Smooth muscle phenotypic modulation--a personal experience.

The idea that smooth muscle cells can exist in multiple phenotypic states depending on the functional demands placed upon them has been around for >5 decades. However, much of the literature today refers to only recent articles, giving the impression that it is a new idea. At the same time, the current trend is to delve deeper and deeper into transcriptional regulation of smooth muscle genes, and much of the work describing the change in biology of the cells in the different phenotypic states does not appear to be known. This loss of historical perspective regarding the biology of smooth muscle phenotypic modulation is what the current article has tried to mitigate.

Transcription factors expressed in olfactory bulb local progenitor cells revealed by genome-wide transcriptome profiling.

The local progenitor population in the olfactory bulb (OB) gives rise to mitral and tufted projection neurons during embryonic development. In contrast, OB interneurons are derived from sources outside the bulb where neurogenesis continues throughout life. While many of the genes involved in OB interneuron development have been characterized, the genetic pathways driving local progenitor cell differentiation in this tissue are largely unknown. To better understand this process, we used transcriptional profiling to monitor gene expression of whole OB at daily intervals from embryonic day 11 through birth, generating a compendium of gene expression encompassing the major developmental events of this tissue. Through hierarchical clustering, bioinformatics analysis, and validation by RNA in situ hybridizations, we identified a large number of transcription factors, DNA binding proteins, and cell cycle-related genes expressed by the local neural progenitor cells (NPCs) of the embryonic OB. Further in silico analysis of transcription factor binding sites identified an enrichment of genes regulated by the E2F-Rb pathway among those expressed in the local NPC population. Together these results provide initial insights into the molecular identity of the OB local NPC population and the transcription factor networks that may regulate their function.

Cellular plasticity of inflammatory myeloid cells in the peritoneal foreign body response.

Implantation of sterile foreign objects in the peritoneal cavity of an animal initiates an inflammatory response and results in encapsulation of the objects by bone marrow-derived cells. Over time, a multilayered tissue capsule develops with abundant myofibroblasts embedded in extracellular matrix. The present study used the transgenic MacGreen mouse to characterize the time-dependent accumulation of monocyte subsets and neutrophilic granulocytes in the inflammatory infiltrate and within the tissue capsule by their differential expression of the csf1r-EGFP transgene, F4/80, and Ly6C. As the tissue capsule developed, enhanced green fluorescent protein-positive cells changed from rounded to spindle-shaped morphology and began to co-express the myofibroblast marker alpha-smooth muscle actin. Expression increased with time: at day 14, 11.13 +/- 0.67% of tissue capsule cells co-expressed these markers, compared with 50.77 +/- 12.85% of cells at day 28. The importance of monocyte/macrophages in tissue capsule development was confirmed by clodronate-encapsulated liposome removal, which resulted in almost complete abrogation of capsule development. These results confirm the importance of monocyte/macrophages in the tissue response to sterile foreign objects implanted in the peritoneal cavity. In addition, the in vivo plasticity of peritoneal macrophages and their ability to transdifferentiate from a myeloid to mesenchymal phenotype is demonstrated.

The peritoneal cavity as a bioreactor for tissue engineering visceral organs: bladder, uterus and vas deferens.

Our objective was to produce avascular, myofibroblast-rich tissue capsules for use as autologous grafts for hollow, smooth muscle-walled visceral organs-bladder, uterus and vas deferens. To produce tissue for grafting, templates of the appropriate shape were implanted in the peritoneal cavities of rats or rabbits. After 2-3 weeks, the templates were removed, the encapsulating myofibroblast-rich tissue harvested and grafted to replace resected segments of bladder, vas deferens or uterus of the same animals in which the tissue was grown. Bladder grafts showed 100% patency after 14 months and had developed a morphology similar to normal bladder. Tubes of myofibroblast tissue grafted unilaterally into resected rabbit vasa deferentia developed a morphology resembling native tissue, with sperm in the ejaculate indicative of normal function. At 12 weeks after grafting, uterine graft tissue had increased in thickness and developed the morphology of normal uterus, with endometrium overlying several layers of smooth muscle cells (myometrium-like) which were interspersed with collagen fibrils; grafted uterine horns supported embryos to the late stages of gestation. This study shows that myofibroblast tissue produced in the peritoneal cavity is sufficiently plastic to permit differentiation of cells into bladder, vas deferens or uterine smooth muscle. As a method for producing autologous graft material for repair/replacement of these organs, this approach has many benefits over conventional and current tissue-engineering strategies.

Engineering tissue tubes using novel multilayered scaffolds in the rat peritoneal cavity.

Our aim was to develop novel scaffolds to engineer tissue tubes of smooth muscle-like cells for autologous grafting. Small diameter tubular poly(lactic acid) scaffolds with randomly distributed, interconnected pores up to 100 mum were produced using a thermally induced phase separation method. The scaffolds were surface modified using various biomolecules via a layer-by-layer deposition technique, and implanted in the peritoneal cavities of rats. Histological analysis of scaffolds 3 weeks after implantation showed fully-developed tissue capsules on their outer surfaces, with macrophage-like cells present throughout the internal spaces. Surfaces coated in Matrigel supported the strongest cellular response whereas multilayer coatings with elastin, collagen I, collagen III, or chitosan outermost showed the lowest levels of cellular interaction. Although differences in capsule thickness and the presence or absence of cellularized layers on the inside and outside surfaces of the scaffolds were observed, none of these biomolecule coatings was able to overcome the foreign body response within the peritoneal cavity, even in the presence of a nonadsorptive HA undercoat.

Vascular grafts and the endothelium.

This article discusses the importance of the endothelium for successful vascular grafts derived from both native arteries and synthetic materials. It also discusses the fundamental strategies to endothelialize synthetic grafts in animal experiments and in the clinic, as well as the use of endothelial progenitor cells (EPCs), bone marrow-derived cells, and mesothelium as endothelial substitutes.

CbrA is a stationary-phase regulator of cell surface physiology and legume symbiosis in Sinorhizobium meliloti.

Sinorhizobium meliloti produces an exopolysaccharide called succinoglycan that plays a critical role in promoting symbiosis with its host legume, alfalfa (Medicago sativa). We performed a transposon mutagenesis and screened for mutants with altered succinoglycan production and a defect in symbiosis. In this way, we identified a putative two-component histidine kinase associated with a PAS sensory domain, now designated CbrA (calcofluor-bright regulator A). The cbrA::Tn5 mutation causes overproduction of succinoglycan and results in increased accumulation of low-molecular-weight forms of this exopolysaccharide. Our results suggest the cbrA::Tn5 allele leads to this succinoglycan phenotype through increased expression of exo genes required for succinoglycan biosynthesis and modification. Interestingly, CbrA-dependent regulation of exo and exs genes is observed almost exclusively during stationary-phase growth. The cbrA::Tn5 mutant also has an apparent cell envelope defect, based on increased sensitivity to a number of toxic compounds, including the bile salt deoxycholate and the hydrophobic dye crystal violet. Growth of the cbrA mutant is also slowed under oxidative-stress conditions. The CbrA-regulated genes exsA and exsE encode putative inner membrane ABC transporters with a high degree of similarity to lipid exporters. ExsA is homologous to the Escherichia coli MsbA protein, which is required for lipopolysaccharide transport, while ExsE is a member of the eukaryotic family of ABCD/hALD peroxisomal membrane proteins involved in transport of very long-chain fatty acids, which are a unique component of the lipopolysaccharides of alphaproteobacteria. Thus, CbrA could play a role in regulating the lipopolysaccharide or lipoprotein components of the cell envelope.

Sinorhizobium meliloti bluB is necessary for production of 5,6-dimethylbenzimidazole, the lower ligand of B12.

An insight into a previously unknown step in B(12) biosynthesis was unexpectedly obtained through our analysis of a mutant of the symbiotic nitrogen fixing bacterium Sinorhizobium meliloti. This mutant was identified based on its unusually bright fluorescence on plates containing the succinoglycan binding dye calcofluor. The mutant contains a Tn5 insertion in a gene that has not been characterized previously in S. meliloti. The closest known homolog is the bluB gene of Rhodobacter capsulatus, which is implicated in the biosynthesis of B(12) (cobalamin). The S. meliloti bluB mutant is unable to grow in minimal media and fails to establish a symbiosis with alfalfa, and these defects can be rescued by the addition of vitamin B(12) (cyanocobalamin) or the lower ligand of cobalamin, 5,6-dimethylbenzimidazole (DMB). Biochemical analysis demonstrated that the bluB mutant does not produce cobalamin unless DMB is supplied. Sequence comparison suggests that BluB is a member of the NADH/flavin mononucleotide (FMN)-dependent nitroreductase family, and we propose that it is involved in the conversion of FMN to DMB.

Establishment of metanephros transplantation in mice highlights contributions by both nephrectomy and pregnancy to developmental progression.

BACKGROUND: It has been demonstrated that embryonic kidneys (metanephroi) xenotransplanted into the omentum of adult recipients continue to develop and display immune protection due to their more naïve immune presentation. To date, this has been achieved using rat, pig and human metanephroi, with unilateral nephrectomy (UNX) of recipient rats a requisite of renal development. The aim of this study was to adapt this approach for use in mice and examine the parameters affecting successful onward development in this species. METHODS: Metanephroi at embryonic age (E) 13.5 were transplanted either onto the body wall, abdominal fat pads or omentum of recipient isogenic C57/Bl6 mice using either sutures or polyglycolic acid mesh. Having established greatest success with polyglycolic acid mesh on the body wall, E12.5 and 15.5 days metanephroi from C57/Bl6 mice were then transplanted onto the body wall of control (non-pregnant non-UNX), UNX or 12.5 days post-coitum pregnant isogenic recipients. After 7 days, implanted tissue was harvested and examined using histology and immunohistochemistry for markers of renal maturation. The mean number of S-shaped bodies and glomeruli per section were recorded and statistically analysed for significant differences between all recipient groups and untransplanted metanephroi. The degree of development was scored qualitatively. RESULTS: Transplanted E12.5 metanephroi developed S-shaped bodies and glomeruli in all recipient groups, although there were statistically higher numbers of S-shaped bodies in UNX (n = 2) and pregnant recipients (n = 9) than in control recipients (n = 4). Continued development, as indicated by mature vascularized glomeruli, was only observed in those E15.5 metanephroi transplanted into pregnant recipients (n = 11) with a 15.5-fold increase in S-shaped bodies and 4-fold increase in glomeruli compared with control transplants (n = 12). CONCLUSIONS: We have successfully established metanephros transplantation in mice and demonstrated enhancement of onward development of E12.5 metanephroi in response to both pregnancy and UNX. Using E15.5 metanephroi, continued development only occurred in pregnant recipients, implying pregnancy provides an environment conducive to continued organogenesis. This murine assay, when coupled with transgenically-tagged strains of mice, will allow the investigation of the relative contribution of donor and recipient cells to this process.

Vascular engineering for bypass surgery.

Since the introduction of synthetic vascular grafts in the 1960s, only two-stage endothelial cell seeding has demonstrated any significant improvement over conventional vascular grafts, and its benefits have yet to be demonstrated on a large scale. Tissue engineering is a rapidly expanding field with great potential, but efforts to construct tissue-engineered arterial grafts have, to date, yielded little clinical success. This review explores the latest approaches to the construction of a superior vascular graft, along with its potential for use in the clinic in the future.

Rho and vascular disease.

The Rho family GTPases are regulatory molecules that link surface receptors to organisation of the actin cytoskeleton and play major roles in fundamental cellular processes. In the vasculature Rho signalling pathways are intimately involved in the regulation of endothelial barrier function, inflammation and transendothelial leukocyte migration, platelet activation, thrombosis and oxidative stress, as well as smooth muscle contraction, migration, proliferation and differentiation, and are thus implicated in many of the changes associated with atherogenesis. Indeed, it is believed that many of the beneficial, non-lipid lowering effects of statins occur as a result of their ability to inhibit Rho protein activation. Conversely, the Rho proteins can have beneficial effects on the vasculature, including the promotion of endothelial repair and the maintenance of SMC differentiation. Further identification of the mechanisms by which these proteins and their effectors act in the vasculature should lead to therapies that specifically target only the adverse effects of Rho signalling.

Tissue-engineered blood vessels: alternative to autologous grafts?

Although vascular bypass grafting remains the mainstay for revascularization for ischemic heart disease and peripheral vascular disease, many patients do not have healthy vessels suitable for harvest. Thus, prosthetic grafts made of synthetic polymers were developed, but their use is limited to high-flow/low-resistance conditions because of poor elasticity, low compliance, and thrombogenicity of their synthetic surfaces. To fill this need, several laboratories have produced in vivo or in vitro tissue-engineered blood vessels using molds or prosthetic or biodegradable scaffolds, but each artificial graft has significant problems. Recently, conduits have been grown in the peritoneal cavity of the same animals in which they will be grafted, ensuring no rejection, in the short time of 2 to 3 weeks. Remodeling occurs after grafting such that the tissue is almost indistinguishable from native vessels. This conduit is derived from cells of bone marrow origin, opening new possibilities in vascular modeling and remodeling.

Rho expression and activation in vascular smooth muscle cells.

Phenotypic modulation of smooth muscle cells (SMC) involves dramatic changes in expression and organization of contractile and cytoskeletal proteins, but little is known of how this process is regulated. The present study used a cell culture model to investigate the possible involvement of RhoA, a known regulator of the actin cytoskeleton. In rabbit aortic SMC seeded into primary culture at moderate density, Rho activation was high at two functionally distinct time-points, first as cells modulated to the "synthetic" phenotype, and again upon confluence and return to the "contractile" phenotype. Rho expression increased with time, such that maximal expression occurred upon return to the contractile state. Transient transfection of synthetic state cells with constitutively active RhoA (Val14RhoA) caused a reduction in cell size and reorganization of cytoskeletal proteins to resemble that of the contractile phenotype. Actin and myosin filaments were tightly packed and highly organised while vimentin localised to the perinuclear region; focal adhesions were enlarged and concentrated at the cell periphery. Conversely, inhibition of endogenous Rho by C3 exoenzyme resulted in complete loss of contractile filaments without affecting vimentin distribution; focal adhesions were reduced in size and number. Treatment of synthetic state SMC with known regulators of SMC phenotype, heparin and thrombin, caused a modest increase in Rho activation. Long-term confluence and serum deprivation induced cells to return to a more contractile phenotype and this was augmented by heparin and thrombin. The results implicate RhoA for a role in regulating SMC phenotype and further show that activation of Rho by heparin and thrombin correlates with the ability of these factors to promote the contractile phenotype.

Dog peritoneal and pleural cavities as bioreactors to grow autologous vascular grafts.

OBJECTIVE: The purpose of this study was to grow "artificial blood vessels" for autologous transplantation as arterial interposition grafts in a large animal model (dog). METHOD AND RESULTS: Tubing up to 250 mm long, either bare or wrapped in biodegradable polyglycolic acid (Dexon) or nonbiodegradable polypropylene (Prolene) mesh, was inserted in the peritoneal or pleural cavity of dogs, using minimally invasive techniques, and tethered at one end to the wall with a loose suture. After 3 weeks the tubes and their tissue capsules were harvested, and the inert tubing was discarded. The wall of living tissue was uniformly 1-1.5 mm thick throughout its length, and consisted of multiple layers of myofibroblasts and matrix overlaid with a single layer of mesothelium. The myofibroblasts stained for alpha-smooth muscle actin, vimentin, and desmin. The bursting strength of tissue tubes with no biodegradable mesh scaffolds was in excess of 2500 mm Hg, and the suture holding strength was 11.5 N, both similar to that in dog carotid and femoral arteries. Eleven tissue tubes were transplanted as interposition grafts into the femoral artery of the same dog in which they were grown, and were harvested after 3 to 6.5 months. Eight remained patent during this time. At harvest, their lumens were lined with endothelium-like cells, and wall cells stained for alpha-actin, smooth muscle myosin, desmin and smoothelin; there was also a thick "adventitia" containing vasa vasorum. CONCLUSION: Peritoneal and pleural cavities of large animals can function as bioreactors to grow myofibroblast tubes for use as autologous vascular grafts.

In vivo engineering of blood vessels.

The inadequacy of conventional synthetic grafts has led to efforts to construct a superior vascular graft. In vivo tissue engineering is one approach to this problem that has been investigated for half a century and enables the construction of autogenous vascular prostheses. Three types of in vivo engineering are explored: remodelling of implanted scaffolds, fibrocollagenous tubes, and the artificial artery generated in the peritoneal cavity. Scaffolds designed to be remodelled may be synthetic or biological and have been remodelled in animal models to form vasoactive neoarteries with arterial morphology. The differences in vascular remodelling ability, particularly spontaneous endothelialisation, between animal models and humans may impair the effectiveness of this approach in the clinic. Fibrocollagenous tubes such as the Sparks Mandril have demonstrated poor performance in the clinic and are prone to aneurysm formation. The artificial artery generated in the peritoneal cavity is a novel addition to the ranks of in vivo engineered vascular prostheses and combines many of the best features of scaffolds designed to be remodelled and fibrocollagenous tubes. However, understanding and manipulating the vascular remodelling process will be the key to producing the ideal arterial prosthesis.

Advances in vascular tissue engineering.

Coronary and peripheral artery bypass grafting is commonly used to relieve the symptoms of vascular deficiencies, but the supply of autologous artery or vein may not be sufficient or suitable for multiple bypass or repeat procedures, necessitating the use of other materials. Synthetic materials are suitable for large bore arteries but often thrombose when used in smaller arteries. Suitable replacement grafts must have appropriate characteristics, including resistance to infection, low immunogenicity and good biocompatability and thromboresistance, with appropriate mechanical and physiological properties and cheap and fast manufacture. Current avenues of graft development include coating synthetic grafts with either biological chemicals or cells with anticoagulatory properties. Matrix templates or acellular tubes of extracellular matrix (such as collagen) may be coated or infiltrated with cultured cells. Once placed into the artery, these grafts may become colonised by host cells and gain many of the properties of normal artery. "Tissue-engineered blood vessels" may also be formed from layers of human vascular cells grown in culture. These engineered vessels have many of the characteristics of arteries formed in vivo. "Artificial arteries" may be also be derived from peritoneal granulation tissue in body "bioreactors" by adapting the body's natural wound healing response to produce a hollow tube.

Striking complexity of lipopolysaccharide defects in a collection of Sinorhizobium meliloti mutants.

Although the role that lipopolysaccharide (LPS) plays in the symbiosis between Sinorhizobium meliloti and alfalfa has been studied for over a decade, its function in this process remains controversial and poorly understood. This is largely due to a lack of mutants affected by its synthesis. In one of the definitive studies concerning this issue, Clover et al. (R. H. Clover, J. Kieber, and E. R. Signer, J. Bacteriol. 171:3961-3967, 1989) identified a series of mutants with putative LPS defects, judged them to be symbiotically proficient on Medicago sativa, and concluded that LPS might not have a symbiotic function in S. meliloti. The mutations in these strains were never characterized at the molecular level nor was the LPS from most of them analyzed. We have transduced these mutations from the Rm2011 background from which they were originally isolated into the sequenced strain Rm1021 and have characterized the resulting strains in greater detail. We found the LPS from these mutants to display a striking complexity of phenotypes on polyacrylamide electrophoresis gels, including additional rough LPS bands and alterations in the molecular weight distribution of the smooth LPS. We found that some of the mutants contain insertions in genes that are predicted to be involved in the synthesis of carbohydrate components of LPS, including ddhB, lpsB, lpsC, and lpsE. The majority, however, code for proteins predicted to be involved in a wide variety of functions not previously recognized to play a role in LPS synthesis, including a possible transcription elongation factor (GreA), a possible queuine synthesis protein, and a possible chemotaxis protein. Furthermore, using more extensive assays, we have found that most of these strains have symbiotic deficiencies. These results support more recent findings that alterations in LPS structure can affect the ability of S. meliloti to form an effective symbiosis.

Blockade of vascular smooth muscle cell proliferation and intimal thickening after balloon injury by the sulfated oligosaccharide PI-88: phosphomannopentaose sulfate directly binds FGF-2, blocks cellular signaling, and inhibits proliferation.

Percutaneous transluminal coronary angioplasty is a frequently used interventional technique to reopen arteries that have narrowed because of atherosclerosis. Restenosis, or renarrowing of the artery shortly after angioplasty, is a major limitation to the success of the procedure and is due mainly to smooth muscle cell accumulation in the artery wall at the site of balloon injury. In the present study, we demonstrate that the antiangiogenic sulfated oligosaccharide, PI-88, inhibits primary vascular smooth muscle cell proliferation and reduces intimal thickening 14 days after balloon angioplasty of rat and rabbit arteries. PI-88 reduced heparan sulfate content in the injured artery wall and prevented change in smooth muscle phenotype. However, the mechanism of PI-88 inhibition was not merely confined to the antiheparanase activity of this compound. PI-88 blocked extracellular signal-regulated kinase-1/2 (ERK1/2) activity within minutes of smooth muscle cell injury. It facilitated FGF-2 release from uninjured smooth muscle cells in vitro, and super-released FGF-2 after injury while inhibiting ERK1/2 activation. PI-88 inhibited the decrease in levels of FGF-2 protein in the rat artery wall within 8 minutes of injury. PI-88 also blocked injury-inducible ERK phosphorylation, without altering the clotting time in these animals. Optical biosensor studies revealed that PI-88 potently inhibited (Ki 10.3 nmol/L) the interaction of FGF-2 with heparan sulfate. These findings show for the first time the capacity of this sulfated oligosaccharide to directly bind FGF-2, block cellular signaling and proliferation in vitro, and inhibit injury-induced smooth muscle cell hyperplasia in two animal models. As such, this study demonstrates a new role for PI-88 as an inhibitor of intimal thickening after balloon angioplasty. The full text of this article is available online at http://www.circresaha.org.

Expression of heparan sulphate N-deacetylase/N-sulphotransferase by vascular smooth muscle cells.

Heparan sulphate is an important mediator in determining vascular smooth muscle cell (SMC) phenotype. The sulphation pattern of the heparan sulphate chains is critical to their function. We have examined the initial step in the biosynthesis of the sulphated domains mediated by the enzyme heparan sulphate N-deacetylase/N-sulphotransferase (NDST). Rabbit aortic SMC in primary culture exhibited NDST enzyme activity and expressed NDST-1 in their Golgi apparatus, with maximal expression in SMC 2 days after dispersal in primary culture confirmed by Western blot analysis. Endothelial cells, macrophages and fibroblasts expressed NDST-1 but had generally less intense staining than SMC, although SMC expression decreased with culture. The uninjured rat aorta also showed widespread expression of NDST-1. After balloon de-endothelialisation, NDST-1 could not be detected in SMC of the neointima in the early stages of neointimal formation, but was re-expressed at later time points (after 12 weeks). In human coronary arteries, SMC of the media and the diffuse intimal thickening expressed NDST-1, while SMC in the atherosclerotic plaque were negative for NDST-1. We conclude that SMC may regulate their heparan sulphate sulphation at the level of expression of the enzyme heparan sulphate NDST in a manner related to their phenotypic state.

Chronic intracellular infection of alfalfa nodules by Sinorhizobium meliloti requires correct lipopolysaccharide core.

Our analyses of lipopolysaccharide mutants of Sinorhizobium meliloti offer insights into how this bacterium establishes the chronic intracellular infection of plant cells that is necessary for its nitrogen-fixing symbiosis with alfalfa. Derivatives of S. meliloti strain Rm1021 carrying an lpsB mutation are capable of colonizing curled root hairs and forming infection threads in alfalfa in a manner similar to a wild-type strain. However, developmental abnormalities occur in the bacterium and the plant at the stage when the bacteria invade the plant nodule cells. Loss-of-function lpsB mutations, which eliminate a protein of the glycosyltransferase I family, cause striking changes in the carbohydrate core of the lipopolysaccharide, including the absence of uronic acids and a 40-fold relative increase in xylose. We also found that lpsB mutants were sensitive to the cationic peptides melittin, polymyxin B, and poly-l-lysine, in a manner that paralleled that of Brucella abortus lipopolysaccharide mutants. Sensitivity to components of the plant's innate immune system may be part of the reason that this mutant is unable to properly sustain a chronic infection within the cells of its host-plant alfalfa.