by GFP-tagged A-type lamins.

The behavior of chimeric proteins consisting of A-type lamins and green fluorescent protein (GFP) was studied to investigate the localization and dynamics of nuclear lamins in living cells. Cell line CHO-K1 was transfected with cDNA constructs encoding fusion proteins of lamin A-GFP, lamin Adelta10-GFP, or lamin C-GFP. In the interphase nucleus lamin-GFP fluorescence showed a perinuclear localization and incorporation into the lamina for all three constructs. Our findings show for the first time that the newly discovered lamin A 10 protein is localized to the nuclear membrane. The GFP-tagged lamins were processed and behaved similarly to the endogenous lamin molecules, at least in cells that expressed physiological levels of the GFP-lamins. In addition to the typical perinuclear localization, in the majority of transfected cells each individual A-type lamin-GFP revealed an extensive collection of branching intra- and trans-nuclear tubular structures, which showed a clear preference for a vertical orientation. Time-lapse studies of 3-D reconstructed interphase cells showed a remarkable stability in both number and location of these structures over time, while the lamina showed considerable dynamic movements, consisting of folding and indentation of large parts of the lamina. Fluorescence recovery after bleaching studies revealed a low protein turnover of both tubular and lamina-associated lamins. Repetitive bleaching of intranuclear areas revealed the presence of an insoluble intranuclear fraction of A-type lamins. Time-lapse studies of mitotic cells showed that reformation of the lamina and the tubular structures consisting of A-type lamins did not occur until after cytokinesis was completed.

Localization of smoothelin in avian smooth muscle and identification of a vascular-specific isoform.

Smoothelin is a smooth muscle-specific protein of minor abundance first identified via a monoclonal antibody obtained using an avian gizzard extract as antigen. Dual labelling of ultrathin sections with antibodies to smoothelin together with antibodies to other smooth muscle proteins showed that smoothelin was co-distributed with filamin and desmin in the cytoskeleton domain of the smooth muscle cell. From the finding that smoothelin, unlike desmin, was readily extracted by Triton X-100 as well as under conditions that solubilized myosin, beta-actin and filamin, we conclude that smoothelin is most likely associated with the actin cytoskeleton. Western blot analysis of gizzard smooth muscle tissue revealed an immunoreactive protein band with an apparent molecular weight of 59 kDa that separated into 3-4 isolated variants, while avian vascular muscle showed a polypeptide band of 95 kDa. These results point to the presence of specific isoforms in visceral and vascular smooth muscles. The 59 kDa isoform was shown to be distinct from the 60 kDa filamin-binding protein, described by Maekawa and Sakai (FEBS Lett. 221, 68-72, 1987). As compared to other smooth muscle markers, such as calponin and SM22, smoothelin appeared very late during differentiation in the chick gizzard, on about the 18th embryonic day.

Differentiation of smooth muscle cells in human blood vessels as defined by smoothelin, a novel marker for the contractile phenotype.

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.

Smoothelin expression characteristics: development of a smooth muscle cell in vitro system and identification of a vascular variant.

Recently we described a protein, smoothelin, that has been exclusively found in smooth muscle cells (SMC). The human cDNA has been cloned from a colon cDNA library and the putative protein sequence was deduced. Smoothelin does not belong to a known protein family but shows a partial homology with members of the spectrin family. Transfection studies revealed that smoothelin has an affinity for actin and is either capable of forming filamentous structures or colocalizes with such structures. The protein is expressed in visceral as well as vascular tissues of all vertebrate classes. A study on the distribution of smoothelin in the vascular and placental system showed that smoothelin expression was largely restricted to the muscular pulsating blood vessels. Therefore, we hypothesized that smoothelin is expressed in contractile SMC only (36, 37). No expression of smoothelin was observed in established cell lines of SMC. In tissue explants smoothelin mRNA concentration decreases to undetectable levels within 12 hours after dissection as was in general the case in primary cell cultures. Here we report on continued smoothelin expression for several passages observed in a human prostate primary cell culture system. Smoothelin was demonstrated to colocalize with actin stress fibers but not with desmin filaments. This culture system offers opportunities to study the cytological localization of smoothelin, interactions with other proteins and should provide a system to test the promoter of the smoothelin gene. On immunoblots the molecular weight of smoothelin differed between visceral and vascular smooth muscle tissue with apparent molecular weights of respectively 59 kDa and 94 kDa. There is no evidence for the existence of another gene coding for the 94 kDa smoothelin. Thus, posttranslational modification, alternative splicing and dual promoter control are the alternatives for the expression of two isoforms of smoothelin.

Cytoskeletal characterization of arteriovenous epithelioid cells.

Data on the cytoskeleton of epithelioid cells in arteriovenous anastomosis (AVA) are sparse, but there is evidence that the (myo)-epithelioid cells of the AVAs represent a specialized smooth muscle cell type with less contractile properties. We demonstrated the expression of alpha-smooth muscle actin, smooth muscle myosin, calponin, caldesmon, and caveolin in epithelioid cells of rabbit ear and in human toes, finger tips, and glomus tumors by means of indirect immunofluorescence techniques and immunoelectron microscopy. Epithelioid cells in rabbit ear did not express vimentin, but it was present in human toes, finger tips, and glomus tumors. Epithelioid cells in human toes, finger tips, and glomus tumors did not express desmin, but it was present in rabbit ear. Epithelioid cells did not express cytokeratins. The epithelioid cells examined showed only a weak expression of the protein smoothelin, which occurs exclusively in contractile smooth muscle cells. Immunoelectron microscopical demonstration of (alpha-smooth muscle actin revealed a striking difference in the arrangement of actin filaments in the epithelioid cells as compared to that in the smooth muscle cells of blood vessels. The epithelioid cells contained a loose array of actin filaments, whereas the smooth muscle cells contained tightly packed parallel actin bundles. In the present study we observed a correlation between the lack of contractile marker protein expression in epithelioid cells and the presence of only a few filaments, although the epithelioid cells are alpha-smooth muscle actin positive. The reduced number of contractile elements in the epithelioid cells of rabbit and human anastomoses suggests a lower contractility of epithelioid cells compared to that of the surrounding smooth muscle cells in anastomoses. A second interesting difference between both cell types is the high number of caveolae in epithelioid cells. Immunoelectron microscopy showed a compact distribution of caveolae at the epithelioid cell border, but a more dispersed distribution of caveolae in the cytoplasm of the blood vessel endothelium. The benign glomus tumor was characterized by an expression pattern of cytoskeletal proteins similar to that of epithelioid cells, confirming its description as a benign tumor.

Smoothelin, a novel cytoskeletal protein specific for smooth muscle cells.

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 approximately 40% with a sequence bordering the actin-binding domains of dystrophin, utrophin, beta-spectrin and alpha-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 (approximately 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.

Nuclear lamin expression in chronic hibernating myocardium in man.

Cardiomyocytes of chronic hibernating myocardium are known to undergo structural changes, indicative of dedifferentiation. Amongst these are changes in nuclear shape and chromatin distribution. Nuclear A-type lamins are known to be expressed in a differentiation-related fashion and to contribute to nuclear integrity and chromatin organization. Lamin expression was investigated with immunocytochemical staining procedures in biopsies from patients with chronic hibernating myocardium. The expression of A-type (lamin A and C) were shown to be downregulated during hibernation, while lamin B2 remained present in hibernating cardiomyocytes in a way similar to embryonic muscle cells. All heart muscle cells were shown to be negative for lamin B1. The absence of A-type lamins in chronic hibernating cardiomyocytes could be taken as an additional argument for the dedifferentiation state of these cells. The absence of A-type lamins was accompanied by dispersion of the nuclear heterochromatin, in a way similar to nuclei of embryonic cardiomyocytes.

An alternative splicing product of the lamin A/C gene lacks exon 10.

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.

Integration of titin into the sarcomeres of cultured differentiating human skeletal muscle cells.

Titin is amongst the first sarcomeric proteins to be detected in the process of myofibrillogenesis of striated muscle. During embryogenesis this high molecular weight protein is initially observed in a punctate staining pattern in immunohistochemical studies, while during maturation titin organizes into a cross-striated pattern. The dynamic process of titin assembly up to its integration into the sarcomeres of cultured human skeletal muscle cells has been studied in subsequent stages of differentiation with antibodies to four well-defined titin epitopes. Since in maturated muscle cells these epitopes are clearly distinguishable on the extended titin molecule we wondered how these epitopes reorganize during myofibrillogenesis, and whether such a reorganization would reveal important clues about its supramolecular organization during development. Immunofluorescence staining of postmitotic mononuclear myoblasts indicate that the investigated epitopes of the titin molecule are displayed in a punctate pattern with neighboring, but clearly separate spots in the cytoplasm of the cells. During elongation and fusion of the cells, these titin spots associate with stress fiber-like structures to finally reach their position at either the Z-line, the A-I junction or the A-band. We propose that during this transition the large titin molecule is unfolded, with the amino terminus of the molecule migrating in the direction of the Z-line and the carboxy terminus moving towards the M-line. In maturated, fused myotubes the final cross-striated patterns of all investigated titin epitopes are observed. While this process of unfolding of the titin molecule progresses, other compounds of the Z-line and the A-band migrate to their specific positions in the nascent sarcomere. A-band components such as sarcomeric myosin and C-protein, are also observed as dot-like aggregates during initial stages of muscle cell differentiation and organize into a cross-striated pattern in the sarcomere virtually simultaneously with titin. The Z-line associated component desmin organizes into a cross-striated pattern at a later stage.

Titin expression as an early indication of heart and skeletal muscle differentiation in vitro. Developmental re-organisation in relation to cytoskeletal constituents.

Established myogenic cell lines of different species and tissue origin have been used to study expression and organisation of muscle-specific proteins during differentiation. Furthermore, primary cultures of rat myocard cells were used to examine these same processes during dedifferentiation. In particular, we were interested in the general mechanism that underlies the changes in the supramolecular organisation of titin during in vitro myogenesis. It became obvious that in the differentiating muscle cell cultures the redistribution of desmin, actin and myosin in a typical, differentiation state dependent fashion, always showed a certain delay when compared to titin. The sequence of changes in the assembly of cytoskeletal and sarcomeric structures observed during differentiation of the cell lines was reversed during the process of dedifferentiation in cultured rat myocard cells. These results all indicate that titin is an early marker of myogenic differentiation, both in vivo and in vitro, and the typical reorganisation of this giant molecule is independent of species or muscle cell type.

Putative membrane fatty acid translocase and cytoplasmic fatty acid-binding protein are co-expressed in rat heart and skeletal muscles.

A membrane protein (FAT) homologous to CD36 has recently been implicated in the binding and transport of long-chain fatty acids (FA). Expression of this protein in rat heart, skeletal muscles and in isolated cardiac cells was studied. Changes in expression during development of the heart were also examined. Expression of FAT was compared to that of the cytoplasmic fatty acid-binding protein (H-FABP) to determine whether coexpression, indicative of related biological functions, could be demonstrated. FAT and H-FABP mRNAs showed a similar muscle tissue distribution and similar cellular localization in the heart. During development, heart mRNA levels for both proteins were upregulated in the same way. In conclusion, expression of FAT and H-FABP in muscle tissues and cell-types with high FA metabolism and the upregulation of mRNA levels associated with heart development, when FA utilization increases, support the suggested role of both proteins in FA metabolism.