Neural cell adhesion molecule is expressed by smooth muscle cells during the development of the rat vascular system

Summary The presence and distribution of neural cell adhesion molecule (N-CAM) was examined by light and electron microscopical immunocytochemistry in the descending thoracic aorta, the superior mesenteric artery and mesenteric arteries from fetal and adult rats (embryonic day 15 to post-natal day 90). In embryonic and early post-natal rats, N-CAM immunoreactivity was localized in perivascular nerves, in the smooth muscle cell plasma membrane and basal lamina. In nerves, N-CAM-immunoreactive sites were found associated with both the axon and Schwann cell membranes. N-CAM immunoreactivity was also found associated with the surface of adventitial fibroblast-like cells and with collagen fibrils, in regions where these fibrils were in contact with smooth muscle cells. In mature vessels N-CAM immunoreactivity was found to be restricted to the perivascular innervation and the surface of fibroblast-like cells. These observations indicate that N-CAM is expressed transiently in rat vascular tissues during development and is localized not only on the surface of smooth muscle cells but also in association with extracellular matrix components.     

Extracellular matrix remodeling in the vascular wall.

The extracellular matrix provides a structural framework essential for the functional properties of vessel walls. The three dimensional organization of the extracellular matrix molecules--elastin, collagens, proteoglycans and structural glycoproteins--synthesized during fetal development--is optimal for these functions. Early in life, the vessel wall is subjected to injury: lipid deposition, hypoxia, enzyme secretion and reactive oxygen species production during inflammatory processes, and the extracellular matrix molecules are hydrolyzed by proteases--matrix metalloproteinases, leukocyte elastase, etc. In uninjured arteries and veins, some proteases are constitutively expressed, but through the control of their activation and/or their inhibition by inhibitors, these proteases have a very low activity. During the occurrence of vascular pathologies--atherosclerosis, hypertension, varicosis, restenosis, etc.--the balance between proteases and their inhibitors is temporally destroyed through the induction of matrix metalloproteinase gene expression or the secretion of enzymes by inflammatory cells. Smooth muscle cells, the most numerous cells in vascular walls, have a high ability to respond to injury through their ability to synthesize extracellular matrix molecules and protease inhibitors. However, the three dimensional organization of the newly synthesized extracellular matrix is never functionally optimal. In some other pathologies--aneurysm--the injury overcomes the responsive capacity of smooth muscle cells and the quantity of extracellular matrix decreases. In conclusion, care should be taken to maintain the vascular extracellular matrix reserve and any therapeutic manipulation of the protease/inhibitor balance must be perfectly controlled, because an accumulation of abnormal extracellular matrix may have unforeseen adverse effects.     

Expression of tenascin by vascular smooth muscle cells. Alterations in hypertensive rats and stimulation by angiotensin II.

The extracellular matrix glycoprotein tenascin is associated with remodeling events in many embryonic and pathologic tissues. The expression of tenascin has been investigated by immunohistochemistry in blood vessels of Wistar-Kyoto (normotensive) and spontaneously hypertensive rats. Weak tenascin staining was present throughout the tunica media of large and small arteries from normotensive animals; strong staining was only detectable at branching sites. In arteries from hypertensive animals, foci of strong tenascin staining were scattered throughout the tunica media. The expression of tenascin mRNA and protein by rat aortic smooth muscle cells cultured in serum-free medium was induced by the vasoconstrictor peptide angiotensin II. Transforming growth factor-beta and platelet-derived growth factor also stimulated tenascin mRNA expression. Vascular smooth muscle cells attached specifically to a substratum of tenascin, but remained rounded. Thus, increased focal tenascin expression by vascular smooth muscle cells is associated with hypertension, and may mediate angiotensin II-induced changes in vascular structure in hypertension.     

Redox signaling, vascular function, and hypertension

Accumulating evidence supports the importance of redox signaling in the pathogenesis and progression of hypertension. Redox signaling is implicated in many different physiological and pathological processes in the vasculature. High blood pressure is in part determined by elevated total peripheral vascular resistance, which is ascribed to dysregulation of vasomotor function and structural remodeling of blood vessels. Aberrant redox signaling, usually induced by excessive production of reactive oxygen species (ROS) and/or by decreases in antioxidant activity, can induce alteration of vascular function. ROS increase vascular tone by influencing the regulatory role of endothelium and by direct effects on the contractility of vascular smooth muscle. ROS contribute to vascular remodeling by influencing phenotype modulation of vascular smooth muscle cells, aberrant growth and death of vascular cells, cell migration, and extracellular matrix (ECM) reorganization. Thus, there are diverse roles of the vascular redox system in hypertension, suggesting that the complexity of redox signaling in distinct spatial spectrums should be considered for a better understanding of hypertension.     

Vascular tissue engineering: microtextured scaffold templates to control organization of vascular smooth muscle cells and extracellular matrix

The in vitro construction of tissue-engineered small diameter (<6mm) blood vessels with sufficient strength and mechanical compliance has evaded researchers. We hypothesize that the high spatial organization of the medial layer of vascular smooth muscle cells (VSMCs) and their surrounding matrix provides high burst strength, compliance, and stability. We investigated the effect of microfabricated polydimethylsiloxane (PDMS) scaffolds with various groove widths on VSMC organization. We found that the presence of these grooved topographical cues significantly enhanced VSMC aspect ratio, alignment, and oriented remodeling of the underlying extracellular matrix. This study suggests that topographical patterning of tissue scaffolds can influence cellular and matrix spatial organization and could provide a framework for achieving the required organization and physical properties for blood vessels.     

An ultrastructural study of developing extracellular matrix in vitelline blood vessels of the early chick embryo

This investigation was designed to describe the morphological events in embryonic development of peripheral blood vessels (vasculogenesis) and to relate this process to the appearance of extracellular matrix (ECM) during growth and maturation of these tissues. Extraembryonic vitelline vessels of the early chick embryo were chosen for this study and light, transmission, and scanning electron microscopy were carried out on vessels excised from chick embryos (Hamburger-Hamilton stages 8 through 23). Our data show that early (stage 10) vessels are composed of two distinct epithelial layers, an inner layer of presumptive endothelium surrounded by a layer of splanchnopleuric mesoderm. During development, the inner layer gives rise to mature vascular endothelium while splanchnopleuric mesoderm differentiates to form primitive vascular smooth muscle. Ultrastructural studies show the presence of collagen and basal lamina in the extracellular space between these two layers during initiation of endothelial and smooth muscle cytodifferentiation. Furthermore, ruthenium red-positive material is present on basal surfaces of developing vascular endothelium at this time, indicating possible glycosaminoglycans (GAG) or other polyanionic components of the ECM. These data suggest that the sequential production of basal lamina, collagen(s), and/or GAG's by developing peripheral vessel wall epithelia may be critical to their final differentiation.     

Localization of vascular endothelial growth factor-D in malignant melanoma suggests a role in tumour angiogenesis

Expression of angiogenic and lymphangiogenic factors by tumours may influence the route of metastatic spread. Vascular endothelial growth factor (VEGF) is a regulator of tumour angiogenesis, but studies of the inhibition of solid tumour growth by neutralizing anti-VEGF antibodies indicated that other angiogenic factors may be involved. VEGF-D may be an alternative regulator because like VEGF it is angiogenic and it activates VEGF receptor-2 (VEGFR-2), an endothelial cell receptor which is a key signalling molecule in tumour angiogenesis. This study reports the generation of monoclonal antibodies to the receptor-binding domain of VEGF-D and the use of these antibodies to localize VEGF-D in malignant melanoma. VEGF-D was detected in tumour cells and in vessels adjacent to immunopositive tumour cells, but not in vessels distant from the tumours. These findings are consistent with a model in which VEGF-D, secreted by tumour cells, activates endothelial cell receptors and thereby contributes to the regulation of tumour angiogenesis and possibly lymphangiogenesis. In addition, VEGF-D was detected in the vascular smooth muscle, but not the endothelium, of vessels in adult colon. The endothelium of these vessels was negative for VEGFR-2 and VEGFR-3. As VEGF receptors can be up-regulated on endothelium in response to vessel damage and ischaemia, these findings of a specific localization of VEGF-D in smooth muscle of the blood vessels suggest that VEGF-D produced by vascular smooth muscle could play a role in vascular repair by stimulating the proliferation of endothelial cells.     

Vascular smooth muscle cells from injured rat aortas display elevated matrix production associated with transforming growth factor-beta activity.

The arterial response to injury is characterized by a short period of increased proliferation and migration of vascular smooth muscle cells, followed by an extended period of extracellular matrix accumulation in the intima. Transforming growth factor-beta (TGF-beta) has been implicated as a causative factor in the formation of extracellular matrix in this process, which leads to progressive thickening of the intima, known as intimal hyperplasia. In vitro analysis of vascular smooth muscle cells harvested from normal rat aortas and from aortas injured 14 days earlier showed that both types of cells attached equally well to culture dishes but that the initial spreading of the cells was increased in cells derived from injured vessels. Cells from the injured arteries produced more fibronectin and proteoglycans into the culture medium than the cells from normal arteries and contained more TGF-beta 1 mRNA. TGF-beta 1 increased proteoglycan synthesis by normal smooth muscle cells, and the presence of a neutralizing anti-TGF-beta 1 antibody reduced proteoglycan synthesis by the cells from injured arteries in culture. Fibronectin synthesis was not altered by these treatments. These results indicate that the accumulation of extracellular matrix components in neointimal lesions is at least partially caused by autocrine TGF-beta activity in vascular smooth muscle cells.     

Heterogeneity of myosin antigenic expression in vascular smooth muscle in vivo

Rabbit antisera elicited against purified human nonmuscle (platelet) and smooth muscle (uterine myometrium) myosins identified distinct species of myosin when frozen sections of a variety of mammalian tissues were examined by immunofluorescence microscopy. Antiplatelet myosin antiserum specifically stained several nonmuscle cell types including epithelial, some connective tissue, and all vascular endothelial (arterial, venous, capillary) cells. Antismooth muscle myosin antiserum stained only smooth muscle and no other cell types. Neither antiserum reacted with rat cardiac (ventricular) or skeletal muscle cells. Antismooth muscle myosin antiserum staining was detectable in medial vascular smooth muscle in all vessels examined from rat, bovine, human, and guinea pig sources (including elastic and muscular arteries, arterioles, venules, and veins). Although antiplatelet myosin antiserum did not stain nonvascular smooth muscle or vascular smooth muscle in muscular arteries, arterioles, venules, or veins, it did uniformly and specifically stain medial vascular smooth muscle in elastic arteries. This staining of elastic arteries was abolished by absorption of antiplatelet myosin antiserum with purified platelet myosin but not uterine myosin. Similarly, the reactivity of antismooth muscle myosin antiserum was abolished by incubation with uterine but not platelet myosin. The differences in staining patterns observed with antiplatelet myosin antiserum and antismooth muscle myosin antiserum in elastic arteries versus other blood vessels suggests a heterogeneity of antigenic expression in vascular smooth muscle myosin. The most likely explanations for this heterogeneity are the presence of different gene products (myosin isozymes) or a posttranslational alteration (possibly conformational) of a single myosin species. Heterogeneity in this important component of the contractile apparatus of vascular smooth muscle may have significant implications for the physiology and pathophysiology of the vessel wall.     

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.     

Human vascular smooth muscle in culture. Growth and ultrastructure.

Mixed primary cultures of endothelial and smooth muscle cells were obtained from human umbilical cord vessels after prolonged collagenase digestion of their luminal surfaces. Morphologically homogeneous populations of vascular smooth muscle were then selectively isolated and subcultured for up to 16 weeks. Ultrastructurally, cultured cells were characterized by the presence of bundles of myofilaments with dense bodies similar to native umbilical vessel smooth muscle. Mature cultures developed a distinctive topographical organization consisting of bands of parallel cells and intertwined, multilayered areas. Elaborate intercellular attachments formed along contiguous cell surfaces. Large amounts of extracellular material accumulated, including amorphous substance, elastic fiber microfibrils, and 250- to 300-A,faintly-banded fibrils. In older cultures, focal proliferation, extracellular material secretion and cellular degeneration produced nodular protrusions somewhat resembling atherosclerotic lesions in vivo. Endothelium and smooth muscle cultured from this readily available human source may provide useful comparative material for pathophysiologic studies of vascular disease.     

罗格列酮对CD34修饰脱细胞血管支架体内生长的影响

背景：早期研制的脱细胞血管基质支架上预载CD34+抗体会促进其再内皮化,但同时会加重支架内血管内膜增生。国内外研究证实过氧化物酶增殖体受体γ激动剂罗格列酮在体外可抑制平滑肌细胞增生及迁移,可减少血管损伤处内膜增生。 目的：进一步验证过氧化物酶增殖体受体γ激动剂罗格列酮对CD34抗体修饰脱细胞血管支架体内移植后平滑肌细胞生长及内膜增生的影响。 方法：获取新鲜兔颈动脉,应用光化学偶联法将CD34抗体固定到去细胞光氧化的血管支架上,构建抗体修饰的组织工程血管。将制备的血管分别移植于实验兔的颈动脉上,其中对照组予以移植单纯光氧化处理的脱细胞血管,CD34组予以CD34抗体预载的血管,罗格列酮组移植CD34抗体预载的血管并予喂养罗格列酮。 结果与结论：移植后10 d：对照组移植血管内皮样细胞数量稀少,CD34组和罗格列酮组可见较多的内皮样细胞覆盖;CD34组血管内膜较罗格列酮组厚,α-SMA染色显示CD34组血管平滑肌细胞数量较后者为多,其差异有显著性意义。移植后30 d：CD34组和罗格列酮组血管内皮样细胞基本覆盖管腔全层,对照组内皮样细胞数量仍较少;另外,CD34组血管内膜及管壁中可见大量的平滑肌样细胞及细胞外基质沉积,而罗格列酮组血管结构中平滑肌样细胞数量相对较少,内膜增生亦较轻。提示CD34修饰脱细胞血管支架可促进其内皮细胞的增生,罗格列酮可抑制血管支架中平滑肌细胞的增殖,减少内膜增生。     

Mechanical properties of tissue-engineered vascular constructs produced using arterial or venous cells

There is a clinical need for better blood vessel substitutes, as current surgical procedures are limited by the availability of suitable autologous vessels and suboptimal behavior of synthetic grafts in small caliber arterial graft (<5 mm) applications. The aim of the present study was to compare the mechanical properties of arterial and venous tissue-engineered vascular constructs produced by the self-assembly approach using cells extracted from either the artery or vein harvested from the same human umbilical cord. The production of a vascular construct comprised of a media and an adventitia (TEVMA) was achieved by rolling a continuous tissue sheet containing both smooth muscle cells and adventitial fibroblasts grown contiguously in the same tissue culture plate. Histology and immunofluorescence staining were used to evaluate the structure and composition of the extracellular matrix of the vascular constructs. The mechanical strength was assessed by uniaxial tensile testing, whereas viscoelastic behavior was evaluated by stepwise stress-relaxation and by cyclic loading hysteresis analysis. Tensile testing showed that the use of arterial cells resulted in stronger and stiffer constructs when compared with those produced using venous cells. Moreover, cyclic loading demonstrated that constructs produced using arterial cells were able to bear higher loads for the same amount of strain when compared with venous constructs. These results indicate that cells isolated from umbilical cord can be used to produce vascular constructs. Arterial constructs possessed superior mechanical properties when compared with venous constructs produced using cells isolated from the same human donor. This study highlights the fact that smooth muscle cells and fibroblasts originating from different cell sources can potentially lead to distinct tissue properties when used in tissue engineering applications.     

Circulating progenitors contribute to angiogenesis, vascular repair, and lesion formation

Atherosclerosis is responsible for more than half of all deaths in western countries. Numerous studies have reported that exuberant accumulation of smooth muscle cells plays a principal role in the pathogenesis of vascular diseases. It has been assumed that smooth muscle cells derived from the adjacent medial layer migrate, proliferate and synthesize extracellular matrix. Although much effort has been devoted, targeting migration and proliferation of medial smooth muscle cells, no effective therapy to prevent occlusive vascular remodeling has been established. Recently, we reported that bone marrow cells substantially contribute to the pathogenesis of vascular diseases, in models of post-angioplasty restenosis, graft vasculopathy and hyperlipidemia-induced atherosclerosis. It was suggested that bone marrow cells may have the potential to give rise to vascular progenitor cells that home in the damaged vessels and differentiate into smooth muscle cells or endothelial cells, thereby contributing to vascular repair, remodeling, and lesion formation. This article overviews recent findings on circulating vascular precursors and describes potential therapeutic strategies for vascular diseases, targeting mobilization, homing, differentiation and proliferation of circulating progenitor cells.     

Role of lysosomes in mesenteric arterial lesions of renal hypertensive rats

The acid phosphatase activities of arterial cells in the mesenteric arteries of renal hypertensive rats were investigated by both light and electron microscopy. Light microscopically, strongly positive acid phosphatase reactions were confirmed in endothelial cells, intimal cells, medial cells and adventitial cells of the mesenteric arteries, together with considerable deposition of fibrinoid substance in the intima and media in contrast to the appearance in control rats. Electron microscopically, lysosomes with acid phosphatase-positive reaction products were increased in number in endothelial cells, intimal smooth muscle cells, medial smooth muscle cells and adventitial neutrophils or macrophages. The lysosomes in intimal smooth muscle cells and those which were extracellularly discharged were responsible for lysis of the fibrinoid substance. In the media, acid phosphatase-positive lysosomes of medial cells and extracellularly discharged matrix lysosomes with acid phosphatase-positive reactions were also involved in the hydrolysis of necrotic substances and extracellular matrix. These acid phosphatase-positive reactions were diminished both light and electron microscopically in endothelial cells, intimal cells, medial muscle cells and adventitial cells in the regions of healing arteries where fibrinoid substance had been degradated and the intima showed cellulofibrous thickening. The possible role of this acid phosphatase activation for the clearance of cell debris as well as exudative substances in the healing of damaged arterial tissue was discussed.     

ROLE OF LYSOSOMES IN MESENTERIC ARTERIAL LESIONS OF RENAL HYPERTENSIVE RATS

The acid phosphatase activities of arterial cells in the mesenteric arteries of renal hypertensive rats were investigated by both light and electron microscopy. Light microscopically, strongly positive acid phosphatase reactions were confirmed in endothelial cells, intimal cells, medial cells and adventitial cells of the mesenteric arteries, together with considerable deposition of fibrinoid substance in the intima and media in contrast to the appearance in control rats. Electron microscopically, lysosomes with acid phosphatase-positive reaction products were increased in number in endothelial cells, intimal smooth muscle cells, medial smooth muscle cells and adventitial neutrophils or macrophages. The lysosomes in intimal smooth muscle cells and those which were extracellularly discharged were responsible for lysis of the fibrinoid substance. In the media, acid phosphatase-positive lysosomes of medial cells and extracellularly discharged matrix lysosomes with acid phosphatase-positive reactions were also involved in the hydrolysis of necrotic substances and extracellular matrix. These acid phosphatase-positive reactions were diminished both light and electron microscopically in endothelial cells, intimal cells, medial muscle cells and adventitial cells in the regions of healing arteries where fibrinoid substance had been degradated and the intima showed cellulofibrous thickening. The possible role of this acid phosphatase activation for the clearance of cell debris as well as exudative substances in the healing of damaged arterial tissue was discussed.     

Development and evaluation of a novel decellularized vascular xenograft

Although autogenous saphenous vein remains the standard for coronary and infrapopliteal bypass, many patients do not have a suitable vein. Attempts at developing a small-caliber vascular graft have failed largely due to occlusion, neointimal hyperplasia, or aneurismal degradation. We have designed and characterized a novel small-caliber vascular xenograft that may overcome these failure modes. To reduce immune reactions, porcine common carotid arteries were decellularized by enzymatic and detergent treatments. Histology and electron microscopic examination showed complete removal of cellular components while the extracellular matrix structure remained intact. To reduce thrombogeneity, decellularized vascular grafts were covalently linked with heparin. The efficiency of heparin linkage was demonstrated with toluidine blue staining and the antithrombogeneity of the heparin-treated grafts was demonstrated with a clot time test. Mechanical testing of the graft was performed. Decellularized-heparin-treated grafts were similar in compliance to fresh vessels and burst testing showed grafts to withstand pressures exceeding 10 times physiologic blood pressure. There was no difference in suture retention strength between fresh vessels and decellularized-heparin-treated grafts. Decellularized, heparinized grafts were implanted in dogs as carotid artery bypass grafts and showed smooth muscle cells densely populating the wall, and endothelial cells lining the lumen by two months. This study provides a new strategy to develop a small-caliber vascular graft with excellent mechanical properties, antithrombogeneity, and tissue compatibility.