Junctional transfer in cultured vascular endothelium: I. Electrical coupling

Vascular endothelial cultures are composed of flat, polygonal monolayer cells which retain many of the growth, metabolic and physiological characteristics of the intimal endothelium. However, intercellular gap and tight junctions, which are thought to perform important roles in normal intimal physiology, are reduced in complexity and extent in culture. We have used electrophysiological techniques to test confluent (3- to 5-day) primary cultures of calf aortic (BAEC) and umbilical cord vein (BVEC) endothelium for junctional transfer of small ions. Both cell types are extensively electrically coupled. The passive electrical properties of the cultured cells were calculated from the decrease in induced membrane potential deflections with distance from an intracellular, hyperpolarizing electrode. Data analyses were based on a thin-sheet model for current flow (Bessel function). The generalized space constants (lambda) were 208.6 microns (BAEC) and 288.9 microns (BVEC). The nonjunctional (6.14 and 8.72 X 10(8) omega) and junctional (3.67 and 3.60 X 10(6) omega) resistances were similar for the BAEC and BVEC, respectively. We detected no statistically significant differences in the resistance estimates for the two cell types. In vivo ultrastructural studies have suggested that aortic endothelium has more extensive gap junctions than venous endothelium. We have found that these ultrastructural differences are reduced in culture. The lack of any significant difference in electrical coupling capability suggests that cultured BAEC and BVEC have functionally similar junctional characteristics.     

Dual Benefit of Reduced Cx43 on Atherosclerosis in LDL Receptor-Deficient Mice

Paracrine cell-to-cell interactions are crucial events during atherogenesis, however, little is known on the role of gap junctional communication during this process. We recently demonstrated increased expression of Cx43 in intimal smooth muscle cells and in a subset of endothelial cells covering the shoulder of atherosclerotic plaques. The purpose of this study was to examine the role of Cx43 in the development of atherosclerosis in vivo. Atherosclerosis-susceptible LDL receptor-deficient (LDLR^?/?) mice were intercrossed with mice heterozygous for Cx43 (Cx43^+/?mice). Male mice with normal (Cx43^+/+LDLR^?/?) or reduced (Cx43^+/?LDLR^?/?) Cx43 level of 10 weeks old were fed a cholesterol-rich diet (1.25%) for 14 weeks. Both groups of mice showed similar increases in serum lipids and body weight. Interestingly, the progression of atherosclerosis was reduced by 50% (P < 0.01) in the thoraco-abdominal aorta and in the aortic roots of Cx43^+/?LDLR^?/?mice compared with Cx43^+/+LDLR^?/?littermate controls. In addition, atheroma in Cx43^+/?LDLR^?/?mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells, important features associated, in human, with stable atherosclerotic lesions. Thus, reducing Cx43 expression in mice provides beneficial effects on both the progression and composition of the atherosclerotic lesions.     

Primary cultures of rat aortic endothelial and smooth muscle cells: I. An in vitro model to study xenobiotic-induced vascular cytotoxicity

Summary Primary cultures of rat vascular endothelial and smooth muscle cells were developed as models to study xenobiotic-induced cytotoxicity. Endothelial and smooth muscle cells were isolated by enzymatic digestion and mechanical dissociation of rat thoracic aortae. Optimal cell growth and minimal fibroblast contamination in cultures of both cell types were obtained in Medium 199 supplemented with 10% fetal bovine serum. Cultured cells were characterized by distinctive morphologic features and growth patterns. Intercellular endothelial cell junctions were selectively stained with silver nitrate. Endothelial cells also exhibited a nonthrombogenic surface, as reflected by platelet-binding studies. Confluent cultures of smooth muscle cells, but not endothelial cells, contracted in response to norepinephrine (10 μM). Cultures of both cell types were exposed to acrolein (2, 5 or 50 ppm), an environmental pollutant, for 4 24 h. Morphologic damage, lactate dehydrogenase release, and cellular thiol content were used as indices of cytotoxicity. Acrolein-induced enzyme leakage and morpholgic alterations were dose- and time-dependent and more pronounced in cultures of smooth muscle cells than in endothelial cells. The total thiol content of endothelial cells exposed to acrolein (50 ppm) for 24 h was not significantly different from that of respective controls. In contrast, the content of treated smooth muscle cells was higher than that of controls. These observations show that primary cultures of vascular cells provide a useful model to evaluate xenobiotic-induced cytotoxicity. The information obtained using a cell culture system may be complemented by the use of other in vivo and in vitro models to determine the mechanisms by which xenobiotics cause vascular cell injury.     

Effects of cholesterol oxidase on cultured vascular smooth muscle cells

Summary Cholesterol oxidase (3-hydroxy-steroid oxidase) catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives. The purpose of the present study was to investigate its effects on cultured vascular smooth muscle cells. Cultured rabbit aortic smooth muscle cells were morphologically altered after exposure to cholesterol oxidase in the presence of culture medium containing 10% fetal calf serum. If fetal calf serum was absent, cells were unaffected by the treatment. The extent of morphological change of the smooth muscle cells was dependent upon the time of exposure to the enzyme and the concentration of cholesterol oxidase employed. After moderate treatment with cholesterol oxidase, cells excluded trypan blue. Further, a specific mitochondrial marker DASPMI (dimethyl aminostyryl-methyl-pyridiniumiodine) which was used as a fluorescent index of cell viability, revealed that cell viability was unchanged after moderate cholesterol oxidase treatment. Nile red, a hydrophobic probe which selectively stains intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with cholesterol oxidase. Cellular nile red fluorescence intensity increased linearly with the time and concentration of cholesterol oxidase treatment. These results demonstrate that cholesterol oxidase alters lipid deposition in the cell and changes cell morphology. The primary site of action of cholesterol oxidase appears to be independent of the cell membrane itself and instead is dependent upon the lipid content in the surrounding culture media. These changes occur prior to the cytotoxic effects of extensive oxidation. Because oxidized cholesterol may play an important role in the pathogenesis of atherosclerosis, our results have implications for intracellular accumulation of lipids in smooth muscle cells during the atherosclerotic lesion.     

Hypertriglyceridemic serum from magnesium-deficient rats induces proliferation and lipid accumulation in cultured vascular smooth muscle cells

An important characteristic of hyperlipemia associated with magnesium deficiency in rats is the postprandial accumulation of triglyceride-rich lipoproteins. The present investigation was performed to determine the effect of serum from magnesium-deficient animals on cultured vascular smooth muscle cells (VSMC). Sera were obtained from control and magnesium-deficient rats fed adequate or deficient diets for 8 days. Magnesium-deficient animals were hypertriglyceridemic compared with control rats, but their total cholesterolemia was not significantly modified. Pooled sera from control and magnesium-deficient animals were added to the culture medium at various concentrations. The maximum of proliferation for both control and magnesium-deficient sera was reached when they were added at 6% to the culture medium and on day 4 after the begining of incubation. Medium containing serum from magnesium-deficient rats stimulated the cell proliferation as monitored by cell count and [³H]thymidine incorporation. Staining of VSMC with Oil red O and measuring lipids have shown a marked lipid accumulation (triglycerides) in cells incubated with serum obtained from magnesium-deficient animals compared with serum from control rats. These results indicate that serum from magnesium-deficient rats contains factors that stimulate proliferation of arterial medial cells and that hyperlipemia associated with magnesium-deficiency may cause lipid accumulation in vascular cells.     

Metabolic control analysis of the Warburg-effect in proliferating vascular smooth muscle cells

Summary The accumulation and proliferation of vascular smooth muscle cells (VSMC) within the vessel wall is an important pathogenic feature in the development of atherosclerosis. Glucose metabolism has been implicated to play an important role in this cellular mechanism. To further elucidate the role of glucose metabolism in atherogenesis, glycolysis and its regulation have been investigated in proliferating VSMC. Platelet derived growth factor (PDGF BB)-induced proliferation of VSMCs significantly stimulated glucose flux through glycolysis. Further evaluating the enzymatic regulation of this pathway, the analysis of flux:metabolite co-responses revealed that anaerobic glycolytic flux is controlled at different sites of gycolysis in proliferating VSMCs, being consistent with the concept of multisite modulation. These findings indicate that regulation of glycolytic flux in proliferating VSMCs differs from traditional concepts of metabolic control of the Embden–Meyerhof pathway.     

Permeance of novikoff hepatoma gap junctions: Quantitative video analysis of dye transfer

Summary Fluorescent dyes are commonly used to study permeable (gap) junctions, but only rarely have quantitative values for junctional dye permeability been determined. In the present study, junctional permeance (PA, i.e., the product of the junctional permeability coefficient,P, times the junctional area,A) to Lucifer Yellow CH (LY) has been obtained for pairs of Novikoff hepatoma cells. Dye was microinjected into one cell and the subsequent transfer monitored by a SIT camera and recorded on video tape. The intensities of fluorescence in the injected and recipient cell were measured using a Digisector (Microworks) digitizing board and an Apple II Plus computer to analyze the video records. These changes in intensity, along with an estimate of volume of the spherical cells, were used to calculate the junctional permeance (PA) of cell pairs according to Fick's diffusion equation. Junctional permeances show considerable variation ranging from 0.08×10^–11 to 27.0×10^–11 cm³/sec. Using the meanPA and a previous estimate of the mean number of junctional channels per interface in the Novikoff cultures, a value for diffusion coefficient of LY through gap junctions is calculated to be about 1.4×10^–6 cm²/sec. There is a general proportionality between meanPA and cell volume for hepatoma cell pairs of a certain size range. Such a relationship between cell volume and junctional capacity suggests one source of variation ofPA. Other possible sources, e.g., related to position in the cell cycle, are discussed.     

The extracellular matrix and the control of proliferation of vascular endothelial and vascular smooth muscle cells

In this short review we describe the observations which have led us to conclude that one of the most important components involved in modulating cell proliferation in vitro, and probably in vivo as well, may be the extrac-cellular matrix upon which cells rest.     

Transforming growth factor-beta: Signal transduction via protein kinase C in cultured embryonic vascular smooth muscle cells

Summary Transforming growth factor-beta (TGF-β), an ubiquitous regulatory peptide, has diverse effects on the differentiation and behavior of vascular smooth muscle cells (VSMC). However, the molecular mechanism through which TGF-α exerts its effects remains obscure. We investigated the phosphoinositide/protein kinase C [PKC] signaling pathway in the action of TGF-β on cultured embryonic avian VSMC of differing lineage: a) thoracic aorta, derived from the neural crest; and b) abdominal aorta, derived from mesenchyme. The second messenger responsible for activation of PKC is sn-1,2-diacylglycerol [DAG]; TGF-β increased the mass amounts of DAG in the membranes of neural crest-derived VSMC concurrent with translocation of PKC from the soluble to the membrane fraction, but TGF-β had no effect on the DAG or PKC of mesenchyme-derived VSMC. TGF-β potentiated the growth of platelet-derived growth factor (PDGF)-treated, neural crest-derived VSMC; but abolished PDGF-induced growth of mesenchymal cells. It is concluded that molecular and functional responses of VSMC to TGF-β are heterogeneous and are functions of the embryonic lineage of the VSMC.     

A morphometric study of vascular smooth muscle cells in culture

Summary Cultured arterial smooth muscle cells derived from different times in culture, different passages, and different species were evaluated by a combination of transmission electron microscopy and morphometry. The morphometric studies focused on point counting and monitored the following cellular components: lysosomes, myofilaments, mitochondria, ribosomes, and rough endoplasmic reticulum (RER). Percent volume composition values for the organelles involved in protein synthesis, namely ribosomes and RER, show significant fluctuations with time. Consistent with these observations, the cells showed increasing myofilaments during the early weeks in culture, which subsequently decreased significantly. The data also indicate that rabbit cells in culture may become synthetically quiescent with time and the distribution of cellular components is altered with each succeeding passage. Cultured calf (bovine) cells exhibit similar activity periods compared to rabbit but show a significantly higher lysosomal and lower myofilament content than rabbit. Calf cells could not be maintained for longer than 21 days in the absence of ascorbate, whereas ascorbate affects the ultrastructure of rabbit cells less dramatically. Age, passage, and donor, among others, are important considerations for studying in vitro smooth muscle cells. With proper morphologic and morphometric monitoring, these smooth muscle cell culture systems can be important tools in the study of aging or pathologic processes, or both. This work was presented as partial fulfillment for the degree of Ph.D. This work was supported by National Institutes of Health Grants HL-13262, HL-19717, and AG-00001.     

Porcine aortic organ culture: A model to study the cellular response to vascular injury

Summary Organ cultures of porcine thoracic aorta were studied to define the characteristics of this system as a model to study the reaction of endothelial cells (ECs) and the underlying smooth muscle cells (SMCs) to injury. Both nonwounded and wounded cultures the latter having had part of the endothelial surface gently denuded with a scalpel blade, were studied over a 7 d period by scanning and transmission electron microscopy. The results showed that the nonwounded ECs underwent a shape change from elongated to polygonal within 24 h in culture. In both nonwounded and wounded explants there was cell proliferation beneath the nondenuded endothelium so that by 7 d several layers of cells were present showing features of the secretory type of SMCs. This proliferation, however, did not occur if the endothelium was totally removed from the aorta. There was also evidence of gaps between the surface ECs, and by 7 d lamellipodia of cells beneath the surface were present in these gaps. Occasionally, elongated cells were seen to be present on the surface of the endothelium. In the wounded organ culture, cell migration and proliferation occurred extending from the wound edge and producing a covering of cells on the denuded area. There were also multilayered cells beneath the surface similar to the nonwounded area. Occasional foam cells were seen in the depth of the multilayered proliferating cells. The results indicate that organ culture of porcine thoracic aorta is a good model to study the reaction of ECs and underlying SMCs to injury. This work was supported by a grant from the Ontario Heart Foundation.     

Lymphatic endothelial and smooth-muscle cells in tissue culture

Summary Endothelial and smooth-muscle cells from bovine mesenteric lymphatic vessels have been collected and cultured in vitro. The endothelial cells grew as a monolayer exhibiting a “cobblestone” appearance with individual cells tending to be more flattened at confluence than their blood vascular counterparts. Approximately 30% of these cells expressed Factor VIII antigen compared with bovine mesenteric artery or human umbilical-vein endothelium in which the majority of cells were positive. The lymphatic smooth-muscle cells exhibited focal areas of multilayering and were Factor VIII negative. The availability of lymphatic endothelial and smooth-muscle cells in culture will provide a new tool for the investigation of the biological properties of the lymphatic vessels and their role in homeostasis. Supported by the Medical Research Council of Canada, Grant MA-7925     

Effects of oxidative modification of cholesterol in isolated low density lipoproteins on cultured smooth muscle cells

Summary It has been proposed that low density lipoprotein (LDL) must undergo oxidative modification before it can participate in atherosclerosis. The present paper studied the effect of cholesterol oxidation in LDL on cultured vascular smooth muscle cells. LDL was oxidized by cholesterol oxidase (3--hydroxy-steroid oxidase) which catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives. Cholesterol oxidase treatment of LDL did not result in lipid peroxidation. Cultured rabbit aortic smooth muscle cells were morphologically changed following exposure to cholesterol oxidized LDL. Nile red, a hydrophobic probe which can selectively stain intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with oxidized or non-oxidized LDL cholesterol. LDL which did not undergo oxidation of its cholesterol had no effect on the cells. However, cellular nile red fluorescence intensity was increased as the pre-incubation time of cholesterol oxidase with LDL increased. This was supported by HPLC analysis which revealed that the oxidized cholesterol content of treated cells increased. These findings suggest that cholesterol oxidation of LDL can alter lipid deposition in the cells and change cell morphology. The oxidation of cholesterol in vivo may play an important role in the modification of LDL which could contribute to the generation of the lipid-laden foam cells.     

Tight junction development between cultured hepatoma cells: Possible stages in assembly and enhancement with dexamethasone

Freeze-fracture and thin-section methods were used to study tight junction formation between confluent H4-II-E hepatoma cells that were plated in monolayer culture in media with and without dexamethasone, a synthetic glucocorticoid. Three presumptive stages in the genesis of tight junctions were suggested by these studies: (1) “formation zones” (smooth P-fracture face ridges deficient in intramembranous particles), apparently matched across a partially reduced extracellular space, develop between adjacent cells; (2) linear strands and aggregates of 9–11 nm particles collect along the ridges of the formation zones. The extracellular space was always reduced when these structures were found matched with pits in gentle E-face depressions; (3) the linear arrays of particles on the ridges associate within the membranes to form the fibrils characteristic of mature tight junctions. The formation zones resemble tight junctions in terms of size, complexity and the patterns of membrane ridges. Although some of the beaded particle specialization may actually be gap junctions, it is unlikely that all can be interpreted in this way. No other membrane structures were detected that could represent developmental stages of tight junctions. Dexamethasone (at 2 × 10^?6 M) apparently stimulated formation of tight junctions. Treated cultures had a greater number of formation zones and mature tight junctions, although no differences in qualitative features of the junctions were noted.     

The use of confocal microscopy in the investigation of cell structure and function in the heart,vascular endothelium and smooth muscle cells

In recent years, fluorescence microscopy imaging has become an important tool for studying cell structure and function. This non invasive technique permits characterization, localisation and qualitative quantification of free ions, messengers, pH, voltage and a pleiad of other molecules constituting living cells. In this paper, we present results using various commercially available fluorescent probes as well as some developed in our laboratory and discuss the advantages and limitations of these probes in confocal microscopy studies of the cardiovascular system.     

Increased endothelial and vascular smooth muscle cell adhesion on nanostructured titanium and CoCrMo

In the body, vascular cells continuously interact with tissues that possess nanostructured surface features due to the presence of proteins (such as collagen and elastin) embedded in the vascular wall. Despite this fact, vascular stents intended to restore blood flow do not have nanoscale surface features but rather are smooth at the nanoscale. As the first step towards creating the next generation of vascular stent materials, the objective of this in vitro study was to investigate vascular cell (specifically, endothelial, and vascular smooth muscle cell) adhesion on nanostructured compared with conventional commercially pure (cp) Ti and CoCrMo. Nanostructured cp Ti and CoCrMo compacts were created by separately utilizing either constituent cp Ti or CoCrMo nanoparticles as opposed to conventional micron-sized particles. Results of this study showed for the first time increased endothelial and vascular smooth muscle cell adhesion on nanostructured compared with conventional cp Ti and CoCrMo after 4 hours' adhesion. Moreover, compared with their respective conventional counterparts, the ratio of endothelial to vascular smooth muscle cells increased on nanostructured cp Ti and CoCrMo. In addition, endothelial and vascular smooth muscle cells had a better spread morphology on the nanostructured metals compared with conventional metals. Overall, vascular cell adhesion was better on CoCrMo than on cp Ti. Results of surface characterization studies demonstrated similar chemistry but significantly greater root-mean-square (rms) surface roughness as measured by atomic force microscopy (AFM) for nanostructured compared with respective conventional metals. For these reasons, results from the present in vitro study provided evidence that vascular stents composed of nanometer compared with micron-sized metal particles (specifically, either cp Ti or CoCrMo) may invoke cellular responses promising for improved vascular stent applications.     

培养大鼠主动脉血管平滑肌细胞产生巨噬细胞集落刺激因子及其受体的表达

本研究用培养大鼠主动脉血管平滑肌细胞（ＶＳＭＣｓ）,结果如下：（１）用生物活性检测法发现ＶＳＭＣｓ无血清条件培养液可刺激巨噬细胞集落形成,其作用能被抗巨噬细胞集落刺激因子（ＭＣＳＦ）抗体抑制;（２）用免疫细胞化学技术证明ＶＳＭＣｓ存在ＭＣＳＦ受体;（３）用Ｎｏｒｔｈｅｒｎ ｂｌｏｔ技术证明ＶＳＭＣｓ有ＭＣＳＦ及其受本的ｍＲＮＡ表达,血清刺激使两者表达明显增强。本研究首次报道了培养大鼠主动脉ＶＳＭＣ     

Impact of the tissue factor pathway inhibitor gene on apoptosis in human vascular smooth muscle cells

Tissue factor pathway inhibitor (TFPI) plays a vitally important role in the blood coagulation pathway. Recent studies indicated that TFPI induces apoptosis in vascular smooth-muscle cells (VSMCs) in animals. The present study investigated whether the TFPI gene could also induce apoptosis in human vascular smooth-muscle cells (hVSMCs). Such cells were isolated from human umbilical arteries and subsequently transfected with pIRES-TFPI plasmid (2 μg/mL). MTT assaying and cell counting were applied to measure cell viability and proliferation, RT-PCR was utilized to analyze TFPI gene expression in the cells. Apoptosis was analyzed by fluorescence activated cell sorting (FACS). Several key proteins involved in apoptosis were examined through Western blotting. It was shown that TFPI gene transfer led to its increased cellular expression, with a subsequent reduction in hVSMC proliferation. Further investigation demonstrated that TFPI gene expression resulted in lesser amounts of procaspase-3, procaspase-8 and procascase-9, and an increased release of mitochondrial cytochrome c (cyt-c) into cytoplasm, thereby implying the involvement of both extrinsic and intrinsic pathways in TFPI gene-induced apoptosis in hVSMCs.     

Predominant role of type 1 IP3 receptor in aortic vascular muscle contraction

Inositol 1,4,5-trisphosphate receptor (IP₃R) plays a crucial role in generating Ca²⁺ signaling and three subtypes of IP₃R have been identified. In spite of a high degree of similarity among these subtypes, their effects on spatio-temporal Ca²⁺ patterns are specific and diverse; therefore the physiological significance of the differential expression levels of IP₃R subtypes in various tissues remains unknown. Here, we examined the relative contribution of the specific subtype of IP₃Rs to the agonist-induced Ca²⁺ signaling and contraction in IP₃R-deficient vascular smooth muscle cells and found that IP₃R1 deficient cells exclusively showed less sensitivity to the agonist, compared to those from the other genotypes. We also found that IP₃R1 dominantly expressed in vascular aortae on a consistent basis, and that phenylephrine (PE)-induced aortic muscle contraction was reduced specifically in IP₃R1-deficient aortae. Taken together, we concluded that IP₃R1 plays a predominant role in the function of the vascular smooth muscle in vivo.     

Neuron-like differentiation of adipose tissue-derived stromal cells and vascular smooth muscle cells

Adipose tissue-derived stromal cells (ADSC) have previously been shown to possess stem cell properties such as transdifferentiation and self-renewal. Because future clinical applications are likely to use these adult stem cells in an autologous fashion, we wished to establish and characterize rat ADSC for pre-clinical tests. In the present study, we showed that rat ADSC expressed stem cell markers CD34 and STRO-1 at passage 1 but only STRO-1 at passage 3. These cells could also be induced to differentiate into adipocytes, smooth muscle cells, and neuron-like cells, the latter of which expressed neuronal markers S100, nestin, and NF70. Isobutylmethylxanthine (IBMX), indomethacin (INDO), and insulin were the active ingredients in a previously established neural induction medium (NIM); however, here we showed that IBMX alone was as effective as NIM in the induction of morphological changes as well as neuronal marker expression. Finally, we showed that vascular smooth muscle cells could also be induced by either NIM or IBMX to differentiate into neuron-like cells that expressed NF70.