Modulation of microvascular growth and morphogenesis by reconstituted basement membrane gel in three-dimensional cultures of rat aorta: A comparative study of angiogenesis in Matrigel,collagen, fibrin,and plasma clot

Summary Rings of rat aorta cultured in Matrigel, a reconstituted gel composed of basement membrane molecules, gave rise to three-dimensional networks composed of solid cellular cords and occasional microvessels with slitlike lumina. Immunohistochemical and ultrastructural studies showed that the solid cords were composed of endothelial sprouts surrounded by nonendothelial mesenchymal cells. The angiogenic response of the aortic rings in Matrigel was compared to that obtained in interstitial collagen, fibrin, or plasma clot. Morphometric analysis demonstrated that the mean luminal area of the microvascular sprouts and channels was significantly smaller in Matrigel than in collagen, fibrin, or plasma clot. The percentage of patent microvessels in Matrigel was also markedly reduced. Autoradiographic studies of³H-thymidine-labeled cultures showed reduced DNA synthesis by developing microvessels in Matrigel. The overall number of solid endothelial cords and microvessels was lower in Matrigel than in fibrin or plasma clot. A mixed cell population isolated from Matrigel cultures formed a monolayer in collagen or fibrin-coated dishes but rapidly reorganized into a polygonal network when plated on Matrigel. The observation that gels composed of basement membrane molecules modulate the canalization, proliferation, and organization into networks of vasoformative endothelial cells in three-dimensional cultures supports the hypothesis that the basement membrane is a potent regulator of microvascular growth and morphogenesis. This work was supported by grants from the W. W. Smith Charitable Trust and grants CA14137 and HL43392 from the National Institutes of Health, Bethesda, MD.     

Isolation and characterization of microvascular endothelial cells from chicken fat pads

Summary Microvascular endothelial cells from abdominal fat pads of 6-wk-old broiler chickens were isolated to provide anin vitro system to study their physiological functions. The isolation procedure produced clumps of 10–30 cells, which attached to culture vessels in 4 h and attained confluency in 2 wk. At confluency, cells had a cobblestone appearance but were not contact inhibited and detached from the bottom of the culture vessel 2 wk after reaching confluency. The cells internalized acetylated low density lipoproteins, a characteristic of endothelial cells. This property was used to obtain pure endothelial cell cultures using the cell sorter. When cultured over Matrigel, a reconstituted matrix, the cells aligned themselves into chordlike structures and formed branching microvessels. Cells plated on type I collagen-coated culture flasks occasionally formed chordlike structures and proliferated at a faster rate than cells plated on Matrigel. Cells cultured on laminin-coated plates were slender and had long cytoplasmic extensions however, cells cultured on uncoated plastic had fibroblastic morphology. These properties are similar to those described for microvessel endothelial cells isolated from tissues of other species.     

IL-5-dependent conversion of normodense human eosinophils to the hypodense phenotype uses 3T3 fibroblasts for enhanced viability, accelerated hypodensity, and sustained antibody-dependent cytotoxicity

Human peripheral blood-derived eosinophils were assessed for their viability, density, and functional properties after 7 days of culture with purified mouse IL-5 and mouse 3T3 fibroblasts. Whereas none of the eosinophils remained viable after 7 days of culture in the absence of IL-5, 38 +/- 12% and 61 +/- 14% (n = 6, mean +/- SD) of the eosinophils survived in the presence of 1 pM IL-5 alone or 1 pM IL-5 in the presence of 3T3 fibroblasts, respectively (p less than 0.05). Direct contact between the fibroblasts and the eosinophils was not needed for this enhanced IL-5-dependent viability. After 7 days, 66 +/- 7% (n = 6) of the cocultured eosinophils were viable when the two cell types were separated by a 0.4-microns filter. As assessed by density-gradient centrifugation after 7 days of IL-5 exposure, all of the original normodense eosinophils became hypodense. The time course of this conversion was accelerated by the presence of 3T3 fibroblasts. Enhanced helminthic cytotoxicity was maintained by the 7-day cultured eosinophils only if they had been cocultured with fibroblasts. Eosinophils killed 10 +/- 11% (n = 5), 48 +/- 17%, and 31 +/- 15% of the larvae when they were cultured for 7 days in IL-5 alone, in IL-5 in direct contact with 3T3 fibroblasts, or in IL-5 with filter separation of the fibroblasts and the eosinophils, respectively. The ability of IL-5 to induce progenitor cells to differentiate selectively into eosinophils, and of 3T3 fibroblasts to facilitate the IL-5-mediated conversion of normodense eosinophils to hypodense eosinophils with increased viability and antibody-dependent cytotoxicity suggests a role for both hematopoietic and tissue factors in determining the presence and pathobiologic function of activated hypodense eosinophils in patients with hypereosinophilic conditions.     

Cultured endothelial cells derived from the human iliac arteries

Cells derived from the endothelium of human iliac arteries were cultured in vivo. The cells were isolated, grown, and subcultured in HEPES buffered Medium 199 supplemented with 20% heat inactivated human whole blood serum, human alpha-thrombin, and commercial endothelial cell growth supplement derived from bovine brain. The cells were viable in culture for 8 to 10 passages at a split ratio of 1:3. After the 10th passage, the cells began to enlarge and their growth rate was reduced. No cultures were viable after the 12th passage. The cells were determined to be of endothelial origin by their morphology at confluence; their ultrastructural characteristics, including the presence of Weibel-Palade bodies; the production and release of factor VIII-related antigen; and by their maintenance of a surface that prevented platelet attachment. The cultured arterial endothelial cells released prostacyclin in response to challenge with thrombin and protamine sulfate but not in response to bradykinin or the platelet-derived growth factor. Although the cultures described in this report were derived from patients with varying degrees of atherosclerotic disease, there were no significant differences in morphological or physiological parameters among these cultures or in comparison with commonly studied cells derived from human umbilical veins.     

Effects of selected vasoactive substances on adenylate cyclase activity in brain,isolated brain microvessels,and primary cultures of brain microvessel endothelial cells

The specific activity of adenylate cyclase was assayed in homogenates of gray matter, freshly isolated and primary cultured microvessel endothelial cells from bovine cerebral cortex. Specific activities for the tissues were 14.6±2.1, 15.6±2.7, and 8.4±1.5 pmol cAMP/mg protein/min±SD for gray matter, cultured microvessels, and freshly isolated microvessels, respectively. Adenylate cyclase associated with gray matter and cultured microvessels was sensitive to histamine and selected catecholamines. Perhaps due to metabolic deficiencies, adenylate cyclase of freshly isolated microvessels exhibited little or no response to either the catecholamines or histamine. Angiotensin II stimulated adenylate cyclase of both freshly isolated and cultured microvessels but had no effect on gray matter. Bradykinin did not stimulate cAMP generation in any of the tissues. Overall results support the role of cAMP in regulating brain microvessel functions and suggest that primary cultures of brain microvessels may be useful in examining cAMP-mediated biochemical pathways at the blood-brain barrier.     

Primary culture of microvascular endothelial cells from bovine retina

Summary To provide an in vitro system for studying retinal capillary function we have developed methods for isolation and culture of microvascular endothelial cells from retina. Retinal microvessels were isolated by homogenization of the retina and collection of the microvessels onto nylon mesh. Treatment of the isolated microvessels with collagenase and dispase followed by Percoll gradient centrifugation yielded endothelial cells that were largely free of pericytes. A homogeneous population of endothelial cells that were capable of at least six population doublings was obtained by plating onto a fibronectin coated substrate in plasma derived serum. The endothelial origin of these cells was confirmed by the presence of Factor VIII antigen, angiotensin converting enzyme activity, numerous tight junctions, and a cell surface that did not bind platelets. A second cell type, which did not exhibit these cell markers and which is presumably the intramural pericyte, was obtained when the isolated microvessels were plated on tissue culture grade plastic in fetal bovine serum. Supported by Research Grants 5R01-EY03772 and 5R01-ES02380 from the U.S. Public Health Service (G. W. G.) and Established Investigator Award 31-107 from the American Heart Association (A. L. B.).     

Cultured endothelial cells derived from the human iliac arteries

Summary Cells derived from the endothelium of human iliac arteries were cultured in vivo. The cells were isolated, grown, and subcultured in HEPES buffered Medium 199 supplemented with 20% heat inactivated human whole blood serum, human alpha-thrombin, and commercial endothelial cell growth supplement derived from bovine brain. The cells were viable in culture for 8 to 10 passages at a split ratio of 1:3. After the 10th passage, the cells began to enlarge and their growth rate was reduced. No cultures were viable after the 12th passage. The cells were determined to be of endothelial origin by their morphology at confluence; their ultrastructural characteristics, including the presence of Weibel-Palade bodies; the production and release of factor VIII-related antigen; and by their maintenance of a surface that prevented platelet attachment. The cultured arterial endothelial cells released prostacyclin in response to challenge with thrombin and protamine sulfate but not in response to bradykinin or the platelet-derived growth factor. Although the cultures described in this report were derived from patients with varying degrees of atherosclerotic disease, there were no significant differences in morphological or physiological parameters among these cultures or in comparison with commonly studied cells derived from human umbilical veins. The above work was supported by Grant CA28540 from the National Institutes of Health and by a grant from The Council for Tobacco Research, USA.     

Medium perfusion enables engineering of compact and contractile cardiac tissue

We hypothesized that functional constructs with physiological cell densities can be engineered in vitro by mimicking convective-diffusive oxygen transport normally present in vivo. To test this hypothesis, we designed an in vitro culture system that maintains efficient oxygen supply to the cells at all times during cell seeding and construct cultivation and characterized in detail construct metabolism, structure, and function. Neonatal rat cardiomyocytes suspended in Matrigel were cultured on collagen sponges at a high initial density (1.35 x 10(8) cells/cm(3)) for 7 days with interstitial flow of medium; constructs cultured in orbitally mixed dishes, neonatal rat ventricles, and freshly isolated cardiomyocytes served as controls. Constructs were assessed at timed intervals with respect to cell number, distribution, viability, metabolic activity, cell cycle, presence of contractile proteins (sarcomeric alpha-actin, troponin I, and tropomyosin), and contractile function in response to electrical stimulation [excitation threshold (ET), maximum capture rate (MCR), response to a gap junctional blocker]. Interstitial flow of culture medium through the central 5-mm-diameter x 1.5-mm-thick region resulted in a physiological density of viable and differentiated, aerobically metabolizing cells, whereas dish culture resulted in constructs with only a 100- to 200-microm-thick surface layer containing viable and differentiated but anaerobically metabolizing cells around an acellular interior. Perfusion resulted in significantly higher numbers of live cells, higher cell viability, and significantly more cells in the S phase compared with dish-grown constructs. In response to electrical stimulation, perfused constructs contracted synchronously, had lower ETs, and recovered their baseline function levels of ET and MCR after treatment with a gap junctional blocker; dish-grown constructs exhibited arrhythmic contractile patterns and failed to recover their baseline MCR levels.     

In vitro differentiation of myoblasts from skeletal muscle of rainbow trout

Substrata, plating densities and tissue culture media were compared for their effects on the proliferation and differentiation of myoblasts from skeletal muscle of rainbow trout. Mononuclear cells were isolated from the lateralis muscle of 4–11-month-old trout and plated on to glass coverslips coated with fibronectin, laminin or Matrigel. Cell proliferation was estimated by determining the density of nuclei on successive days in culture, and myoblast differentiation was detected by immunostaining cultures with the myosin-specific monoclonal antibody MF20. Mononuclear cell proliferation was highest for cells cultured on fibronectin or laminin and lowest for cells cultured on Matrigel, but the total number of nuclei in myosin-positive cells did not differ between substrata. The percentage of nuclei in myosin-positive myocytes and myotubes was significantly higher for cells cultured on Matrigel. The proportion of cells adhering to Matrigel and undergoing differentiation increased with plating density. Of three media tested, Dulbecco's Modified Eagle Medium (DMEM), RPMI 1640 (RPMI), Leibovitz's L-15 (L-15) supplemented with 1 or 10% fetal bovine serum (FBS), a significantly greater proportion of the myoblasts differentiated when cells were cultured in L-15+ 10% FBS. These results suggest that culturing trout muscle-derived cells on a substratum of Matrigel at a high density and maintaining cells in L-15+ 10% FBS provide the conditions that maximize the proportion of cells that actively synthesize muscle myosin and facilitate trout myoblast differentiation in vitro .     

Reduced viability of human vascular endothelial cells cultured on Matrigel™

Optimal vascular homeostasis requires efficient control of both proliferation and elimination of vascular endothelial cells. Programmed cell death, or apoptosis, is the main mechanism controlling cell elimination, and it is an essential component of vascular formation. Human vascular endothelial cells die in vitro, if prevented from obligatory survival factors like growth factors or attachment and cell spreading, but very little is known about the mechanisms controlling endothelial cell elimination. Signaling from the extracellular matrix affects the behavior and functions of human umbilical vein endothelial cells (HUVECs), and we have recently demonstrated the beneficial effects of plating on the reconstituted extracellular matrix Matrigel™, on the inducible nitric oxide production of freshly isolated HUVECs. In this work we observed that cultured HUVECs formed typical capillary-like structures on Matrigel, but unexpectedly, after 24–48 hours their viability was gradually lost. Viability was measured with an assay based on mitochondrial reduction of reagent XTT. No decrease in viability was seen in freshly isolated HUVECs or in cultured fibroblasts during this time. It is known that cells often turn into apoptosis if they receive conflicting information from their surroundings, and apparently signaling from Matrigel to HUVECs, while at their in vitro proliferating phenotype, resulted in launching of the apoptotic machinery. Thus, proliferating and differentiated phenotypes of endothelial cells seemed to have different sensitivity to signals that induce apoptosis. J. Cell. Physiol. 176:92–98, 1998. © 1998 Wiley-Liss, Inc.     

Transient receptor potential canonical channels are required for in vitro endothelial tube formation

In endothelial cells Ca(2+) entry is an essential component of the Ca(2+) signal that takes place during processes such as cell proliferation or angiogenesis. Ca(2+) influx occurs via the store-operated Ca(2+) entry pathway, involving stromal interaction molecule-1 (STIM1) and Orai1, but also through channels gated by second messengers like the transient receptor potential canonical (TRPC) channels. The human umbilical vein-derived endothelial cell line EA.hy926 expressed STIM1 and Orai1 as well as several TRPC channels. By invalidating each of these molecules, we showed that TRPC3, TRPC4, and TRPC5 are essential for the formation of tubular structures observed after EA.hy926 cells were plated on Matrigel. On the contrary, the silencing of STIM1 or Orai1 did not prevent tubulogenesis. Soon after being plated on Matrigel, the cells displayed spontaneous Ca(2+) oscillations that were strongly reduced by treatment with siRNA against TRPC3, TRPC4, or TRPC5, but not siRNA against STIM1 or Orai1. Furthermore, we showed that cell proliferation was reduced upon siRNA treatment against TRPC3, TRPC5, and Orai1 channels, whereas the knockdown of STIM1 had no effect. On primary human umbilical vein endothelial cells, TRPC1, TRPC4, and STIM1 are involved in tube formation, whereas Orai1 has no effect. These data showed that TRPC channels are essential for in vitro tubulogenesis, both on endothelial cell line and on primary endothelial cells.     

Assessment of sperm characteristics post-thaw and response to calcium ionophore in relation to fertility in Swedish dairy AI bulls

The present study examined the relationship between bull sperm characteristics post-thawing, after swim-up, and after challenge to calcium ionophore in relation to fertility (56-d nonreturn rates) after artificial insemination (AI). Spermatozoa from 25 semen batches derived from 15 Swedish Red and White AI bulls were evaluated with regard to post-thaw motility, membrane integrity, and migration through a swim-up procedure. The swim-up separated spermatozoa were assessed in terms of sperm concentration, viability and capacitation status as well as their response to exogenous calcium ionophore (A23187). Acrosome reactions were evaluated by fluorescence microscopy and flow cytometry. Sperm motility and viability post-thawing were significantly correlated with fertility. For the swim-up separated semen, significant correlations to nonreturn rates were found for concentration, viability, number of viable spermatozoa and sperm capacitation status (Pattern F and Pattern B). The only parameter significantly correlated to fertility after the ionophore challenge was the percentage of acrosome-reacted spermatozoa with remaining equatorial fluorescence, as assessed by fluorescence microscopy, but not by flow cytometry. The regression analysis showed that combining the results of sperm membrane integrity assessment post-thawing with those of capacitation status after swim-up provided the best prediction of fertility. The accuracy of prediction did not improve when these parameters were combined with the percentage of spermatozoa in which acrosome reaction was induced by ionophore challenge.     

猫骨髓间充质干细胞的体外分离培养、纯化、鉴定

目的:分离、培养、纯化家猫的骨髓间充质干细胞,并对获得细胞的表面标志物进行鉴定,为进一步利用骨髓间充质干细胞的细胞移植实验奠定基础。方法:采用全骨髓贴壁法体外分离、培养、纯化家猫骨髓间充质干细胞,通过多次更换培养液获得较纯化的骨髓间充质干细胞,倒置相差显微镜下对细胞形态进行观察;根据第1、3、5、7、9代细胞的镜下增殖情况绘制出生长曲线;通过流式细胞仪检测细胞表面标志抗原CD34、CD44和CD90的表达率。结果:在倒置相差显微镜下观察,分离培养的骨髓间充质干细胞贴壁呈梭形或纺锤形;原代细胞生长丛集成片,5~7 d达到融合,进行传代;培养到第三代以后,细胞出现相对均匀的梭形扁平外观,迅速增殖的细胞呈涡流样排列;第3、5代骨髓间充质干细胞增殖能力强于第7、9代;采用流式细胞仪分析结果显示细胞的CD34、CD44和CD90阳性率分别为17.5%、97.9%和91%,这与骨髓间充质干细胞表面抗原的表达一致。结论:分离培养的细胞具有骨髓间充质干细胞特性,成分相对单一,第3、5代细胞纯度高,增殖能力强,适用于进一步的实验研究。     

A subpopulation of peritoneal macrophages form capillarylike lumens and branching patterns in vitro

OBJECTIVE: We have previously shown that monocytes/macrophages (MC/Mph) influence neovascularization by extracellular matrix degradation, and by direct incorporation into growing microvessels. To date, neither the phenotype of these cells, nor the stages of their capillary-like conversion were sufficiently characterized. METHODS: We isolated mouse peritoneal Mph from transgenic mice expressing fluorescent proteins either ubiquitously, or specifically in the myelocytic lineage. These Mph were embedded in Matrigel which contained fluorescent protease substrates, exposed to an MCP-1 chemotactic gradient, and then examined by confocal microscopy after various intervals. RESULTS: Within 3 hrs after gel embedding, we detected TIMP-1 and MMP-12 dependent proteolysis of the matrix surrounding Mph, mostly in the direction of high concentrations of MCP-1. After 2 days, Mph developed intracellular vacuoles containing degradation product. At 5 days these vacuoles were enlarged and/or fused to generate trans-cellular lumens in approximately 10% of cells or more (depending on animal's genetic background). At this stage, Mph became tubular, and occasionally organized in three-dimensional structures resembling branched microvessels. CONCLUSION: Isolated mouse peritoneal Mph penetrate Matrigel and form tunnels via a metalloprotease-driven proteolysis and phagocytosis. Following a morphological adjustment driven by occurrence, enlargement and/or fusion process of intracellular vacuoles, similar to that described in bona fide endothelium, a subpopulation of these cells end up by lining a capillary-like lumen in vitro. Thus we show that adult Mph, not only the more primitive 'endothelial progenitors', have functional properties until now considered defining of the endothelial phenotype.     

Role of protein kinase C in the control of vascular prostacyclin: study of phorbol esters effect in bovine aortic endothelium and smooth muscle

In bovine aortic endothelial cells, phorbol 12-myristate, 13-acetate induced a smaller stimulation of prostacyclin release than ionophore A23187: the combination of both agents was highly synergistic. The responses of the bovine aortic smooth muscle were very different in the 2 preparations studied. In media explants cultured for short periods, neither phorbol 12-myristate, 13-acetate, nor A23187, alone or in combination, were able to increase prostacyclin release, whereas serotonin was an effective stimulus. In cultured smooth muscle cells, outgrown from the explants, phorbol 12-myristate, 13-acetate increased prostacyclin release to the same levels as A23187 or serotonin. It is concluded that increased cytosolic Ca++ level and protein kinase C activity induce a synergistic stimulation of endothelial prostacyclin. On the other hand, the phenotypic modulation of the arterial smooth muscle, from a contractile to a synthetic state, seems to be associated with a profound change in the control of prostacyclin.     

Inducible nitric oxide and prostacyclin productions are differently controlled by extracellular matrix and cell density in human vascular endothelial cells

Both cell-matrix and cell-cell interactions are important regulators of the function of most human cells. In this study we investigated how these interactions controlled the production of vasodilators nitric oxide (NO), and prostacyclin (PGI₂), in freshly isolated human umbilical vein endothelial cells (HUVECs). On the reconstituted extracellular matrix (ECM) Matrigel freshly isolated HUVECs treated with interleukin-1β, lipopolysaccharide, and interferon-γ, produced more NO, but less PGI₂, than on gelatin substratum. High cell density was essential for inducibility of NO production in cells plated on gelatin substratum, but not on ECM. In cells plated on gelatin substratum at low cell density, which mimicked conventional HUVEC culturing conditions, both inducible NO production and the inducible NO synthase (iNOS) mRNA levels, detected by competitive RT-PCR, were low. However, inducible PGI₂ production remained high in these cells. Highest inducible NO productions were observed in HUVECs that presumably had best maintained their original differentiated phenotype. Thus our data imply that the inducible NO and PGI₂ productions of freshly isolated HUVECs were differently controlled by the extracellular matrix and cell density. Our data suggest that both cell-matrix and cell-cell interactions may have a strong influence on the proinflammatory cytokine responses of human vascular endothelial cells. J. Cell. Biochem. 64:538–546. © 1997 Wiley-Liss, Inc.     

Thrombin-induced release of von Willebrand factor from endothelial cells is mediated by phospholipid methylation. Prostacyclin synthesis is independent of phospholipid methylation

The biochemical events that lead to thrombin-stimulated release of von Willebrand factor and prostacyclin synthesis in cultured endothelial cells are examined. Treatment of human umbilical vein endothelial cells with thrombin results in an instantaneous increase in phospholipid methylation which can be blocked by 3-deazaadenosine, a methyltransferase inhibitor. 3-Deazaadenosine also blocks the thrombin-induced Ca2+ influx into endothelial cells and the release of von Willebrand factor, indicating that these processes are coupled. The phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) and the Ca2+ ionophore A23187 both bypass the phospholipid methylation and directly stimulate Ca2+ influx and von Willebrand factor release. In contrast to the stimulus-induced von Willebrand factor release, the thrombin-induced prostacyclin synthesis cannot be blocked by 3-deazaadenosine. Similarly, incubation of endothelial cells with EDTA has no influence on the thrombin-induced prostacyclin synthesis, and PMA has no stimulatory effect on prostacyclin synthesis. These observations indicate that thrombin induces different metabolic responses in endothelial cells: phospholipid methylation followed by a Ca2+ influx, which subsequently leads to release of von Willebrand factor, and liberation of arachidonic acid from phospholipids for prostacyclin formation, which is independent of phospholipid methylation and Ca2+ influx.     

Multidrug Resistance-Related Transport Proteins in Isolated Human Brain Microvessels and in Cells Cultured from These Isolates

Abstract: The multidrug transporter, P-glycoprotein (Pgp), at the blood-brain barrier is thought to be important for limiting access of toxic agents to the brain, but controversy surrounds its cellular location, whether on endothelium or on adjacent astrocyte foot processes. In the present study, the distribution of protein and mRNA for Pgp and for another transporter, multidrug resistance-associated protein (MRP), is compared with that for the endothelial marker, platelet-endothelial cell adhesion molecule-1 (PECAM-1) and for the astrocyte-derived glial fibrillary acidic protein (GFAP) in microvessels isolated from human brain and in cells grown from these microvessels. Activities of the multidrug transporters are assessed in the cultured cells from the effects of transport inhibitors on intracellular [³H]vincristine accumulation. The isolated microvessels show strong immunocytochemical staining for Pgp and PECAM-1 and little or no staining for GFAP and MRP, and they contain mRNAs detectable by RT-PCR encoding only Pgp and PECAM-1, but not GFAP or MRP. Thus, Pgp may well be synthesised and expressed on cells within the microvessels rather than on adherent astrocyte foot processes. In cells grown from the microvessels, although PECAM-1 remains, Pgp expression decreases and MRP appears. Evidence suggests these multidrug transporters are functionally active in the cultured cells.     

Role of protein kinase C in the control of vascular prostacyclin: Study of phorbol esters effect in bovine aortic endothelium and smooth muscle

In bovine aortic endothelial cells, phorbol 12-myristate, 13-acetate induced a smaller stimulation of prostacyclin release than ionophore A23187: the combination of both agents was highly synergistic. The responses of the bovine aortic smooth muscle were very different in the 2 preparations studied. In media explants cultured for short periods, neither phorbol 12-myristate, 13-acetate, nor A23187, alone or in combination, were able to increase prostacyclin release, whereas serotonin was an effective stimulus. In cultured smooth muscle cells, outgrown from the explants, phorbol 12-myristate, 13-acetate increased prostacyclin release to the same levels as A23187 or serotonin. It is concluded that increased cytosolic Ca⁺⁺ level and protein kinase C activity induce a synergistic stimulation of endothelial prostacyclin. On the other hand, the phenotypic modulation of the arterial smooth muscle, from a contractile to a synthetic state, seems to be associated with a profound change in the control of prostacyclin.     

Variant endothelial cells from human carotid artery in culture

Summary Endothelial cells were cultured from the carotid artery with thickened intima comprised of two to five layers of smooth muscle cells, isolated from a 19-yr-old female, who died from a accident. The cells were grown and subcultured in Medium 199 supplemented with 20% heat inactivated fetal bovine serum. The cells are still viable at present, the 22nd passage. The cultured cells were found to have the following characteristics: existence of Factor VIII-related surface angiten and prostacyclin synthesis slightly less than that for typical endothelial cells. The most outstnading feature was the formation by an individual cell of a single ring, and composite ring formed by two to five cells. Neither the synthesis of an angiotensin converting enzyme nor that of a Weibel-Palade body could be detected by electron microscopy. The cultured cells possessed only a few characteristics specific for typical endothelial cells and were designated as variant endothelial cells.