Transient receptor potential vanilloid 1-mediated expression and secretion of endothelial cell-derived calcitonin gene-related peptide

Calcitonin gene-related peptide (CGRP), the principal transmitter in sensory nerves, could also be expressed in vascular endothelium. Transient receptor potential vanilloid 1(TRPV1), which modulates the synthesis and release of CGRP in sensory nerves, is also present in endothelial cells. The present study tested whether TRPV1 modulates the release and synthesis of CGRP in endothelial cells, and evaluated the protective effect of endothelial cell-derived CGRP. Human umbilical vein endothelial cells (HUVECs) were treated with capsaicin or hyperthermia. The level of CGRP mRNA was detected by RT-PCR, and protein level was measured by radioimmunoassay. Endothelial cell injury was induced by lysophosphatidylcholine, and evaluated by cell viability and lactate dehydrogenase activity. HUVECs expressed CGRP, both alpha- and beta-subtype. Capsaicin increased the level of CGRP in the culture medium, and up-regulated the expression of CGRP in endothelial cells. Hyperthermia also increased the level of CGRP mRNA. These effects were abolished by capsazepine, a competitive antagonist of TRPV1. Capsaicin significantly attenuated the endothelial cell damage induced by LPC, which was prevented and aggravated by capsazepine or CGRP(8-37,) antagonist of CGRP receptor. These results indicate that TRPV1 also regulates the expression and secretion of endothelial cell-derived CGRP, which affords protective effects on endothelial cells.     

Calcitonin gene-related peptide vasodilation of human pulmonary vessels

Human calcitonin gene-related peptide (CGRP) is localized to sensory neurons in pulmonary vessels and is a potent vasodilator. We have characterized the effects of CGRP in human pulmonary vessels and localized the receptors for this peptide by autoradiography. Fresh human lung tissue was obtained from eight patients undergoing surgery and small (200-400 microns ID) pulmonary arteries and veins were dissected free of surrounding connective and pulmonary tissue. Pairs of vessels were studied and in one of each pair the endothelium was left intact and from the other of each pair the endothelium was removed by gentle abrasion. For functional studies arteries (n = 9) and veins (n = 9) were suspended in an organ bath, precontracted with 1 microM prostaglandin F2 alpha. CGRP (10 pM to 10 microM) was added in a cumulative manner. CGRP caused a dose-dependent relaxation of endothelium intact human pulmonary arteries and veins with log EC50 values of -8.01 +/- 0.35 and -8.70 +/- 0.40, respectively (not significant). Removal of the endothelium did not diminish the vasodilator potency of CGRP in either vessel. For autoradiographic studies, cryostat sections of the small human pulmonary vessels with or without endothelium were used. 125I-CGRP densely labeled CGRP receptors on vascular smooth muscle and endothelial removal did not have any effect on grain density. We concluded that CGRP is a potent vasodilator of human pulmonary arteries and veins that is not dependent on an intact endothelium. These functional studies correlate with the distribution of CGRP receptors as localized by autoradiography.     

Enhanced expression of endothelin-1 and endothelin-converting enzyme-1 in acute hypoxic rat aorta

Deeply anesthetized male Wistar rats were perfused by Hanks' balanced salt solution bubbled with either 95%air and 5%CO2 (normoxic group) or 95%N2 and 5%CO2 (hypoxic group) from the thoracic aorta for 30 min, and the isolated abdominal aortae from both groups were used for electron microscopy, immunocytochemistry of endothelin (ET)-1 and ET-converting enzyme (ECE)-1, and in situ hybridization of preproET-1 mRNA. A remarkable increase in the number of Weibel-Palade (WP) bodies, storage sites of ET-1 and ECE-1, occurred in the hypoxic group when compared to the normoxic group. Immunoreactivities for ET-1 and ECE-1, and signals for preproET-1 mRNA were seen along the endothelia of both groups, but the intensities were significantly elevated in the hypoxic group. The increase in the number of ECE-1 immunoreactive gold particles was noticed especially in WP bodies in the hypoxic group. These findings indicate the enhancement of preproET-1 synthesis in the aortic endothelial cells as well as the acceleration of ET-1 processing in increased WP bodies in such cells in an experimentally hypoxic condition of the rat aortae.     

Vascular endothelial expression of indoleamine 2,3-dioxygenase 1 forms a positive gradient towards the feto-maternal interface

We describe the distribution of indoleamine 2,3-dioxygenase 1 (IDO1) in vascular endothelium of human first-trimester and term placenta. Expression of IDO1 protein on the fetal side of the interface extended from almost exclusively sub-trophoblastic capillaries in first-trimester placenta to a nearly general presence on villous vascular endothelia at term, including also most bigger vessels such as villous arteries and veins of stem villi and vessels of the chorionic plate. Umbilical cord vessels were generally negative for IDO1 protein. In the fetal part of the placenta positivity for IDO1 was restricted to vascular endothelium, which did not co-express HLA-DR. This finding paralleled detectability of IDO1 mRNA in first trimester and term tissue and a high increase in the kynurenine to tryptophan ratio in chorionic villous tissue from first trimester to term placenta. Endothelial cells isolated from the chorionic plate of term placenta expressed IDO1 mRNA in contrast to endothelial cells originating from human umbilical vein, iliac vein or aorta. In first trimester decidua we found endothelium of arteries rather than veins expressing IDO1, which was complementory to expression of HLA-DR. An estimation of IDO activity on the basis of the ratio of kynurenine and tryptophan in blood taken from vessels of the chorionic plate of term placenta indicated far higher values than those found in the peripheral blood of adults. Thus, a gradient of vascular endothelial IDO1 expression is present at both sides of the feto-maternal interface.     

In vitro effects of endotoxin on bovine and sheep lung microvascular and pulmonary artery endothelial cells

A single infusion of Escherichia coli endotoxin into sheep results in structural evidence of pulmonary endothelial injury, increases in both prostacyclin and prostaglandin E2 (PGE2) in lung lymph, and an increase in pulmonary microvascular permeability. Endotoxin-induced lung endothelial damage can also be induced in vitro, but to date these studies have utilized endothelium from large pulmonary vessels. In the present study, we have grown endothelial cells from peripheral lung vessels of cows and sheep and exposed these microvascular endothelial cells to endotoxin. Controls included lung microvascular endothelium without endotoxin and endothelial cells from bovine and sheep main pulmonary artery with and without addition of endotoxin. We found that endotoxin caused significant increases in release of prostacyclin and PGE2 from both bovine and sheep lung microvascular and pulmonary artery endothelium. Normal bovine and sheep pulmonary artery and bovine lung microvascular endothelium released greater levels of prostacyclin than PGE2 (ng/ng); release of PGE2 from the microvascular cells was greater than from the pulmonary artery endothelium in both species. Exposure of endothelial cells from cow and sheep main pulmonary artery to endotoxin results in endothelial cell retraction and pyknosis, a loss of barrier function, increased release of prostacyclin and PGE2 and eventual cell lysis. In lung microvascular cells, the increases in prostanoids were accompanied by changes in cell shape but occurred in the absence of either detectable alterations in barrier function or cytolysis. Thus, while endotoxin causes alterations to endothelial cells from both large and small pulmonary vessels, the effects are not identical suggesting site specific phenotypic expression of endothelial cells even within a single vessel. To determine whether the response of either the large or small pulmonary vessel endothelial cells in culture mimics most closely the in vivo response of the lung to endotoxin requires further study.     

Culture and characterization of bovine mesenteric lymphatic endothelium

Summary Lymphatic endothelial cells isolated from bovine mesenteric lymphatic vessels were cultured and characterized. Lymphatic endothelial cells grew as a monolayer displaying an elongated morphology in preconfluent primary cultures. When confluent, the cells exhibited a polygonal morphology to form a “cobblestone” pattern previously described for cultured vascular endothelium. All culture lymphatic endothelial cells expressed Factor VIII-related antigen and boundUlex europaeus I lectin. Ultrastructurally, cultured lymphatic endothelium was characterized by the presence of Weibel-Palade bodies as well as the usual cytoplasmic organelles.     

Clonidine induces calcitonin gene-related peptide expression via nitric oxide pathway in endothelial cells

The present study was to determine whether clonidine could induce calcitonin gene-related peptide (CGRP) production and the underlying mechanisms. Human umbilical vein endothelial cells were treated with clonidine and the dose–effect or time–effect relationship of clonidine on CGRP production was examined. Youhimbine (a α₂-adrenoceptor blocker) and l-NAME (an antagonist of nitric oxide synthase, NOS) were chosen to explore the role of α₂-adrenoceptor and nitric oxide pathway in the effect of clonidine on endothelial cell-derived CGRP production. The level of CGRP mRNA or protein was detected by Real Time-PCR or radioimmunoassay. Nitric oxide content was measured by nitroreduction assay. The study showed that clonidine was able to induce CGRP mRNA (α- and β-isoforms) expression in a dose-dependent manner in endothelial cells. The effect of clonidine on endothelial cell-derived CGRP synthesis and secretion was attenuated in the presence of youhimbine. l-NAME treatment could also inhibit clonidine-induced CGRP synthesis and secretion concomitantly with the decreased NO content in culture medium. These results suggest that clonidine could stimulate CGRP synthesis and secretion in endothelial cells through the activation of α₂-adrenoceptor, which is related to the NO pathway.     

Endothelial coordination of cerebral vasomotion via myoendothelial gap junctions containing connexins 37 and 40

Control of cerebral vasculature differs from that of systemic vessels outside the blood-brain barrier. The hypothesis that the endothelium modulates vasomotion via direct myoendothelial coupling was investigated in a small vessel of the cerebral circulation. In the primary branch of the rat basilar artery, membrane potential, diameter, and calcium dynamics associated with vasomotion were examined using selective inhibitors of endothelial function in intact and endothelium-denuded arteries. Vessel anatomy, protein, and mRNA expression were studied using conventional electron microscopy high-resolution ultrastructural and confocal immunohistochemistry and quantitative PCR. Membrane potential oscillations were present in both endothelial cells and smooth muscle cells (SMCs), and these preceded rhythmical contractions during which adjacent SMC intracellular calcium concentration ([Ca(2+)](i)) waves were synchronized. Endothelium removal abolished vasomotion and desynchronized adjacent smooth muscle cell [Ca(2+)](i) waves. N(G)-nitro-l-arginine methyl ester (10 microM) did not mimic this effect, and dibutyryl cGMP (300 muM) failed to resynchronize [Ca(2+)](i) waves in endothelium-denuded arteries. Combined charybdotoxin and apamin abolished vasomotion and depolarized and constricted vessels, even in absence of endothelium. Separately, (37,43)Gap27 and (40)Gap27 abolished vasomotion. Extensive myoendothelial gap junctions (3 per endothelial cell) composed of connexins 37 and 40 connected the endothelial cell and SMC layers. Synchronized vasomotion in rat basilar artery is endothelium dependent, with [Ca(2+)](i) waves generated within SMCs being coordinated by electrical coupling via myoendothelial gap junctions.     

Growth and morphology of rabbit marginal vessel endothelium in cell culture

A procedure for the isolation and cultivation of endothelium from the marginal vessels of the rabbit ear is described. Endothelial cells, isolated by slow perfusion with a trypsin solution, are cultured in minimal essential medium supplemented with 10% fresh rabbit serum for up to 6 mo. In primary culture, marginal vessel endothelial cells grow in an expanding circular pattern with closely apposed cell membranes. Weibel-Palade bodies, subcellular organelles unique to endothelial cells in situ, are present in both primary and in serially cultivated cells (12 passages). In intact skin, Weibel-Palade (W-P) bodies are observed in the perinuclear cytoplasm in close proximity to the cell membrane facing the vascular lumen. 8-16 tubules of 200 A diameter are present in each body. In primary and subcultured cells, W-P bodies of identical size are seen in the vicinity of the Golgi apparatus and in close proximity to the outer cell membrane. At the optimum serum concentration (10%), a cell doubling time of 72-96 h is observed. When growth in normal rabbit serum and in platelet-poor serum is compared, a slower growth rate is observed in the absence of platelets, suggesting that factors released by platelets affect endothelial cell proliferation. However, addition of crude platelet factor does not substitute for complete serum. Fibroblast growth factor is not mitogenic for rabbit marginal vessel endothelium in vitro.     

Molecular and ultrastructural characterization of endothelial cells differentiated from human bone marrow mesenchymal stem cells

In this study characterization of endothelial cells differentiated from human bone marrow mesenchymal stem cells (hBMCs) was investigated in relation to their capillary network formation potential. Differentiation was performed in presence of vascular endothelial growth factor (VEGF) and insulin like growth factor-1 (IGF-1). A panel of cellular and molecular markers was used for characterization of the endothelial cells. The cells were strongly positive for von Willebrand factor (vWF) and vascular endothelial growth factor receptor 2 (VEGFR2) when measured at protein and mRNA levels. Development of endothelial cells was found to be associated with formation of typical organelles such as Weibel Palade (WP) bodies, Cavealae and pinocytic vesicles. Early vessel growth was also evidenced by showing specific junctions between the cells. The migratory and angiogenic properties of the cells were confirmed by showing capillary network formation in vitro. These results indicate that the capacity of endothelial cells differentiated from hBMSCs in formation of vascular system is consistent with molecular and structural development.     

Effects of alpha calcitonin gene-related peptide in human bronchial smooth muscle and pulmonary artery

Although airway and pulmonary vessel tone are regulated predominantly by cholinergic and adrenergic impulses, biologically active peptides such as calcitonin gene-related peptide (CGRP) may significantly influence human smooth muscle tone in normal and pathophysiological states. In the present study, the expression of CGRP and its receptor CGRPR-1 and the biological effect of the peptide were investigated in human airways and pulmonary arteries. Immunohistochemistry revealed the presence of CGRP in human airway nerves and neuro-epithelial cells, whereas the receptor was found in epithelial cells and smooth muscle myocytes of the bronchi and in pulmonary artery endothelium. On precontracted bronchi (3-4 mm in diameter) alpha-CGRP (0.01-10 nM) caused a concentration-dependent contraction on epithelium-denuded bronchi, whereas no significant effect was recorded in bronchi with intact epithelium. In pulmonary arteries (2-6 mm in diameter), alpha-CGRP caused a concentration-dependent relaxation of endothelium intact and denuded vessels. Pre-treatment with indomethacin, but not with l-NAME, prevented the relaxation induced by alpha-CGRP in pulmonary arteries suggesting that prostaglandins but not nitric oxide (NO) are involved in the intracellular signal transduction pathway. The effects induced by alpha-CGRP in bronchi and vessels were prevented by application of the antagonist CGRP((8-37)). In summary, the present studies examined the biological function of CGRP in human airways and demonstrated a constrictory effect of CGRP only in epithelium-denuded airway smooth muscle indicating an alteration of CGRP airway effects in respiratory tract pathological states with damaged epithelium such as chronic obstructive pulmonary disease or bronchial asthma.     

Polarized secretion of IGF-I and IGF-I binding protein activity by cultured aortic endothelial cells

Insulin-like growth factor-I (IGF-I) secretion by the vascular endothelium has been proposed to play a role in the regulation of vascular smooth muscle cell proliferation. Because vascular smooth muscle cells are adjacent to the abluminal surface of the endothelium, we tested the hypothesis that secretion of IGF-I by endothelial cells is polarized. Porcine aortic endothelial cells were cultured on permeable membranes and IGF-I measured by radioimmunoassay. Basal secretion exceeded apical secretion by a ratio of 2.3 ± 0.2:1.0 (P < 0.05). We also identified 35 kDa IGF-I binding protein activity that is preferentially secreted on the basal surface of endothelial cells. We conclude that both IGF-I and IGF-I binding protein activity secretion by endothelial cells is polarized towards the basal surface of the endothelium. A polarized secretion mechanism for IGF-I may be of importance in the normal growth and differentiation of the vasculature as well as in the development of vascular pathology. © 1993 Wiley-Liss, Inc.     

Calcitonin gene-related peptide prevents blood-brain barrier injury and brain edema induced by focal cerebral ischemia reperfusion

Cerebral ischemia is one of the diseases that most compromise the human species. Therapeutic recovery of blood-brain barrier (BBB) disruption represents a novel promising approach to reduce brain injury after stroke. To determine the effects of calcitonin gene-related peptide (CGRP) on the BBB participate in stroke progression, rat cerebral ischemia reperfusion injury was induced by a 2-hour left transient middle cerebral artery occlusion (MCAO) using an intraluminal filament, followed by 46h of reperfusion. CGRP (1μg/ml) at the dose of 3μg/kg (i.p.) was administered at the beginning of reperfusion. Subsequently, 48h after MCAO, arterial blood pressure, infarct volume, water content, BBB permeability, BBB ultrastructure, levels of aquaporin-4 (AQP4) and its mRNA were evaluated. CGRP could reduce arterial blood pressure (P<0.001), infarct volume (P<0.05), cerebral edema (P<0.01), BBB permeability (P<0.05), AQP4 mRNA expression (P<0.05) and AQP4 protein expression (P<0.01). Furthermore, CGRP treatment improved ultrastructural damage of capillary endothelium cells and decreased the loss of the tight junction observed by transmission electronic microscopy (TEM) after 46h of reperfusion. Our findings show that CGRP significantly reduced postischemic increase of brain edema with a 2-hour therapeutic window in the transient model of focal cerebral ischemia. Moreover, it seems that at least part of the anti-edematous effects of CGRP is due to decrease of BBB disruption by improving ultrastructural damage of capillary endothelium cells, enhancing basal membrane, and inhibiting AQP4 and its mRNA over-expression. The data of the present study provide a new possible approach for acute stroke therapy by administration of CGRP.     

Differential responsiveness of vascular endothelial cells to different types of fluid mechanical shear stress

Early atherosclerotic lesions localize preferentially, in arterial regions exposed to low flow, oscillatory flow, or both; however, the cellular basis of this observation remains to be determined. Atherogenesis involves dysfunction of the vascular endothelium, the cellular monolayer lining the inner surfaces of blood vessels. How low flow, oscillatory flow, or both may lead to endothelial dysfunction remains unknown. Over the past two decades, fluid mechanical shear (or frictional) stress has been shown to intricately regulate the structure and function of vascular endothelial cells (ECs). Furthermore, recent data indicate that beyond being merely responsive to shear stress, ECs are able to distinguish among and respond differently to different types of shear stress. This review focuses on EC differential responses to different types of steady and unsteady shear stress and discusses the implications of these responses for the localization of early atherosclerotic lesions. The mechanisms by which endothelial differential responsiveness to different types of flow may occur are also discussed.     

Mesenchymal stem cell marker Stro-1 is a 75 kd endothelial antigen

Stro-1 is the best-known mesenchymal stem cell (MSC) marker. However, previous studies have observed its expression in the endothelium. In the present study we performed immunofluorescence (IF) staining for Stro-1, using endothelial marker vWF as reference. In the liver, both proteins were expressed in the endothelium of the central veins and hepatic sinusoids. In the lung, both were expressed in the endothelium of pulmonary blood vessels, but while vWF was absent in the alveolar capillaries, Stro-1 was present. In the kidney, both were expressed in the endothelium of renal arterial branches, but while vWF was strongly expressed in the glomeruli, Stro-1 only scantly. IF staining in cultured endothelial cells also showed extensive overlaps between Stro-1 and vWF. Western blot analysis with Stro-1 antibody detected a single protein band of 75kd in endothelial cells but not smooth muscle cells, fibroblasts, or B cells. Cancer cell lines PC3, DU145, MCF7, and K562 were also positive. Adipose-derived stem cells (ADSCs) expressed higher levels of Stro-1 when cultured beyond the first passage or when induced to differentiate into endothelial cells. These data, together with previous studies, indicate that Stro-1 is intrinsically an endothelial antigen, and its expression in MSC is probably an induced event.     

Microparticle formation after co-culture of human whole blood and umbilical artery in a novel in vitro model of flow

Cardiovascular disease (CVD) is now the largest killer in western society, and the importance of interactions between vascular endothelium and circulating blood components in disease pathogenesis is well established. Microparticles are a heterogeneous population of <1 μm blood borne particles that arise from blebbing or shedding of cell membranes. The microparticle population includes several classes of apoptotic bodies; however, increased numbers of procoagulant microparticles have been described in plasma from people with CVD. We have previously demonstrated that interactions of monocytes and platelets with isolated inflamed endothelial cells lead to production of pro-coagulant tissue factor bearing microparticles under laminar flow conditions. Here we have investigated microparticle production after perfusion of human whole blood through intact inflamed human umbilical artery. When blood was perfused through umbilical arteries which had been pre-stimulated with tumour necrosis factor (TNFα) for 18 h under flow conditions, there was significantly increased production of microparticles from both platelet and non-platelet sources, in particular from erythrocytes. To determine whether microparticles generated during interactions with inflamed endothelium could induce a pro-inflammatory response in trans, we isolated microparticles by centrifugation after co-culture and incubated with isolated quiescent endothelial cells followed by measurement of reactive oxygen species formation. Microparticles derived from co-culture with inflamed endothelium induced significantly enhanced levels of reactive oxygen species (ROS). These data suggest that presence of an inflamed endothelium causes release of pro-inflammatory microparticles from circulating blood cells, which could contribute to prolonged endothelial activation and subsequent atherosclerotic changes in blood vessels subjected to inflammatory insult.     

Origin of coronary endothelial cells from epicardial mesothelium in avian embryos

It has been established that coronary vessels develop through self-assembly of mesenchymal vascular progenitors in the subepicardium. Mesenchymal precursors of vascular smooth muscle cells and fibroblasts are known to originate from an epithelial-to-mesenchymal transformation of the epicardial mesothelium, but the origin of the coronary endothelium is still obscure. We herein report that at least part of the population of the precursors of the coronary endothelium are epicardially-derived cells (EPDCs). We have performed an EPDC lineage study through retroviral and fluorescent labelling of the proepicardial and epicardial mesothelium of avian embryos. In all the experiments onlythe surface mesothelium was labelled after 3 h of reincubation. However, endothelial cells from subepicardial vessels were labelled after 24-48 h and endothelial cells of intramyocardial vessels were also labelled after 48-96 h of reincubation. In addition, the development of the coronary vessels was studied in quail-chick chimeras, obtaining results which also support a mesothelial origin for endothelial and smooth muscle cells. Finally, quail proepicardial explants cultured on Matrigel showed colocalization of cytokeratin and QH1 (mesothelial and endothelial markers, respectively) after 24 h. These results, taken together, suggest that EPDC show similar competence to that displayed by bipotential vascular progenitor cells [Yamashita et al., Nature 408: 92-96 (2000)] which are able to differentiate into endothelium or smooth muscle depending on their exposure to VEGF or PDGF-BB. It is conceivable that the earliest EPDC differentiate into endothelial cells in response to myocardially-secreted VEGF, while further EPDC would be recruited by the nascent capillaries via PDGFR-beta signalling, giving rise to mural cells.