Expression of alphaVbeta3 integrin in the chick embryo aortic endothelium

The integrin chain alphaV, expressed in association with beta3, by cells of the megakaryocytic/thrombocytic and endothelial lineages is thought to play an important role in angiogenesis. alphaVbeta3 expression by endothelial cells is not constitutive but induced by various stimuli in avian and human models. Here the developmental pattern of alphaVbeta3 expression was analysed in the chick embryo by immunocytochemistry, using a specific monoclonal antibody. On day 2 of development alphaVbeta3 expression was restricted to rare cells in the blood stream, in the embryo proper and in the yolk sac blood islands. AlphaVbeta3 expression by endothelial cells became detectable on day 3 and was restricted to the dorsal aorta. Interestingly it was absent from the intra-aortic hemopoietic clusters (E3.5) which, as we have showed previously, express the alphaIIbbeta3 integrin and display progenitor potentialities. However the endothelium underlying intra-embryonic hemopoietic clusters expressed this integrin. In contrast E6-7 para-aortic hemopoietic foci contained numerous alphaVbeta3 positive cells. Both alphaVbeta3 and alphaIIbbeta3 were expressed in these latter hemopoietic sites, while alphaVbeta3 was still selectively expressed by the aortic endothelium until E6. Thereafter, at E7 the pulmonary artery also expressed it. Since alphaIIbbeta3 is expressed by avian and murine multilineage hemopoietic progenitors, we then studied the hemopoietic potentialities of alphaVbeta3/alphaIIbeta3 double positive cells from embryonic bone marrow differentiating in vitro in erythro-myeloid conditions. Thrombocytic, erythroid and myeloid progenitor potentialities were found within the cell population expressing both beta3 integrins.     

MB1, a quail leukocyte/vascular endothelium antigen: characterization of the lymphocyte-surface form and identification of its secreted counterpart as alpha 2-macroglobulin

A monoclonal antibody (alpha-MB1) binds to a cell surface marker expressed throughout ontogeny and adult life by vascular endothelial and hemopoietic cells of the quail, with the exception of erythrocytes, although it was raised against the heavy chain of quail immunoglobulin M. In addition to an 80 kDa polypeptide accounting for immunoglobulin mu chain, alpha-MB1 stains intensely a 180-kDa band on Western blots of reduced plasma proteins. We have previously characterized MB1 antigens of quail endothelial cells as glycoproteins of apparent molecular masses ranging from 80 to 200 kDa and provided evidence for the participation of vascular endothelium in the secretion of alpha-MB1-positive plasma components. We demonstrate here that this circulating material is the proteinase inhibitor alpha 2-macroglobulin. Furthermore, the MB1 antigens immunoprecipitated from lymphocytes are shown to be essentially similar to their endothelial counterparts, suggesting that the same molecular complex is expressed by all the elements of the hemangioblastic cell lineage. Finally, the cross reactivity of the alpha-MB1 antibody with immunoglobulin mu chain is confirmed and shown to occur via a carbohydrate epitope.     

Demonstration of a phagocytic cell system belonging to the hemopoietic lineage and originating from the yolk sac in the early avian embryo.

It is well established that hemopoietic cells arising from the yolk sac invade the avian embryo. To study the fate and role of these cells during the first 2.5-4.5 days of incubation, we constructed yolk sac chimeras (a chick embryo grafted on a quail yolk sac and vice versa) and immunostained them with antibodies specific to cells of quail hemangioblastic lineage (MB1 and QH1). This approach revealed that endothelial cells of the embryonic vessels are of intraembryonic origin. In contrast, numerous hemopoietic cells of yolk sac origin were seen in embryos ranging from 2.5 to 4.5 days of incubation. These cells were already present within the vessels and in the mesenchyme at the earliest developmental stages analyzed. Two hemopoietic cell types of yolk sac origin were distinguishable, undifferentiated cells and macrophage-like cells. The number of the latter cells increased progressively as development proceeded, and they showed marked acid phosphatase activity and phagocytic capacity, as revealed by the presence of numerous phagocytic inclusions in their cytoplasm. The macrophage-like cells were mostly distributed in the mesenchyme and also appeared within some organ primordia such as the neural tube, the liver anlage and the nephric rudiment. Comparison of the results in the two types of chimeras and the findings obtained with acid phosphatase/MB1 double labelling showed that some hemopoietic macrophage-like cells of intraembryonic origin were also present at the stages considered. These results support the existence in the early avian embryo of a phagocytic cell system of blood cell lineage, derived chiefly from the yolk sac. Cells belonging to this system perform phagocytosis in cell death and may also be involved in other morphogenetic processes.     

A novel method to bursectomize avian embryos and obtain quail----chick bursal chimeras. I. Immunocytochemical analysis of such chimeras by using species-specific monoclonal antibodies

Chick embryos were bursectomized at 5 days of incubation according to a novel surgical technique described in this article. This method yields birds that are able to hatch and are devoid of the physiologic deficiencies resulting from the previously used method, which involved resection of the cloacal and posterior embryonic region. The bursectomized embryos were grafted in situ with a quail bursa of the same age, which thereafter became chimeric through chick host hemopoietic cell invasion. By means of species-specific antibodies, the chimeric condition revealed 1) that the bursal epithelium expresses a unique antigenic determinant (MB1 determinant), until now considered to be an exclusive feature of blood vessel endothelium and hemopoietic cells, and 2) that this determinant appears in bursal epithelium at the time and site of hemopoietic cell invasion. The other point arising from this work concerns the apparent constitutive Ia expression by perifollicular blood capillary endothelial cells in normal and chimeric bursas.     

Embryonic vascular development: immunohistochemical identification of the origin and subsequent morphogenesis of the major vessel primordia in quail embryos

The development of the embryonic vasculature is examined here using a monoclonal antibody, QH-1, capable of labelling the presumptive endothelial cells of Japanese quail embryos. Antibody labelling is first seen within the embryo proper at the 1-somite stage. Scattered labelling of single cells appears ventral to the somites and at the lateral edges of the anterior intestinal portal. The dorsal aorta soon forms a continuous cord at the ventrolateral edge of the somites and continues into the head to fuse with the ventral aorta forming the first aortic arch by the 6-somite stage. The rudiments of the endocardium fuse at the midline above the anterior intestinal portal by the 3-somite stage and the ventral aorta extends craniad. Intersomitic arteries begin to sprout off of the dorsal aorta at the 7-somite stage. The posterior cardinal vein forms from single cells which segregate from somatic mesoderm at the 7-somite stage to form a loose plexus which moves mediad and wraps around the developing Wolffian duct in later stages. These studies suggest two modes of origin of embryonic blood vessels. The dorsal aortae and cardinal veins apparently arise in situ by the local segregation of presumptive endothelial cells from the mesoderm. The intersomitic arteries, vertebral arteries and cephalic vasculature arise by sprouts from these early vessel rudiments. There also seems to be some cell migration in the morphogenesis of endocardium, ventral aorta and aortic arches. The extent of presumptive endothelial migration in these cases, however, needs to be clarified by microsurgical intervention.     

Affiliation between endothelial and intra-embryonic hematopoietic stem cells]

We have investigated the developmental relationship of the hemopoietic and endothelial lineages in the floor of the chicken aorta, a site of hemopoietic progenitor emergence in the embryo proper. We show that, prior to the onset of hemopoiesis, the aortic endothelium uniformly expresses the endothelium-specific membrane receptor VEGF-R2. The onset of hemopoiesis can be precisely determined by detecting the common leukocyte antigen CD45. VEGF-R2 and CD45 are expressed in complementary fashion, namely the hemopoietic clusters in the floor of the aorta are CD45+/VEGF-R2-, while the rest of the aortic endothelium is CD45-/VEGF-R2+. To determine if the hemopoietic clusters are derived from EC, we tagged the E2 endothelial tree with a non-replicative retroviral vector and low density lipoproteins. Twenty four-48 hours later, labelled cells in the vascular tree were found to be either endothelial or hemopoietic but exceptionally both. Another 1-2 days later, groups of labelled cells appear in the dorsal mesentery within the hemopoietic "paraortic foci". Since no CD45+ cells were inserted among endothelial cells at the time of vascular labelling, hemopoietic clusters and foci must be concluded to derive from precursors with an endothelial phenotype.     

The breakup of intravascular microbubbles and its impact on the endothelium

Encapsulated microbubbles (MBs) serve as endovascular agents in a wide range of medical ultrasound applications. The oscillatory response of these agents to ultrasonic excitation is determined by MB size, gas content, viscoelastic shell properties and geometrical constraints. The viscoelastic parameters of the MB capsule vary during an oscillation cycle and change irreversibly upon shell rupture. The latter results in marked stress changes on the endothelium of capillary blood vessels due to altered MB dynamics. Mechanical effects on microvessels are crucial for safety and efficacy in applications such as focused ultrasound-mediated blood–brain barrier (BBB) opening. Since direct in vivo quantification of vascular stresses is currently not achievable, computational modelling has established itself as an alternative. We have developed a novel computational framework combining fluid–structure coupling and interface tracking to model the nonlinear dynamics of an encapsulated MB in constrained environments. This framework is used to investigate the mechanical stresses at the endothelium resulting from MB shell rupture in three microvessel setups of increasing levels of geometric detail. All configurations predict substantial elevation of up to 150 % for peak wall shear stress upon MB breakup, whereas global peak transmural pressure levels remain unaltered. The presence of red blood cells causes confinement of pressure and shear gradients to the proximity of the MB, and the introduction of endothelial texture creates local modulations of shear stress levels. With regard to safety assessments, the mechanical impact of MB breakup is shown to be more important than taking into account individual red blood cells and endothelial texture. The latter two may prove to be relevant to the actual, complex process of BBB opening induced by MB oscillations.     

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.     

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.     

Vasculogenesis in the early quail blastodisc as studied with a monoclonal antibody recognizing endothelial cells

QH1, a monoclonal antibody that recognizes quail endothelial and haemopoietic cells, was applied to quail blastodiscs in toto, in order to analyse by immunofluorescence the emergence of the vascular tree. The first endothelial cells were detected in the area opaca at the headfold stage and in the area pellucida at the 1-somite stage. Single cells then interconnected progressively, especially in the anterior intestinal portal and along the somites building up the linings of the heart and dorsal aortas. This study demonstrates that endothelial cells differentiate as single entities 4 h earlier in development than hitherto detected and that the vascular network forms secondarily. The horseshoe shape of the extraembryonic area vasculosa is also a secondary acquisition. A nonvascularized area persists until later (at least the 14-somite stage) in the region of the regressing primitive streak.     

Immunohistochemical demonstration of Angiopoietin-2 in lymphatic vascular development

The cellular expression of Angiopoietin-2 (Ang2) was studied during lymphatic development in mouse by immunohistochemistry and compared to that of lymphatic endothelial markers. At the earliest stage of lymphvasculogenesis, Prox1-identified lymphatic precursor cells of the cardinal vein displayed an intense immunoreaction for Ang2 in their cytoplasm, implying that Ang2 may adjust lymphatic specification and sprouting from the veins under the control of Prox1. Thereafter, Ang2 was constantly expressed in Prox1 and/or LYVE-1-immunopositive endothelial cells of lymphatic sacs and vessels, ranging from lymphatic capillaries to collectors, throughout embryonic and neonatal development, and the lymphatic endothelial cells simultaneously exhibited immunoreactivity to Tie2, a primary receptor for angiopoietins. These results suggest that lymphatic endothelial cells may regulate lymphatic development via their own Ang2-Tie2 signaling. Ang2 is further immunolocalized in the developing blood vessels including hepatic sinusoids, adrenal medullary vasculature and postnatal pulmonary vessels, thereby indicating that the blood vessels, which undergo vascular remodeling and sudden alteration of blood flow during the development, are also likely to express Ang2. The present study is first to demonstrate Ang2 expression in the lymphatic endothelial cells during development, and consequently Ang2 is regarded as a molecular profile of the developing lymphatic endothelial cells required for lymphatic vascular organization.     

Assembly of trunk and limb blood vessels involves extensive migration and vasculogenesis of somite-derived angioblasts

Vascular development requires the assembly of precursor cells into blood vessels, but how embryonic vessels are assembled is not well understood. To determine how vascular cells migrate and assemble into vessels of the trunk and limb, marked somite-derived angioblasts were followed in developing embryos. Injection of avian somites with the cell-tracker DiI showed that somite-derived angioblasts in unperturbed embryos migrated extensively and contributed to trunk and limb vessels. Mouse-avian chimeras with mouse presomitic mesoderm grafts had graft-derived endothelial cells in blood vessels at significant distances from the graft, indicating that mouse angioblasts migrated extensively in avian hosts. Mouse graft-derived endothelial cells were consistently found in trunk vessels, such as the perineural vascular plexus, the cardinal vein, and presumptive intersomitic vessels, as well as in vessels of the limb and kidney rudiment. This reproducible pattern of graft colonization suggests that avian vascular patterning cues for trunk and limb vessels are recognized by mammalian somitic angioblasts. Mouse-quail chimeras stained with both the quail vascular marker QH1 and the mouse vascular marker PECAM-1 had finely chimeric vessels, with graft-derived mouse cells interdigitated with quail vascular cells in most vascular beds colonized by graft cells. Thus, diverse trunk and limb blood vessels have endothelial cells that developed from migratory somitic angioblasts, and assembly of these vessels is likely to have a large vasculogenic component.     

Brain induces the expression of an early cell surface marker for blood-brain barrier-specific endothelium.

Capillaries derived from the perineural vascular plexus invade brain tissue early in embryonic development. Considerably later they differentiate into blood-brain barrier (BBB)-forming blood vessels. In the chick, the BBB as defined by impermeability for the protein horseradish peroxidase develops around embryonic day 13. We have previously found that brain endothelial cells start to express a number of proteins at around the same time, suggesting that these proteins play a role in BBB function. Here we describe a 74 kd protein defined by the monoclonal antibody HT7 that is expressed on the surface of chick embryonic blood cells and brain endothelial but on no other endothelial cells. This protein is not detectable on early embryonic brain endothelium, but is expressed by these cells on embryonic day 10. It is absent in choroid plexus endothelial cells which represent permeable fenestrated endothelial cells. The antigen is expressed on choroid plexus epithelium which is the site of the blood-cerebrospinal fluid barrier. Since it is also found in basolateral membranes of kidney tubules, it may be involved in specific carrier mechanisms. Embryonic mouse brain tissue transplanted on the chick chorio-allantoic membrane induces the expression of this antigen on endothelial cells derived from the chorio-allantois. Brain tissue can therefore induce in endothelial cells in vivo the expression of a molecule characteristic of brain endothelium.     

Transient Expression of Iron Transport Proteins in the Capillary of the Developing Rat Brain

Iron is essential for normal brain function and its uptake in the developing rat brain peaks during the first two weeks after birth, prior to the formation of the blood–brain barrier (BBB). The first step of iron transport from the blood to the brain is transferrin receptor (TfR)-mediated endocytosis in the capillary endothelial cells. However, the subsequent step from the endothelium into interstitium has not been fully described. The goal of this study was to examine the expression of iron transport proteins by immunodetection and RT–PCR in the developing rat brain. Tf and TfR are transiently expressed in perivascular NG2+ cells of the capillary wall during the early postnatal weeks in the rat brain. However, MTP-1 and hephaestin were expressed in endothelial cells, but not in the NG2+ perivascular cells. Immunoblot analysis for these iron transfer proteins in the developing brain generally confirmed the immunochemical findings. Furthermore, the expression of Tf and TfR in the blood vessels precedes its expression in oligodendrocytes, the main iron-storing cells in the vertebrate brain. RT–PCR analysis for the primary culture of endothelial cells and pericytes revealed that Tf and TfR were highly expressed in the pericytes while MTP-1 and hephaestin were expressed in the endothelial cells. The specific expression of Tf and TfR in brain perivascular cells and MTP-1 and hephaestin in endothelial cells suggest the possibility that trafficking of elemental iron through perivascular cells may be instrumental in the distribution of iron in the developing central nervous system.     

The alpha-isoform of caveolin-1 is a marker of vasculogenesis in early lung development.

Caveolin-1 is a scaffolding protein component of caveolae, membrane invaginations involved in endocytosis, signal transduction, trans- and intracellular trafficking, and protein sorting. In adult lung, caveolae and caveolin-1 are present in alveolar endothelium and Type I epithelial cells but rarely in Type II cells. We have analyzed patterns of caveolin-1 expression during mouse lung development. Two caveolin-1 mRNAs, full-length and a 5' variant that will translate mainly into caveolin-1alpha and -beta isoforms, are detected by RT-PCR at embryonic day 12 (E12) and afterwards in the developing and adult lung. Immunostaining analysis, starting at E10, shows caveolin-1alpha localized in primitive blood vessels of the forming lung, in an overlapping pattern to the endothelial marker PECAM-1, and later in all blood vessels. Caveolin-1alpha is not detected in fetal or neonatal lung epithelium but is detected in adult epithelial Type I cells. Caveolin-1 was previously shown to be expressed in alveolar Type I cells. These data suggest that expression of caveolin-1 isoforms is differentially regulated in endothelial and epithelial cells during lung development. Caveolin-1alpha is an early marker for lung vasculogenesis, primarily expressed in developing blood vessels. When the lung is fully differentiated postnatally, caveolin-1alpha is also expressed in alveolar Type I cells.     

Identification of a widely distributed 90-kDa glycoprotein that is homologous to the Hermes-1 human lymphocyte homing receptor

Homing of recirculating lymphocytes from the blood into the lymphoid tissues is mediated by 90-kDa homing receptors on the lymphocyte cell surface, allowing selective binding to specialized endothelium lining high endothelial venules. This study describes two novel mAb, NKI-P1 and NKI-P2, directed against functional epitopes of a human lymphocyte homing receptor, gp90. Biochemical studies demonstrated that these antibodies recognize a 90-kDa glycoprotein which is similar to the Ag recognized by the mAb Hermes-1. This notion was confirmed by immunohistochemical studies showing identical reaction patterns. Furthermore, it was observed that NKI-P1 and NKI-P2 blocked adhesion of lymphocytes to high endothelial venules. Immunohistochemical, immunofluorescence, and immunoprecipitation studies revealed that gp90 is widely expressed on hemopoietic cells including lymphocytes, macrophages/dendritic cells, myeloid cells, and erythrocytes. The gp90 is also expressed on a number of nonhemopoietic cells such as endothelial cells, certain epithelial cells, and fibroblasts. In addition to its expression on normal cells, gp90 is present on a spectrum of tumor cell lines of lymphoid, monocytic, epithelial, glial, and melanocytic origin. In addition to the 90-kDa product, the antibodies immunoprecipitate several polypeptides in the range of 120 to 200 kDa. Interestingly, it was observed that certain mamma tumor cell-line cells lack the 90-kDa polypeptide indicating the heterogeneous expression of the molecules recognized by the antibodies. These results indicate that the 90-kDa glycoprotein homologues of the Hermes-1 human lymphocyte homing receptor are expressed on hemopoietic tissues as well as on a number of nonhemopoietic tissues and tumor cell lines. Although the function of these molecules in nonlymphoid cells is presently unknown, they might play a role in cell-cell or cell-matrix adhesion.     

Alterations of bone marrow sinus endothelium induced by ionizing irradiation: implications in the homing of intravenously transplanted marrow cells.

The endothelium of bone marrow sinuses is a continuous layer which is selective in its cellular transport. It is not known how selective and massive seeding of hemopoietic progenitor cells after intravenous transplantation of marrow cells occurs. We postulate that the conditioning irradiation could disrupt the endothelial barrier, thus permitting the "homing" of progenitor cells to occur. To demonstrate this phenomenon, we irradiated mice with doses ranging from 100-2000 cGy-total body and studied perfusion-fixed marrow by transmission electron microscopy. The major finding was sloughing and denudation of plasma membrane, particularly on the luminal side of endothelium. Membrane vesiculation was also frequently seen in this border. Moreover, dilatation of the perinuclear space and rough endoplasmic reticulum was commonplace and testified to instability and fragility of the membrane system. Focal cytoplasmic swelling of endothelium was seen reflecting increased permissiveness of the endothelial barrier. Endocytosis and phagocytosis were increased in the marrow; and the endothelium, normally quiescent with regard to phagocytosis, was now overtly phagocytic. A dipogenecity of the adventitial layer was increased as hemopoietic function of marrow decreased. The end result of membrane alterations in the endothelium was the appearance of discontinuities in these cells, which form the essential element of bone marrow-blood barrier. Consequent to these discontinuities, the permissiveness of the endothelial barrier was enhanced and those cellular elements, such as mature, nonreticulated erythrocytes that are normally confined to the vascular space, now appeared in large number in the hemopoietic compartment. With low doses, these findings were transient and repair set in by 1-2 weeks. With higher doses, total disruption of marrow-blood barrier occurred and the process did not seem to be repairable. We conclude that the conditioning irradiation before bone marrow transplantation is essential in disrupting the endothelial barrier and permitting large-scale entry of transplanted cells into the hemopoietic compartment.     

Anticancer drugs induce necrosis of human endothelial cells involving both oncosis and apoptosis

The endothelium is the first physiological barrier between blood and tissues and can be injured by physical or chemical stress, particularly by the drugs used in cancer therapy. We found that four anticancer agents: etoposide, doxorubicin, bleomycin and paclitaxel induced apoptosis in human umbilical vein endothelial cells (HUVECs) (as judged by DNA fragmentation) with a time- and concentration-dependent decrease in bcl-2 protein but without the involvement of p53. As revealed by immunoblotting, bax protein was expressed in HUVECs treated with 1 mg/ml etoposide whereas bcl-2 protein disappeared. Oncosis occurred parallel to apoptosis with the release of lactate dehydrogenase into the supernatant, and, for doxorubicin and etoposide with the inversion of the distribution of angiotensin I-converting enzyme between supernatant and cells. Among the four tested anticancer drugs, only doxorubicin induced an oxidative stress, with significative malondialdehyde production. Thus, human endothelial cells in confluent cultures seem to be in an equilibrium of resistance to apoptosis related to bcl-2 expression; this equilibrium can be disrupted by a chemical stress, such as the antiproliferative drugs known as pro-apoptotic for tumour cells. For doxorubicin and bleomycin, this cellular toxicity can be related to their unwanted effects in human cancer therapy. Low doses of doxorubicin, paclitaxel or etoposide, however, could induce apoptosis of endothelial cells of new vessels surrounding the tumour, thus leading to specific vessel regression with minimal toxic effects for the endothelium of the other vessels. These findings provide evidence of relationships between endothelial toxicity of anticancer drugs and the key role of bcl-2 for resistance of endothelium cells toward apoptosis; moreover lack of p53 and bax in quiescent cells contributes to resistance of endothelial cells to DNA-damaging agents.     

Dysfunction of kidney endothelium after ischemia/reperfusion and its prevention by mitochondria-targeted antioxidant

One of the most important pathological consequences of renal ischemia/reperfusion (I/R) is kidney malfunctioning. I/R leads to oxidative stress, which affects not only nephron cells but also cells of the vascular wall, especially endothelium, resulting in its damage. Assessment of endothelial damage, its role in pathological changes in organ functioning, and approaches to normalization of endothelial and renal functions are vital problems that need to be resolved. The goal of this study was to examine functional and morphological impairments occurring in the endothelium of renal vessels after I/R and to explore the possibility of alleviation of the severity of these changes using mitochondria-targeted antioxidant 10-(6′-plastoquinonyl)decylrhodamine 19 (SkQR1). Here we demonstrate that 40-min ischemia with 10-min reperfusion results in a profound change in the structure of endothelial cells mitochondria, accompanied by vasoconstriction of renal blood vessels, reduced renal blood flow, and increased number of endothelial cells circulating in the blood. Permeability of the kidney vascular wall increased 48 h after I/R. Injection of SkQR1 improves recovery of renal blood flow and reduces vascular resistance of the kidney in the first minutes of reperfusion; it also reduces the severity of renal insufficiency and normalizes permeability of renal endothelium 48 h after I/R. In in vitro experiments, SkQR1 provided protection of endothelial cells from death provoked by oxygen–glucose deprivation. On the other hand, an inhibitor of NO-synthases, L-nitroarginine, abolished the positive effects of SkQR1 on hemodynamics and protection from renal failure. Thus, dysfunction and death of endothelial cells play an important role in the development of reperfusion injury of renal tissues. Our results indicate that the major pathogenic factors in the endothelial damage are oxidative stress and mitochondrial damage within endothelial cells, while mitochondria-targeted antioxidants could be an effective tool for the protection of tissue from negative effects of ischemia.     

Lymphoid tissue- and inflammation-specific endothelial cell differentiation defined by monoclonal antibodies

Endothelial cells play an essential role in immune responses by regulating the entry of leukocytes into lymphoid tissues and sites of inflammation. As an initial approach to analyzing endothelial cell specialization in relation to such immune function, we have produced monoclonal antibodies (MAB) against mouse lymph node endothelium. Three antibodies were selected: MECA-20, recognizing the endothelium of all blood vessels in lymphoid as well as non-lymphoid organs; MECA-217, which stains the endothelium lining large elastic arteries, but among small vessels is specific for post-capillary venules within lymphoid organs and tissues exposed to exogenous antigen, such as skin and uterus; and MECA-325, an antibody that demonstrates specificity for the specialized high endothelial venules (HEV) that control lymphocyte homing into lymph nodes and Peyer's patches. MECA-325 failed to stain vessels in any non-lymphoid organs tested. Immunoperoxidase studies of HEV in lymph node frozen sections, and of isolated high endothelial cells in suspensions, demonstrated that the antigens recognized by all three antibodies are expressed at the cell surface; those defined by MECA-20 and MECA-325 are also present in the cytoplasm. To study the regulation of the antigens defined by these MAB in relation to extra-lymphoid immune reactions, we assessed their expression in induced s.c. granulomas as a model for chronic inflammation. Small vessels in the granulomas were already stained by MECA-217 in the first days of development. In contrast MECA-325 detected postcapillary venules (which frequently displayed the morphologic characteristics of HEV) only from approximately 1 wk, in parallel with the development of a persistent mononuclear cell infiltrate including numerous lymphocytes. The selective appearance of the MECA-325 antigen on vascular endothelium supporting lymphocyte traffic in both lymphoid and extra-lymphoid sites suggests that this antigen may play an important role in the process of lymphocyte extravasation. The demonstration of lymphoid organ- and inflammation-specific microvascular antigens offers direct evidence for a complex specialization of endothelium in relation to immune stimuli, and supports the concept that microvascular differentiation may play an important role in local immune responses.