Synthesis and structure-activity relationship of diarylamide urea derivatives as selective inhibitors of the proliferation of human coronary artery smooth muscle cells

A series of diarylamide urea derivatives were synthesized and evaluated for their inhibitory activities against human coronary artery smooth muscle cells (SMCs) and human coronary artery endothelial cells (ECs). Compound 2o was superior to the lead compound, Tranilast, in terms of its potency of the inhibitory activity and cell selectivity.     

Synthesis and structure-activity relationship of diarylamide derivatives as selective inhibitors of the proliferation of human coronary artery smooth muscle cells

A series of diarylamide derivatives were synthesized and evaluated for their inhibitory activities against human coronary artery smooth muscle cells (SMCs) and human coronary artery endothelial cells (ECs). Compound 2w was superior to the lead compound, Tranilast, in terms of the potency of the activity and cell selectivity.     

Simultaneous isolation of endothelial and smooth muscle cells from human umbilical artery or vein and their growth response to low-density lipoproteins

In the pathogenesis of atherosclerosis the interplay of endothelial cells (ECs) and smooth muscle cells (SMCs) is disturbed. Oxidatively modified low-density lipoproteins (oxLDLs), important stimulators of atherosclerotic plaque formation in vessels, modify the growth response of both cell types. To compare growth responses of ECs and SMCs of the same vessel with oxLDLs, we developed a method to isolate both cell types from the vessel walls of umbilical cords by enzymatic digestion. The method further allowed the simultaneous isolation of venous and arterial cells from a single umbilical cord. In culture, venous ECs showed an elongated appearance compared with arterial ECs, whereas SMCs of artery and vein did not look different. Smooth muscle cells of both vessel types responded to oxLDLs (60 microg/ml) with an increase in their [(3)H]-thymidine incorporation into DNA. On the contrary, ECs of artery or vein decreased [(3)H]-thymidine incorporation and cell number in the presence of oxLDLs (60 microg/ml) of increasing oxidation grade. Thus, human umbilical SMCs and ECs of the same vessel show a disparate growth response toward oxLDLs. But the physiologically more relevant minimal oxLDLs did not decrease proliferation in venous ECs but only in arterial ECs. This difference in tolerance toward minimal oxLDLs should be taken into account while using venous or arterial ECs of umbilical cord for research in atherosclerosis. Further differences of venous and arterial ECs in tolerance toward minimal oxLDLs could be of clinical relevance for coronary artery bypass grafts.     

Avian coronary endothelium is a mosaic of sinus venosus‐ and ventricle‐derived endothelial cells in a region‐specific manner

The origin of coronary endothelial cells (ECs) has been investigated in avian species, and the results showed that the coronary ECs originate from the proepicardial organ (PEO) and developing epicardium. Genetic approaches in mouse models showed that the major source of coronary ECs is the sinus venosus endothelium or ventricular endocardium. To clarify and reconcile the differences between avian and mouse species, we examined the source of coronary ECs in avian embryonic hearts. Using an enhanced green fluorescent protein‐Tol2 system and fluorescent dye labeling, four types of quail‐chick chimeras were made and quail‐specific endothelial marker (QH1) immunohistochemistry was performed. The developing PEO consisted of at least two cellular populations in origin, one was sinus venosus endothelium‐derived inner cells and the other was surface mesothelium‐derived cells. The majority of ECs in the coronary stems, ventricular free wall, and dorsal ventricular septum originated from the sinus venosus endothelium. The ventricular endocardium contributed mainly to the septal artery and a few cells to the coronary stems. Surface mesothelial cells of the PEO differentiated mainly into a smooth muscle phenotype, but a few differentiated into ECs. In avian species, the coronary endothelium had a heterogeneous origin in a region‐specific manner, and the sources of ECs were basically the same as those observed in mice.     

Induction of indoleamine 2,3-dioxygenase in vascular smooth muscle cells by interferon-gamma contributes to medial immunoprivilege

Atherosclerosis and graft arteriosclerosis are characterized by leukocytic infiltration of the vessel wall that spares the media. The mechanism(s) for medial immunoprivilege is unknown. In a chimeric humanized mouse model of allograft rejection, medial immunoprivilege was associated with expression of IDO by vascular smooth muscle cells (VSMCs) of rejecting human coronary artery grafts. Inhibition of IDO by 1-methyl-tryptophan (1-MT) increased medial infiltration by allogeneic T cells and increased VSMC loss. IFN-gamma-induced IDO expression and activity in cultured human VSMCs was considerably greater than in endothelial cells (ECs) or T cells. IFN-gamma-treated VSMCs, but not untreated VSMCs nor ECs with or without IFN-gamma pretreatment, inhibited memory Th cell alloresponses across a semipermeable membrane in vitro. This effect was reversed by 1-MT treatment or tryptophan supplementation and replicated by the absence of tryptophan, but not by addition of tryptophan metabolites. However, IFN-gamma-treated VSMCs did not activate allogeneic memory Th cells, even after addition of 1-MT or tryptophan. Our work extends the concept of medial immunoprivilege to include immune regulation, establishes the compartmentalization of immune responses within the vessel wall due to distinct microenvironments, and demonstrates a duality of stimulatory EC signals versus inhibitory VSMC signals to artery-infiltrating T cells that may contribute to the chronicity of arteriosclerotic diseases.     

Activation of endothelial TRPV4 channels mediates flow-induced dilation in human coronary arterioles: role of Ca2+ entry and mitochondrial ROS signaling

In human coronary arterioles (HCAs) from patients with coronary artery disease, flow-induced dilation is mediated by a unique mechanism involving the release of H(2)O(2) from the mitochondria of endothelial cells (ECs). How flow activates ECs to elicit the mitochondrial release of H(2)O(2) remains unclear. Here, we examined the role of the transient receptor potential vanilloid type 4 (TRPV4) channel, a mechanosensitive Ca(2+)-permeable cation channel, in mediating ROS formation and flow-induced dilation in HCAs. Using RT-PCR, Western blot analysis, and immunohistochemical analysis, we detected the mRNA and protein expression of TRPV4 channels in ECs of HCAs and cultured human coronary artery ECs (HCAECs). In HCAECs, 4α-phorbol-12,13-didecanoate (4α-PDD), a selective TRPV4 agonist, markedly increased (via Ca(2+) influx) intracellular Ca(2+) concentration. In isolated HCAs, activation of TRPV4 channels by 4α-PDD resulted in a potent concentration-dependent dilation, and the dilation was inhibited by removal of the endothelium and by catalase, a H(2)O(2)-metabolizing enzyme. Fluorescence ROS assays showed that 4α-PDD increased the production of mitochondrial superoxide in HCAECs. 4α-PDD also enhanced the production of H(2)O(2) and superoxide in HCAs. Finally, we found that flow-induced dilation of HCAs was markedly inhibited by different TRPV4 antagonists and TRPV4-specific small interfering RNA. In conclusion, the endothelial TRPV4 channel is critically involved in flow-mediated dilation of HCAs. TRPV4-mediated Ca(2+) entry may be an important signaling event leading to the flow-induced release of mitochondrial ROS in HCAs. Elucidation of this novel TRPV4-ROS pathway may improve our understanding of the pathogenesis of coronary artery disease and/or other cardiovascular disorders.     

Variations in transfection efficiency of VEGF165 and VEGF121-cDNA

Little is known about the expression pattern of vascular endothelial growth factor (VEGF) among smooth muscle cells of different arterial regions. Therefore, we have conducted studies aimed at increasing expression of VEGF in cultured human smooth muscle cells (SMCs) from different sites: aorta, umbilical artery, and coronary artery. Two plasmids harboring human VEGF₁₂₁ and VEGF₁₆₅ isoforms, respectively, were constructed and lipotransfected into vascular SMCs, using the Fu-GENE 6. Extensive optimization of transfection conditions were performed prior to this. Different basal levels of VEGF were observed between cell types: from 0.51–0.95 pg/mL/μg protein in umbilical artery, through 2.32–2.39 pg/mL/μg protein in coronary artery, to 5.45–7.52 pg/mL/μg protein in aortic SMCs. Significant differences in responses to transfection were also observed: The increase in VEGF production was most pronounced in umbilical artery SMCs (e.g., with 4 μg VEGF₁₂₁-cDNA/in the wells)—an approximate 600-fold as opposed to an 18-fold increase in aortic SMCs and a 29-fold increase in coronary artery SMCs. In addition, we observed significant increases in proliferation rate of aortic and coronary endothelial cells (ECs), after incubation with conditioned medium from VEGF-transfected SMCs. Observed changes differed in relation to cell origin and isoform.     

Subepicardial endothelial cells invade the embryonic ventricle wall to form coronary arteries

Coronary arteries bring blood flow to the heart muscle. Understanding the developmental program of the coronary arteries provides insights into the treatment of coronary artery diseases. Multiple sources have been described as contributing to coronary arteries including the proepicardium, sinus venosus (SV), and endocardium. However, the developmental origins of coronary vessels are still under intense study. We have produced a new genetic tool for studying coronary development, an AplnCreER mouse line, which expresses an inducible Cre recombinase specifically in developing coronary vessels. Quantitative analysis of coronary development and timed induction of AplnCreER fate tracing showed that the progenies of subepicardial endothelial cells (ECs) both invade the compact myocardium to form coronary arteries and remain on the surface to produce veins. We found that these subepicardial ECs are the major sources of intramyocardial coronary vessels in the developing heart. In vitro explant assays indicate that the majority of these subepicardial ECs arise from endocardium of the SV and atrium, but not from ventricular endocardium. Clonal analysis of Apln-positive cells indicates that a single subepicardial EC contributes equally to both coronary arteries and veins. Collectively, these data suggested that subepicardial ECs are the major source of intramyocardial coronary arteries in the ventricle wall, and that coronary arteries and veins have a common origin in the developing heart.     

27-Hydroxycholesterol causes remodeling in endothelial cell membrane lipid composition comparable to remodeling in the failed vein grafts of CABG patients

Our objective is to determine if vascular remodeling in CABG patients is related to oxysterols, therefore, we compared failed vein grafts from 18 patients, available after a second coronary artery bypass grafting (CABG), with human endothelial cells (ECs). The ECs were cultured in minimum essential medium (MEM) with or without 27-hydroxycholesterol (27OHC), one of the oxysterol products of oxidatively modified low density lipoproteins (ox-LDL), as an agent to alter molecular mechanisms in vascular cells. Significant changes in phospholipid composition, in fatty acid profile and in calcium concentration were found in the failed vein compared to the native saphenous vein from the same (CABG) patient. The failed vein contained significantly less phosphatidylethanolamine, more sphingomyelin, less arachidonic acid, more linoleic acid and more calcium than the native saphenous vein. Comparable changes in phospholipid composition, in fatty acid profile and increased calcium influx were reproduced in ECs cultured in medium containing 27OHC indicating that an oxysterol is an agent that can alter the lipid composition of vascular cell membranes. Our study indicates that a lipid agent, as well as protein agents that have previously been linked to the process of vascular remodeling, may be fundamental to many vascular diseases.     

Irradiation-induced up-regulation of HLA-E on macrovascular endothelial cells confers protection against killing by activated natural killer cells

Background

Apart from the platelet/endothelial cell adhesion molecule 1 (PECAM-1, CD31), endoglin (CD105) and a positive factor VIII-related antigen staining, human primary and immortalized macro- and microvascular endothelial cells (ECs) differ in their cell surface expression of activating and inhibitory ligands for natural killer (NK) cells. Here we comparatively study the effects of irradiation on the phenotype of ECs and their interaction with resting and activated NK cells.

Methodology/Principal Findings

Primary macrovascular human umbilical vein endothelial cells (HUVECs) only express UL16 binding protein 2 (ULBP2) and the major histocompatibility complex (MHC) class I chain-related protein MIC-A (MIC-A) as activating signals for NK cells, whereas the corresponding immortalized EA.hy926 EC cell line additionally present ULBP3, membrane heat shock protein 70 (Hsp70), intercellular adhesion molecule ICAM-1 (CD54) and HLA-E. Apart from MIC-B, the immortalized human microvascular endothelial cell line HMEC, resembles the phenotype of EA.hy926. Surprisingly, primary HUVECs are more sensitive to Hsp70 peptide (TKD) plus IL-2 (TKD/IL-2)-activated NK cells than their immortalized EC counterpatrs. This finding is most likely due to the absence of the inhibitory ligand HLA-E, since the activating ligands are shared among the ECs. The co-culture of HUVECs with activated NK cells induces ICAM-1 (CD54) and HLA-E expression on the former which drops to the initial low levels (below 5%) when NK cells are removed. Sublethal irradiation of HUVECs induces similar but less pronounced effects on HUVECs. Along with these findings, irradiation also induces HLA-E expression on macrovascular ECs and this correlates with an increased resistance to killing by activated NK cells. Irradiation had no effect on HLA-E expression on microvascular ECs and the sensitivity of these cells to NK cells remained unaffected.

Conclusion/Significance

These data emphasize that an irradiation-induced, transient up-regulation of HLA-E on macrovascular ECs might confer protection against NK cell-mediated vascular injury.     

Topological Localization of Monomeric C-reactive Protein Determines Proinflammatory Endothelial Cell Responses

The activation of endothelial cells (ECs) by monomeric C-reactive protein (mCRP) has been implicated in contributing to atherogenesis. However, the potent proinflammatory actions of mCRP on ECs in vitro appear to be incompatible with the atheroprotective effects of mCRP in a mouse model. Because mCRP is primarily generated within inflamed tissues and is rapidly cleared from the circulation, we tested whether these discrepancies can be explained by topological differences in response to mCRP within blood vessels. In a Transwell culture model, the addition of mCRP to apical (luminal), but not basolateral (abluminal), surfaces of intact human coronary artery EC monolayers evoked a significant up-regulation of MCP-1, IL-8, and IL-6. Such polarized stimulation of mCRP was observed consistently regardless of EC type or experimental conditions (e.g. culture of ECs on filters or extracellular matrix-coated surfaces). Accordingly, we detected enriched lipid raft microdomains, the major surface sensors for mCRP on ECs, in apical membranes, leading to the preferential apical binding of mCRP and activation of ECs through the polarized induction of the phospholipase C, p38 MAPK, and NF-κB signaling pathways. Furthermore, LPS and IL-1β induction of EC activation also exhibited topological dependence, whereas TNF-α did not. Together, these results indicate that tissue-associated mCRP likely contributes little to EC activation. Hence, topological localization is an important, but often overlooked, factor that determines the contribution of mCRP and other proinflammatory mediators to chronic vascular inflammation.     

The mysterious origins of coronary vessels

The origin of the coronary vessels remains a mystery. Here we discuss recent studies that address this puzzle, including new work by Tian et al. recently published in Cell Research.We face a growing epidemic of coronary vascular disease. Better understanding of the development of this unique vascular system will allow development of new treatment strategies. The origin of the coronary vessels has been a longstanding mystery. Classical anatomists proposed several potential sources for coronary vessels: the proepicardium (PE), the liver, the sinus venosus (SV) and the endocardium (Figure 1). Several recent reports have used sophisticated molecular and cell biological approaches to address this mystery, but have come to apparently contradictory conclusions. Tian et al.¹ use new lineage-tracing approaches to solve this puzzle, leading to new insights and new questions.Open in a separate windowFigure 1Diagram of E9.5 mouse embryo illustrating the proposed sources of coronary ECs. sv, sinus venosus; pe, proepicardium; li, liver primordium; v, ventricle; a, atrium.Initial studies in avian embryos, based on clonal retroviral labeling, dye labeling and quail-chick interspecies chimeras, indicated that coronary vascular smooth muscle and endothelial cells (vSMCs and ECs) derive from extracardiac sources. Most studies pinpointed the PE, a transient embryonic outgrowth of the septum transversum, as the cell source². PE cells transit to the heart, where they undergo an epithelial to mesenchymal transition (EMT). Based on these data, the predominant view from the early 1990s through the mid-2000s was that coronary vessels formed through a vasculogenic process from PE-derived mesenchymal cells. However, not all studies were in agreement. For example, Poelmann et al.³ reached a different conclusion and identified the nearby liver primoridium as the cell source. This study concluded that ECs and precursors formed small vessels that initially connected to the SV and then to subepicardial cells overlying the myocardium, which subsequently penetrated the myocardium to form the coronary vessels.The mainstream view of coronary artery formation from PE-derived ECs has been re-evaluated over the past decade through the use of Cre-LoxP genetic lineage-tracing approaches in mice^4,5,6,7. Several different mouse Cre lines that label populations within the PE were developed. Although these lines generally robustly label coronary vSMCs, they label a low fraction of coronary ECs (generally < 10%). Superficially, this suggests a divergence between avian and mammalian systems, but detailed comparison suggests that the results may be entirely consistent: the avian data indicate that some coronary ECs arise from the PE but the fraction of ECs that originate from PE was not determined. Both avian and murine studies could therefore be interpreted to suggest that a small fraction of coronary ECs arise from PE. A recent study further pointed out that PE contains heterogeneous cell populations, and some of these subpopulations (e.g., Sema3d⁺) contribute more robustly to coronary ECs than others (e.g., Tbx18⁺)⁷. Some lineages traced from the PE also contributed to ECs in the SV and endocardium, providing alternative routes whereby PE may give rise to coronary ECs. This study did not define the fraction of coronary ECs labeled by any of these subpopulations, therefore an estimate of the extent that these additional PE subpopulations contribute to coronary ECs is currently unavailable.Red-Horse et al.⁸ recently re-examined the endothelial lining of the SV as the origin of coronary ECs. Consistent with the study by Poelmann et al.³ in avian embryos, Red-Horse et al. observed that the first vessels of the heart tube connect to the SV. Elegant clonal labeling experiments using an EC-specific, tamoxifen-induced Cre (Cdh5-CreERT2) showed that labeling of single cells around E7.5 yielded descendant “clones” of ECs. At this point in development, PE cells do not express CDH5 and therefore these clones do not originate from this source. Most clones (74%) included SV ECs. However, its relationships with extracardiac structures, such as the liver primordium, were not investigated. Interestingly, SV ECs express venous markers, but descendant ECs belong to arterial and venous lineages. Based on these data, Red-Horse et al. concluded that most coronary ECs arise by angiogenic sprouting of SV ECs onto the developing heart, where they dedifferentiate, proliferate, form the coronary plexus, and subsequently redifferentiate into coronary arteries, capillaries and veins. While these data are compelling, to what extent this mechanism contributes to the coronary vasculature cannot be determined from this study.Wu et al.⁹ used a different lineage-tracing strategy to study coronary vessel origins and reached a different conclusion. This study was based on both constitutive and inducible Cre alleles driven by endocardium-specific Nfatc1 regulatory elements, which do not label PE, epicardium or SV prior to E10.5. By clonal analysis, Nfatc1-lineage cells differentiated to both artery and veins. Quantitative analysis showed that Nfatc1-labeled ECs form most intramyocardial coronary ECs (predominantly arteries) and a minority of supepicardial coronary ECs (predominantly veins). The clonal analysis of Red-Horse et al.⁸ also identified endocardial budding as a source of coronary vessels. Their data showed that fewer clones (24%) contained endocardial cells compared to SV cells, leading to the conclusion that endocardium makes a lesser contribution compared to the SV. However, this assumes equivalent labeling by Cdh5-CreERT2 under conditions where tamoxifen levels were limited. The frequency of endocardial cell labeling under these conditions may have been lower, for example if endocardial cells express lower levels of CreERT2.Tian et al.¹ studied coronary vessel development using Apln^CreERT2, a new lineage-tracing tool that selectively labels newly forming vessels but not established vessels or endocardium. Well-executed morphological and lineage-tracing experiments provide strong evidence that Apln^CreERT2 pulse activation at E11.5 labels nearly all subepicardial and intramyocardial coronary vessels of the ventricular free walls. Pulse labeling at this time labeled only rare ECs in the ventricular septum, suggesting that these vessels arise from ECs that express Apln^CreERT2 only after E11.5 and not from labeled ECs already present in the ventricular free walls. The endocardium appears to be an excellent candidate source for ECs in the ventricular septum. Clonal labeling experiments further demonstrated that at the single cell level, Apln⁺ ECs, named subepicardial ECs, retain the potential to differentiate into both arteries and veins.What is the relationship between subepicardial ECs and the proposed sites of origin for coronary ECs (PE, SV, endocardium, and liver primordium)? Using in vitro organ culture, Tian et al.¹ show that these cells are generated from the SV and subsequently extend onto the ventricles. Ventricles (containing ventricular endocardium) did not generate these cells in this system, leading the authors to conclude that they arise from the SV. However, the in vitro system does not yield robust coronary vessel formation, and it is entirely possible that certain developmental processes, such as endocardial budding or epicardial differentiation, are inactive under these conditions. Thus, we can conclude that some Apln⁺ ECs arise from SV, but the possibility of their origin also from other sources such as endocardium, PE, or liver primordium cannot be excluded.In summary, coronary ECs arise from multiple sources, and the balance between sources likely differs by anatomic region. While many studies on coronary vessel origins appear to reach conflicting conclusions, careful considerations of the experimental approaches and their limitations suggest models consistent with most published data. For instance, perhaps endocardial budding generates most intramyocardial coronaries, while angiogenic sprouting from the SV generates most subepicardial coronaries and a subset of intramyocardial coronaries. PE cells may contribute to a fraction of both EC populations, and give rise to most of the supporting smooth muscle cells. The Apln⁺ subepicardial ECs may represent a key common intermediate formed from all of these sources. Evaluating the contribution of each proposed cell source to this population will be important to understand the origins and growth patterns of coronary vessels. Further progress will depend on carefully quantitating the contribution of various EC sources to coronary vessel subtypes stratified by anatomic location.Understanding the origins of coronary vessels has implications for therapeutic strategies for coronary artery diseases, as each cell source suggests distinct mechanisms. For instance, SV angiogenic sprouting would direct us to investigate the signals that induce SV EC dedifferentiation and then redifferentiation into artery and vein ECs. PE-derived ECs might be induced by enhancing adult epicardial EMT and EC differentiation, while an endocardial EC source would prompt us to understand the signals that regulate the endocardial budding and differentiation process. The work of Tian et al. and the many other studies summarized herein are yielding insights into the mystery of coronary vessel origins. Solving this puzzle will yield rich rewards.     

Liquid chromatographic-mass spectrometric determination of cyclooxygenase metabolites of arachidonic acid in cultured cells

A liquid chromatographic-electrospray ionization-mass spectrometric (LC-ESI-MS) technique was developed to simultaneously determine the cyclooxygenase metabolites of arachidonic acid (6-keto-PGF(1alpha), PGD(2), PGE(2), PGF(2alpha), and PGJ(2)) produced by cultured cells. Samples were separated on a C(18) column with water-acetonitrile mobile phase, ionized by electrospray, and detected in the positive mode. Selected ion monitoring (SIM) of m/z 353, 335, 335, 319, and 317 were used for quantifying 6-keto-PGF(1alpha), PGD(2), PGE(2), PGF(2alpha), and PGJ(2), respectively. Prostaglandins were detected at concentrations as low as 1 pg (S/N=3) on the column. The method was used to determine the production of PGs from bovine coronary artery endothelial cells (ECs) and human prostate cancer cells (PC-3) with different degree of invasiveness. Bradykinin (10(-6) M) stimulated a marked increase in the production of 6-keto-PGF(1alpha), PGE(2), and PGF(2alpha) and a small increase of PGD(2) by ECs. 6-Keto-PGF(1alpha) was the major metabolite in these cells. The production of PGE(2) was threefold higher and PGD(2) was twofold higher in PC-3-S (invasive) cells than in PC-3-U (non-invasive) cells.     

RU486 antagonizes the inhibitory effect of peroxisome proliferator-activated receptor alpha on interleukin-6 production in vascular endothelial cells

Peroxisome proliferator-acitivated receptor alpha (PPARalpha) is a member of nuclear receptor superfamily. Recent studies have shown that the activators for PPARalpha inhibit the expression of some inflammatory molecules in vascular endothelial cells (ECs) and vascular smooth muscle cells, indicating the anti-inflammatory roles of PPARalpha on vascular walls. In this investigation, we showed that RU486, already proved to be an active anti-glucocorticoid and anti-progesterone agent, blocked the inhibition of tumor necrosis factor (TNF)-alpha-stimulated interleukin-6 (IL-6) production by the PPARalpha activator fenofibrate in human umbilical vein ECs. Transient transfection of bovine aortic ECs with an IL-6 promoter construct demonstrated that RU486 blocked the inhibitory effect of fenofibrate on TNF-alpha-induced IL-6 promoter activity. By fluorescence microscopy, RU486 was found to prevent fenofibrate-induced nuclear translocation of PPARalpha. Thus, RU486 has an antagonizing effect on PPARalpha-mediated down-regulation of IL-6 in vascular ECs. This effect may be exerted by its interference with the nuclear translocation of PPARalpha.     

Effects of epoxyeicosatrienoic acids on polymorphonuclear leukocyte function

During periods of ischemia and vascular injury, factors are released which recruit monocytes and polymorphonuclear leukocytes (PMNs) to the site of injury by promoting adherence to the endothelium and transmigration across the endothelial cell (EC) layer. During coronary artery stenosis, we have shown that the endothelium-derived, cytochrome P450 metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs), are elevated. Therefore, we examined if the EETs could stimulate PMN adherence to cultured ECs. Pretreatment of ECs with EETs for either 30 min or 4 hr did not alter the adherence of 51Cr-labelled PMNs to ECs while phorbol myristate acetate (PMA) produced a 4-fold increase in PMN adherence. The combination of EETs and PMA did not significantly augment or diminish PMA-induced PMN adherence to ECs. When ECs and 51Cr-labelled PMNs were coincubated, treatment with EETs alone did not alter PMN adherence. However, when EETs and PMA were added together during the coincubation of ECs and 51Cr-labelled PMNs, the EETs produced a concentration-related decrease in PMN adherence. Microscopic analysis of the culture media bathing the cells revealed aggregates of the labeled PMNs. We examined the effects of the EETs on PMN aggregation. 8,9-EET (10, 50, and 100 microM) increased PMN aggregation (7 +/- 3, 35 +/- 10, and 65 +/- 11%) and intracellular calcium by 1.7 +/- 0.5, 4.7 +/- 1.4, and 6.8 +/- 2.3-fold above basal. 5,6-, 11,2- and 14,15-EETs also stimulated aggregation. FMLP stimulated the production of superoxide; however, 8,9-EET did not. These observations indicate that the decrease in PMN adherence observed in the coincubation experiment is the result of EET-induced PMN aggregation. Given the increase in EET production during coronary artery stenosis, these data may provide insight into their potential biological significance during myocardial ischemia and vascular injury.     

Improved adenovirus vectors for infection of cardiovascular tissues

To identify improved adenovirus vectors for cardiovascular gene therapy, a library of adenovirus vectors based on adenovirus serotype 5 (Ad5) but carrying fiber molecules of other human serotypes, was generated. This library was tested for efficiency of infection of human primary vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Based on luciferase, LacZ, or green fluorescent protein (GFP) marker gene expression, several fiber chimeric vectors were identified that displayed improved infection of these cell types. One of the viruses that performed particularly well is an Ad5 carrying the fiber of Ad16 (Ad5.Fib16), a subgroup B virus. This virus showed, on average, 8- and 64-fold-increased luciferase activities on umbilical vein ECs and SMCs, respectively, compared to the parent vector. GFP and lacZ markers showed that approximately 3-fold (ECs) and 10-fold (SMCs) more cells were transduced. Experiments performed with both cultured SMCs and organ cultures derived from different vascular origins (saphenous vein, iliac artery, left interior mammary artery, and aorta) and from different species demonstrated that Ad5.Fib16 consistently displays improved infection in primates (humans and rhesus monkeys). SMCs of the same vessels of rodents and pigs were less infectable with Ad5.Fib16 than with Ad5. This suggests that either the receptor for human Ad16 is not conserved between different species or that differences in the expression levels of the putative receptor exist. In conclusion, our results show that an Ad5-based virus carrying the fiber of Ad16 is a potent vector for the transduction of primate cardiovascular cells and tissues.     

Endothelial cell lineages of the heart

During early gastrulation, vertebrate embryos begin to produce endothelial cells (ECs) from the mesoderm. ECs first form primitive vascular plexus de novo and later differentiate into arterial, venous, capillary, and lymphatic ECs. In the heart, the five distinct EC types (endocardial, coronary arterial, venous, capillary, and lymphatic) have distinct phenotypes. For example, coronary ECs establish a typical vessel network throughout the myocardium, whereas endocardial ECs form a large epithelial sheet with no angiogenic sprouting into the myocardium. Neither coronary arteries, veins, and capillaries, nor lymphatic vessels fuse with the endocardium or open to the heart chamber. The developmental stage during which the specific phenotype of each cardiac EC type is determined remains unclear. The mechanisms involved in EC commitment and diversity can however be more precisely defined by tracking the migratory patterns and lineage decisions of the precursors of cardiac ECs. Work carried out by the authors is supported in part by the NIH.     

Protection of xenogeneic cells from human complement-mediated lysis by the expression of human DAF, CD59 and MCP

CD59 and membrane cofactor protein (MCP, CD46) are widely expressed cell surface glycoproteins that protect host cells from the effect of homologous complement attack. cDNAs encoding human CD59 and MCP cloned from Chinese human embryo were separately transfected into NIH/3T3 cells resulting in the expression of human CD59 and MCP protein on the cell surface. The functional properties of expressed proteins were studied. When the transfected cells were exposed to human serum as a source of complement and naturally occurring anti-mouse antibody, they were resistant to human complement-mediated cell killing. However, the cells remained sensitive to rabbit and guinea pig complement. Human CD59 and MCP can only protect NIH/3T3 cells from human complement-mediated lysis. These results demonstrated that complement inhibitory activity of these proteins is species-selective. The cDNAs of CD59 and MCP were also separately transfected into the endothelial cells (ECs) of the pigs transgenic for the human DAF gene to investigate a putative synergistic action. The ECs expressing both DAF and MCP proteins or both DAF and CD59 proteins exhibited more protection against cytolysis by human serum compared to the cells with only DAF expressed alone.