Susceptibility of endothelial cells derived from different blood vessels to common viruses

Summary We examined whether endothelial cells derived from different blood vessels vary in their susceptibility to viral infection. Five common viral pathogens of humans (herpes simplex 1, measles, mumps, echo 9, and coxsackie B4 viruses) were evaluated for growth in endothelial cells derived from bovine fetal pulmonary artery thoracic aorta, and vena cava. All five viruses replicated in each type of endothelial cell. There were apparent differences in the quantities of measles and mumps viruses produced in pulmonary artery endothelium compared with thoracic aorta and vena cava when endothelial cells were obtained from different animals. However when pulmonary artery endothelial cells were compared with vena cava cells from the same animal, growth of each virus was similar in the two cell types. Four of the viruses replicated in the various endothelial cells without producing appreciable changes in cell morphology. These results indicate that endothelial cells from different blood vessels are equally susceptible to the human viruses evaluated, and that viral replication can occur without major alteration in cell morphology. Endothelial cells could serve as permissive cells permitting viruses to leave the circulation and initiate infection in adjacent tissues, including subendothelial smooth muscle cells. This work was supported by Public Health Service grants HL28220, HL 29492, and HL 24914 from the National Heart, Lung and Blood Institute, Bethesda, MD.     

Isolation of a morphologically and functionally distinct smooth muscle cell type from the intimal aspect of the normal rat aorta. Evidence for smooth muscle cell heterogeneity

Summary Recent studies indicate that the neointima of injured rat arteries is composed of a subpopulation of smooth muscle cells (SMCs) distinct from medial smooth muscle cells. However, SMC diversity in normal adult aorta has remained elusive. This study characterizes two morphologically and functionally distinct SMC types isolated from different anatomic regions of the normal rat aorta. Rat aortic medial smooth muscle cells (MSMCs) were isolated from the media after removal of the intimal and adventitial cells. Rat aortic intimal smooth muscle cells (ISMCs) were isolated from the intimal aspect of everted rat aortas. The two cell types were characterized morphologically and immunohistochemically and were compared for their capacity to contract collagen gels in response to endothelin-1. MSMCs were spindle-shaped and grew in hills and valleys showing features previously described for vascular SMCs. Conversely, ISMCs displayed a polygonal and epithelioid shape, grew mainly as a monolayer, and had a higher proliferative rate. Both cell types expressed alpha-smooth muscle actin and were negative for Factor VIII-RAg. ISMCs produced large amounts of a laminin and type IV collagen-rich extracellular matrix which had a characteristic pericellular distribution. ISMCs, but not MSMCs, rapidly contracted collagen gels in response to endothelin-1. This study indicates that the normal rat aorta contains two types of SMCs located in anatomically distinct regions of the vessel wall. Because of their functional characteristics, the SMCs isolated from the intimal aspect of the aorta may play an important role in physiologic as well as pathologic conditions.     

Expression of connexin43 gap functions between cultured vascular smooth muscle cells is dependent upon phenotype

The smooth muscle cell is the predominant cell type of the arterial media. In the adult vascular system, smooth muscle cells are found primarily in the contractile phenotype, but following injury or during atherosclerotic plaque formation the secretory synthetic phenotype is expressed. Recently it has been shown that gap junction connexin43 messenger RNA levels are six times higher in cultured smooth muscle cells in the synthetic phenotype than in intact aorta. We have modulated rabbit aortic smooth muscle cells in culture between the synthetic phenotype and one resembling the contractile phenotype, and correlated gap junction expression with phenotype. A dual labelling technique with antibodies against smooth muscle myosin and a synthetic peptide constructed to match a portion of the connexin43 gap junction protein was used for these experiments. Gap junctions are numerous between synthetic phenotype cells but few are observed between contractile cells. Rat aortic smooth muscle cells were also cultured and the growth and structure of gap junctions followed in the synthetic phenotype by use of freeze-fracture electron microscopy and immunohistochemical techniques. Junctional plaques are similar in structure to those observed in cardiac muscle, their size and number increasing with time in culture. The increased numbers of gap junctions between synthetic phenotype smooth muscle cells may be important during vessel development, following injury, or in atherosclerotic plaque formation.     

Fine morphological aspects of the secretory process in arterial smooth muscle cells. II. Role of microtubules

Preliminary results are presented regarding the role of the microtubular system in the secretory process in the rabbit aorta and pulmonary trunk smooth muscle cells. The results are based on electron microscopic findings of the effect of colchicine in vivo. On colchicine treatment an accumulation of secretory granules versus a vacuolar type of dilatation of the rough endoplasmic reticulum cisternae distinguishes two groups of smooth muscle cells. It is suggested that two secretory pathways operate in these cells, and that the cell microtubular status plays a key role in triggering one or the other pathway.     

Importance of the phenotypic state of vascular smooth muscle cells on the binding and the mitogenic activity of endothelin

Smooth muscle cells of the rabbit aorta, when grown in vitro, express distinguishable forms of phenotypes (contractile and synthetic). On contractile cells, ET-1 specifically bound to a single class of high affinity (KD = 128 pM) and high capacity (Bmax = 66,000 sites/cell) binding sites. But, whereas affinity of [125I]-ET-1 was not significantly affected by phenotypic modulation, synthetic cells displayed a 10-fold lower [125I]-ET-1 binding capacity than contractile smooth muscle cells. Similarly, the mitogenic effect of ET-1 on smooth muscle cells was considerably lower for synthetic than for contractile cells. The ET-1 receptor on primary cells was recognized by sarafotoxin S6b and the different ET-related peptides with an order of potency [ET-1 greater than S6b greater than ET-3 greater than Big ET-1 much greater than ET(16-21)] identical to that inducing smooth muscle cell growth. Therefore, these data indicate that the binding and the mitogenic effects of ET-1 on smooth muscle cells might be of different magnitudes depending on the phenotypic state of these cells.     

Differentiation Between Herpes Simplex Virus Type 1 and Type 2 Strains by Immunoelectroosmophoresis

A method has been elaborated to differentiate between herpes simplex type 1 and type 2 viruses by immunoelectroosmophoresis. With rabbit immune sera cross-absorbed with heterologous virus antigen, a distinct difference was shown between the two virus types. Herpes simplex type 1 virus tested against cross-absorbed type 1 antiserum gave two precipitin lines. Herpes simplex type 2 virus gave one precipitin line when tested against cross-absorbed homologous serum. When the viral antigens were tested against cross-absorbed heterologous immune sera, no or only very weak precipitin reactions were observed. The test is easy and rapid, requires relatively small quantities of antigen and antibody, and is suitable for typing of herpes simplex virus in diagnostic routine work.     

A comparative study on the metabolic activation of 3,4-benzo[a]pyrene to mutagens by aorta smooth muscle cells of rat and rabbit

The mutagenic activation of benzo[a]pyrene (BaP) after exposure to aorta smooth muscle cells of different origin was examined. Three test systems with different genetic endpoints--sister-chromatid exchange (SCE), gene mutation at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus and unscheduled DNA synthesis (UDS)--were used. Treatment of rat and rabbit aorta smooth muscle cells with BaP (1-6 micrograms/ml) resulted in a significant increase of SCEs, HGPRT mutations and UDS. So smooth muscle cells are capable of converting BaP to metabolites with a DNA-damaging action. In order to investigate the relation between the formation of mutagenic BaP metabolites and the susceptibility to atherosclerosis we compared the mutagenic potential of BaP using aorta smooth muscle cells of different species (rat, rabbit) and locations (thoracic and abdominal aorta). Rabbits and abdominal aortas are more susceptible to atherosclerosis than rats and thoracic aortas. The SCE, HGPRT and UDS assays revealed that smooth muscle cells of different origin possessed the same metabolic potential towards BaP. There was no correlation between the mutagenic potency of BaP metabolites and the susceptibility to atherosclerosis. As smooth muscle cells have a low metabolic capacity towards BaP, probably other factors in addition to the metabolic capacity of smooth muscle cells are responsible for species and tissue differences in susceptibility to atherosclerosis.     

Regional functional heterogeneity of aortic smooth muscle cells in rats

The aorta is a magistral artery, which has been traditionally looked upon as a vessel whose properties are invariable throughout its length. However, in the most recent decade, there have been accumulated data that provide evidence that different aorta sections arise from different embryonic origins and that the population of smooth muscle cells making up the vessel’s wall is, consequently, heterogenic. Tracing the fate of smooth muscle cells, the basic components of the vessel, with the aid of genetic marking methods revealed that the cells’ response to various factors is largely determined by the embryonic origin of a certain cell population. However, functional differences between the smooth muscle cells making up different aorta sections remain poorly understood. The aim of the current work was to compare the functional characteristics of the populations of aortic wall smooth muscle cells obtained from the aorta sections differing by their embryonic origin. Towards this end, we obtained smooth muscle cell cultures from the three aorta sections of linear rats, namely, the neural crest derived ascending thoracic aorta, the somites derived descending thoracic aorta, and splanchnic mesoderm derived abdominal aorta. Using immunocytochemistry and Western blotting, the cells from the different regions of aorta were compared on the basis of smooth muscle actin, vimentin, and SM22 content in them. Cell proliferation rate was estimated using the growth curves method. We have demonstrated that the three smooth muscle cell populations arising from different embryonic origins differ in their morphological characteristics as well as by smooth muscle actin and SM22 content. We have shown that smooth muscle cells from the ascending aorta proliferate more actively than the corresponding cells from the descending thoracic aorta. Thus, the functional properties of the populations of rat aortic smooth muscle cells are different and depend on the embryonic origin of the aorta section from which they were obtained.     

Properties of a clonal muscle cell line from rat heart

Two clonal cell lines have been derived from the thoracic aorta of embryonic rats. Both of these cell lines, at some stage of their development possess membranes capable of generating overshooting action potentials spontaneously. Contiguous cells of each of these lines are electrically coupled. Ultrastructural analysis consistently reveals the presence of rows of pinocytotic vesicles, a well-developed rough endoplasmic reticulum, massive tracts of thin filaments oriented parallel to the longitudinal axis of the cell and randomly dispersed intermediate sized filaments. The specific activities of the enzymes myokinase and creatine phosphokinase (CPK) increase 3- to 5-fold after growth has ceased. These two cell lines synthesize a muscle type CPK isoenzyme after the cessation of cell division. It is concluded that these cell lines proliferate as myoblasts and develop into cells which phenotypically resemble smooth muscle. A third clonal cell line, from fetal rat aorta, with properties of both smooth and skeletal muscle, is also described.     

大鼠主动脉内皮细胞和平滑肌细胞的原代培养及生物学特性比较

目的培养大鼠主动脉平滑肌细胞和内皮细胞,细胞纯化与鉴定,比较生物学特性的差异。方法采用血管环贴壁法培养动脉内皮细胞,组织块贴壁法培养动脉平滑肌细胞,并采用有限稀释法挑选内皮细胞单克隆,免疫细胞荧光鉴定二者的特异性标志,相差显微镜观察二者单个细胞及细胞群体在形态上的差异性,CCK-8试剂盒检测细胞的增殖,比较二者对胰酶消化,粘附,冻存后复苏的情况。结果血管环贴壁法成功培养血管内皮细胞,组织块培养法成功培养出血管平滑肌细胞,内皮细胞能够形成单克隆集落,培养的细胞均表达相应的特异性标志,内皮细胞增殖速度和平滑肌细胞有差异,内皮细胞对胰酶的耐受性较差,内皮细胞粘附所需时间短,对冻存后的耐受性较好。结论组织块贴壁法适合内皮细胞和平滑肌细胞的培养,有限稀释法能够纯化原代培养的内皮细胞,大鼠主动脉平滑肌细胞和内皮细胞在细胞形态、增殖、粘附、对胰酶的反应、冻存后复苏均存在差异。     

The Complete Amino Acid Sequence of Actins from Bovine Aorta, Bovine Heart, Bovine Fast Skeletal Muscle, and Rabbit, Slow Skeletal Muscle

Complete amino acid sequences for four mammalian muscle actins are reported: bovine skeletal muscle actin, bovine cardiac actin, the major component of bovine aorta actin, and rabbit slow skeletal muscle actin. The number of different actins in a higher mammal for which full amino acid sequences are now available is therefore increased from two to five. Screening of different smooth muscle tissues revealed in addition to the aorta type actin a second smooth muscle actin, which appears very similar if not identical to chicken gizzard actin. Since the sequence of chicken gizzard actin is known, six different actins are presently characterized in a higher mammal.
The two smooth muscle actins—bovine aorta actin and chicken gizzard actin—differ by only three amino acid substitutions, all located in the amino-terminal end. In the rest of their sequences both smooth muscle actins share the same four amino acid substitutions, which distinguish them from skeletal muscle actin. Cardiac muscle actin differs from skeletal muscle actin by only four amino acid exchanges. No amino acid substitutions were found when actins from rabbit fast and slow skeletal muscle were compared.
In addition we summarize the amino acid substitution patterns of the six different mammalian actins and discuss their tissue specificity. The results show a very close relationship between the four muscle actins in comparison to the nonmuscle actins. The amino substitution patterns indicate that skeletal muscle actin is the highest differentiated actin form, whereas smooth muscle actins show a noticeably closer relation to nonmuscle actins. By these criteria cardiac muscle actin lies between skeletal muscle actin and smooth muscle actins.     

Characterization of myosin heavy chains in cultured aorta smooth muscle cells. A comparative study

Myosin heavy chains (MHCs) from rat aorta smooth muscle cells were analyzed prior to and after these cells were placed into cell culture using sodium dodecyl sulfate-5% polyacrylamide gels, immunoblots, and two-dimensional peptide maps of tryptic digests. Rat aorta smooth muscle cells prior to culture were found to contain two MHCs (mass = 204 and 200 kDa) which cross-reacted with antibodies raised to smooth muscle myosin, but not with antibodies raised to platelet myosin. Tryptic peptide maps of these two MHCs showed no major differences when compared to each other and to maps of vas deferens and uterus smooth muscle MHCs. When rat aorta smooth muscle cells were placed into culture, the MHCs isolated from the cell extracts differed, depending on whether the cells were rapidly growing or postconfluent. Extracts from log-phase cultures contained predominantly MHCs that migrated more rapidly than smooth muscle myosin in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (mass = 196 kDa) and cross-reacted with antibodies raised to platelet myosin, but not to smooth muscle myosin. Tryptic peptide maps of this MHC were very similar to those obtained with MHCs from non-muscle sources such as platelets and fibroblasts. In contrast, extracts from postconfluent rat aorta cell cultures contained three MHCs (mass = 204, 200, and 196 kDa). Using immunoblots and peptide maps, the fastest migrating MHC was found to be identical to the 196-kDa non-muscle MHC, while the two slower migrating MHCs had the same properties as aorta smooth muscle MHCs prior to culture. These results suggest that smooth muscle cells grown in primary culture contain predominantly (greater than 80%) non-muscle myosin while actively growing, but at a postconfluent stage, contain more equivalent amounts of smooth muscle and non-muscle myosins.     

The complete amino acid sequence of actins from bovine aorta, bovine heart, bovine fast skeletal muscle, and rabbit slow skeletal muscle. A protein-chemical analysis of muscle actin differentiation.

Complete amino acid sequences for four mammalian muscle actins are reported: bovine skeletal muscle actin, bovine cardiac actin, the major component of bovine aorta actin, and rabbit slow skeletal muscle actin. The number of different actins in a higher mammal for which full amino acid sequences are now available is therefore increased from two to five. Screening of different smooth muscle tissues revealed in addition to the aorta type actin a second smooth muscle actin, which appears very similar if not identical to chicken gizzard actin. Since the sequence of chicken gizzard actin is known, six different actins are presently characterized in a higher mammal. The two smooth muscle actins--bovine aorta actin and chicken gizzard actin--differ by only three amino acid substitutions, all located in the amino-terminal end. In the rest of their sequences both smooth muscle actins share the same four amino acid substitutions, which distinguish them from skeletal muscle actin. Cardiac muscle actin differs from skeletal muscle actin by only four amino acid exchanges. No amino acid substitutions were found when actins from rabbit fast and slow skeletal muscle were compared. In addition we summarize the amino acid substitution patterns of the six different mammalian actins and discuss their tissue specificity. The results show a very close relationship between the four muscle actins in comparison to the nonmuscle actins. The amino substitution patterns indicate that skeletal muscle actin is the highest differentiated actin form, whereas smooth muscle actins show a noticeably cloer relation to nonmuscle actins. By these criteria cardiac muscle actin lies between skeletal muscle actin and smooth muscle actins.     

糖尿病和非糖尿病动脉粥样硬化兔模型的建立

目的建立兔动脉粥样硬化和糖尿病动脉粥样硬化模型并比较其动脉粥样硬化病变的特点。方法四氧嘧啶静脉推注诱发糖尿病后,行腹主动脉球囊损伤术拉伤内皮并饲高脂饲料建立糖尿病动脉粥样硬化兔模型,非糖尿病动脉粥样硬化兔模型静脉推注生理盐水,余处理相同。喂养10周做腹主动脉造影和腹主动脉内超声后处死,取腹主动脉横切片做HE染色和免疫组化,比较两组兔主动脉内膜/中膜比值及巨噬细胞、平滑肌细胞含量,以评价动脉粥样硬化病变的程度和性质。结果所有兔胸主动脉粥样硬化病变明显轻于腹主动脉;糖尿病动脉粥样硬化兔腹主动脉壁特别是近血管腔处巨噬细胞浸润明显多于动脉粥样硬化兔,而平滑肌细胞含量显著减少。结论糖尿病动脉粥样硬化兔的腹主动脉粥样硬化病变内有更加活跃的炎症细胞浸润,提示病变性质更加不稳定。     

Human smooth muscle VLA-1 integrin: purification, substrate specificity, localization in aorta, and expression during development

A membrane glycoprotein complex was isolated and purified from human smooth muscle by detergent solubilization and affinity chromatography on collagen-Sepharose. The complex was identified as VLA-1 integrin and consisted of two subunits of 195 and 130 kD in SDS-PAGE. Liposomes containing the VLA-1 integrin adhered to surfaces coated with type I, II, III, and IV collagens, Clq subcomponent of the first component of the complement, and laminin. The liposomes specifically adhered to these proteins in a Ca2+, Mg2(+)-dependent manner, but did not bind to gelatin, fibronectin, and thrombospondin substrates. The expression of VLA-1 integrin in different human tissues and cell types, and during aorta smooth muscle development was studied by SDS-PAGE, and subsequent quantitative immunoblotting was performed with antibodies recognizing alpha 1 and beta 1 subunits of the VLA-1 integrin. A high level of VLA- 1 integrin expression was an exceptional feature of smooth muscles. Fibroblasts, endothelial cells, keratinocytes, striated muscles, and platelets contained trace amounts of VLA-1 integrin. In the 10-wk-old human fetal aorta, VLA-1 integrin was found only in smooth muscle cells whereas mesenchymal cells, surrounding aortic smooth muscle cells, were VLA-1 integrin negative. By the 24th wk of gestation, the amount of VLA- 1 integrin was significantly reduced in the aortic media (4.3-fold for alpha 1 subunit and 2.5-fold for beta 1 subunit) compared with that in the 10-wk-old aortic smooth muscle cells. After birth, the expression of VLA-1 integrin increased and in the 1.5-yr-old child aorta the VLA-1 integrin level was almost the same as in adult aortic media. Smooth muscle cells from intimal thickening of adult aorta express five times less alpha 1 subunit of VLA integrin that smooth muscle cells from adult aortic media. In primary culture of aortic smooth muscle cells, the content of the VLA-1 integrin was dramatically reduced and subcultured cells did not contain VLA-1 integrin at all.     

Myosin heavy-chain isoforms in adult and developing rabbit vascular smooth muscle

Two monoclonal antibodies specific for smooth muscle myosin (designated SM-E7 and SM-A9) and one monoclonal anti-(human platelet myosin) antibody (designated NM-G2) have been used to study myosin heavy chain composition of smooth muscle cells in adult and in developing rabbit aorta. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Western blotting experiments revealed that adult aortic muscle consisted of two myosin heavy chains (MCH) of smooth muscle type, named MHC-1 (205 kDa), and MHC-2 (200 kDa). In the fetal/neonatal stage of development, vascular smooth muscle was found to contain only MHC-1 but not MHC-2. Non-muscle myosin heavy chain, which showed the same electrophoretic mobility as the slower migrating MHC, was expressed in an inverse manner with respect to MHC-2, i.e. it was detectable only in the early stages of development. The distinct pattern of smooth and non-muscle myosin isoform expression during development may be related to the different functional properties of smooth muscle cells during vascular myogenesis.