CD44 and tenascin play critical roles in growth and vascular development of the chick chorioallantoic membrane and are targets of cigarette smoke

Chorioallantoic membranes (CAMs) were used to determine which extracellular matrix molecules play essential roles in growth and vascular development in vivo and whether expression of critical molecules is affected by cigarette smoke exposure. Treatment of CAMs on day 5 of development with antibodies to CD44 or tenascin, but not to other matrix molecules, inhibited CAM growth and affected various aspects of blood vessel development including normal growth and branching of vessels, migration of vessels, and formation and differentiation of the capillary plexus. DNA synthesis was inhibited by antibodies to both C44 and tenascin which probably accounted for many of the phenotypic changes observed in treated CAMs. CD44 was located on all cells in day 5 CAMs, and tenascin, while present throughout the CAM, was especially abundant around large, non-migratory mesodermal blood vessels and endodermal cells that were positioned away from the direction of blood vessel migration. These data suggest that while tenascin is required for normal blood vessel migration, high levels of tenascin inhibit migration. The different distributions of CD44 and tenascin in CAMs and the observation that antibodies to either CD44 or tenascin produced similar phenotypes indicate that CD44 and tenascin were not functionally redundant. Mainstream smoke solutions, which produce a phenotype similar to that seen with anti-tenascin and anti-CD44, inhibited expression of CD44 mRNA and increased tenascin mRNA expression. 3-Ethylpyridine, a chemical in cigarette smoke that produced changes in CAM development similar to anti-CD44 and anti-tenascin treatment, also increased tenascin mRNA expression, but did not affect CD44. Together these data show that tenascin and CD44 play critical roles in early growth and vascular development of the CAM and support the idea that 3-ethylpyridine in mainstream smoke impairs CAM growth and vascular development by targeting expression of tenascin. 3-Ethylpyridine is generally regarded as safe and is used in many consumer products including food and tobacco.     

Effect of local shell conductance on the vascularisation of the chicken chorioallantoic membrane

The vascularisation of the chorioallantoic membrane (CAM) of avian embryos is influenced by environmental oxygen partial pressure (P(O(2))) on a global level: incubation at high P(O(2)) reduces the density of pre- and post-capillary vessels of the CAM and decelerates the thinning of the blood-gas barrier, and vice versa. This study investigates the effects of local P(O(2)) on vascular development during the formative period of days ten to fifteen, by making half of the egg hypoxic and the other half hyperoxic. The densities of arterioles, venules and capillaries were reduced under the hypoxic side, compared to untreated eggs, but not significantly changed on the hyperoxic side. Harmonic mean thickness of the tissue barrier and total CAM blood volume were not affected by the treatments. Vascular development of the CAM was therefore only partly influenced by local P(O(2)).     

Angiogenic activity in injured rat corneas as assayed on the chick chorioallantoic membrane

The temporal appearance of an angiogenic effect in chemically cauterized rat corneas was determined by studying the responses that they induced in the vessels of the chick chorioallantoic membrane (CAM). Injured rat corneas were grafted to the CAM from 90 minutes to 7 days after cautery. As controls, uninjured rat corneas and corneas of healthy rats cauterized immediately after death were also grafted. The vascular responses to the grafts were graded in a masked fashion by stereoscopic biomicroscopy on a five-tiered scale, by evaluations of projected colored photographs on the same scale, and by histologic examination of the grafts. Separate coefficients of angiogenesis were determined for the stereoscopic and photographic evaluations. We detected significant differences between corneas of healthy rats that were uninjured or cauterized chemically immediately after death and those that were cauterized in the living rat. Uninjured corneas and corneas cauterized postmortem elicited a mild vascular response in the CAM, as reflected by low coefficients of angiogenesis. Whereas blood vessels were not detected in corneas injured postmortem, some normal corneas vascularized but only after being on the CAM for at least 7 days. The coefficients of angiogenesis of corneas that were cauterized during life were significantly higher than those of both control groups prior to grafting after comparable times on the CAM. Corneas vascularized on the CAM included those that were cauterized as soon as 90 minutes prior to grafting. The strongest vascular responses, as reflected by coefficient of angiogenesis and the frequency of histologically confirmed nucleated avian erythrocytes within intracorneal blood vessels, were found with corneas that were grafted to the CAM 3 days after chemical cauterization. Corneas that vascularized on the CAM were associated with a prominent leukocytic infiltrate suggestively derived from the chick embryo. The results suggest that chemically cauterized rat corneas contain a chemoattractant for polymorphonuclear leukocytes within 90 minutes of injury and that such polymorphonuclear leukocytes or other components of the injured corneas possess the ability to stimulate angiogenesis on the CAM.     

Patterns of vascular sprouting in the postnatal development of the cerebral cortex of the rat

Light microscopic histochemistry for alkaline phosphatase was employed in a study of the development of vascular sprouting, with respect to time and distribution, in the rat cerebral cortex. Sprouts were counted in the full thickness of the cerebral cortex at each day from birth to 21 days of age. Several distinct bursts of sprouting activity were observed at specific times and levels of cortex. From birth to 4 days of age, sprouting was intense in the superficial third of the cortex. At 7 to 8 days, a burst of sprouting was found which was greatest in the middle third. Additional bursts of sprouting appeared at 10 and 14 days. Developing vessels with characteristics of arteries, capillaries, or sprouts were alkaline-phosphatase positive, while veins were not. It is concluded that alkaline phosphatase is a useful marker for identification of both mature and immature vasculature, as it reveals patent and nonpatent vessels, and the sprouts which are precursors of the mature vascular bed. New vessels developing in the cortex arise mainly from blind sprouts of capillaries, evidently in response to the metabolic demands imposed by the maturational process. At birth, the majority of intracortical vessels are capillaries. By 10 days of age, most perforating vessels from the surface have taken on arterial or venous characteristics. The findings are discussed in connection with morphological and biochemical differentiation and the pattern of vascularization in the mature cerebral cortex.     

鸡胚尿囊绒毛膜法研究细胞间黏附分子-1在血管生成中的作用

目的　探讨细胞间黏附分子 1(intercellularadhesionmolecule 1,ICAM 1)在血管生成中的作用。　方法采用鸡胚尿囊绒毛膜 (chorioallantoicmembrane ,CAM)法进行在体血管生成实验。　结果　 1 10d鸡胚的尿囊绒毛膜经ICAM 1作用 3d后 ,明胶海绵周围放射状走行的微血管非常明显 ,似车辐 ,显微镜下明胶海绵内有垂直长入的微血管 ,明胶海绵周边CAM间充质内微血管数目显著多于对照组 (P <0 0 1)。 2 6d鸡胚的尿囊绒毛膜经Anti ICAM 1作用 3d后 ,明胶海绵周围放射状走行的微血管极不明显 ,显微镜下明胶海绵内几乎没有新生的微血管 ,明胶海绵周边CAM间充质内微血管数目显著少于对照组 (P <0 0 1)。　结论　结果提示 1 ICAM 1有诱导微血管生成的作用 ;2 ICAM 1参与胚胎的血管生成。     

Angiogenesis in lipoma: An experimental study in the chick embryo chorioallantoic membrane

Lipoma is one of the most common benign mesenchymal tumors. Its ability to trigger an angiogenic response is a critical step for its growth. Because adipose tissue serves as an important conduit for the vasculature, it is conceivable that the angiogenic properties of this tissue may modulate the growth of the vasculature in a paracrine manner. We investigated in vivo the angiogenic potential of bioptic fragments of human lipoma by using the chick embryo chorioallantoic membrane (CAM), a useful model for such an investigation. The angiogenic response in pathological and control implants was assessed on histologic sections by a morphometric method, 96 h after grafting. Results showed that pathological samples were surrounded by numerous allantoic vessels with a radially arranged pattern around the implant. The vascular counts in the CAMs treated with lipoma implants were comparable to that of FGF-2. The role played in vasoproliferative response by angiogenic cytokines (FGF-2, VEGF) released by adipocytes, by endogenous cytokines, such as FGF-2, stored in the CAM extracellular matrix and by angiogenic growth factors released by perivascular mononuclear cells around the newly-formed blood vessels, were supported by this study.     

Early morphological changes in blood capillaries of mouse duodenal villi induced by X-irradiation

The mouse has been used extensively as a model for radiobiological studies. In particular, the cellular compartments of the intestinal villi have been examined, in an effort to gain an understanding of the gastrointestinal disturbances which follow radiotherapy of the abdomen. The response of the blood vessels has been, however, largely neglected. This paper examines the early response of the duodenal capillaries to an X-ray dose of 10 Gy using conventional light and transmission electron microscopy. The villous capillaries were examined at 6 h, 1 day and 3 days after treatment. The results showed that the capillaries responded to X-irradiation within 6 h. exhibiting marked vasodilation similar to that observed in acute inflammation. Significantly there was no ultrastructural evidence of endothelial cell disruption or loss of junctional attachment between the cells, but the characteristic fenestrae of these vessels were less apparent than in the controls. One day after treatment the capillaries had become constricted, with many vessels totally non-patent. The cytoplasm and nuclei of the endothelial cells showed changes consistent with vascular damage, such as nuclear shape alterations and luminal cytoplasmic projections. Three days after treatment there was variation in the capillary patency, as some vessels showed signs of incipient necrosis whilst others were relatively normal in appearance. The results suggest that the early vascular response typically involves a phase of vasodilation followed by constriction within the first 24 h after treatment, a finding consistent with the radiation response of skin capillaries in what has been described as 'transient erythema'. The ultrastructural changes associated with the phasic changes in patency did not suggest large scale endothelial death, but rather alteration of the functional capacity of the vessels which may in turn affect the other cell populations in the villi.     

Inhibiting MMP activity prevents the development of endometriosis in the chicken chorioallantoic membrane model

BACKGROUND: Matrix metalloproteinases (MMPs) are essential for extracellular matrix remodelling and may contribute to the development of endometriosis. Transplantation of endometrium onto the chicken chorioallantoic membrane (CAM) results in endometriosis-like lesion formation, a process that requires extensive tissue remodelling. We investigated the expression of a wide range of MMPs in menstrual endometrium, endometriosis-like lesions in CAMs, in peritoneal endometriosis and in endometriosis in the rectovaginal space, as well as the function of MMPs in early lesion formation in the CAM model. METHODS: Expression of MMPs was evaluated by immunohistochemistry and MMP function was studied in the CAM by inhibiting MMP activity during lesion formation. RESULTS: Nearly all MMPs were present in all tissues studied. No significant differences in the expression of a majority of MMPs were found in endometriosis-like lesions in CAMs when compared with human endometriosis. Inhibition of MMP-1, -2, -3, -7 and -13 activities significantly impaired endometriosis-like lesion formation in CAMs. CONCLUSIONS: The MMP expression profiles of experimentally induced endometriosis in CAMs and human endometriosis are similar. The prevention of endometriosis-like lesion formation in the CAM by inhibiting MMP activity strongly suggests that MMPs have a function in the early development of endometriotic lesions.     

B16-F10 melanoma cells contribute to the new formation of blood vessels in the chick embryo chorioallantoic membrane through vasculogenic mimicry

Grafting of mammalian cells and tissues to the chick embryo chorioallantoic membrane (CAM) is a well-established experimental system to evaluate many different parameters of tumor growth, and B16-F10 murine melanoma cell line has been successfully used to study metastatic process in the CAM assay. The aim of this study was to demonstrate the capability of B16-F10 melanoma cells to contribute to the new formation of host blood vessels through a vasculogenic mimicry mode. Results have shown that B16-F10 melanoma cells are able to form in 4 days macroscopic tumor masses and induce a strong angiogenic response comparable to that of a well-known angiogenic cytokine, namely fibroblast growth factor-2. Moreover, tumor cells are able to cross the chorionic epithelium, and to move beneath in the mesenchyme to form tumor masses immunoreactive to specific antibodies anti-S100 and anti-MART-1/Melan-A. Finally, we have shown that CAMs new-formed blood vessels are lined by both pigmented melanoma cells and cells immunoreactive to MART-1/Melan-A and PAS, suggesting the occurrence of a vasculogenic mimicry process.     

Corneal neovascularization as studied by scanning electron microscopy of vascular casts

Rat corneas were cauterized chemically, and the induced neovascularization was studied by scanning electron microscopy of vascular casts. Casts were prepared by filling the pericorneal and intracorneal vessels with an acrylic monomer by an intracardiac injection. The initial response to injury was a vasodilation of pericorneal vessels and the appearance of impressions in the walls of veins and venules consistent with those of marginating leukocytes. The first new vascular buds emerged from the pericorneal venules and capillaries at 27 hours after injury. These sprouts lengthened and multiplied by 72 hours to produce a rich anastomosing plexus. New vessels were not seen arising from arteries and arterioles. By 7 days, numerous channels reached the cautery site and, by 13 days, many of the redundant intracorneal vessels had regressed leaving large looping channels connecting either with a pericorneal artery or a vein. The casts of those continuous with the artery had surface features suggesting arterial or arteriolar differentiation, whereas the smooth surfaces of the remaining channels were consistent with those of veins or capillaries. Transmission electron microscopy confirmed this differentiation by documenting intracorneal vessels with the smooth muscle and prominent endothelial cell nuclei which characterize arteries and arterioles. Vascular casts have several advantages in the study of neovascularization. They depict the three-dimensional characteristics of new vessel formation and reflect the vascular and cellular events in the accompanying acute inflammatory response; define more readily than histologic sections the time that the first new buds appear; emphasize capillaries and venules as the predominant source of new vessels; and suggest that certain new intracorneal vessels assume morphologic features of arteries or arterioles, whereas others retain capillary or venous characteristics.     

不同鼻咽癌细胞株对鸡胚尿囊膜模型血管生成影响的比较研究

目的比较不同鼻咽癌细胞株在鸡胚尿囊膜模型上促进血管生成能力的差异。方法选用6日龄鸡胚,开窗后分别种植5-8F、6-10B及CNE-2三种鼻咽癌细胞株,三种细胞株细胞数为2×106/鸡胚者各一组,细胞数为5×105/鸡胚者各一组,另一组为对照组（PBS）,每组10个鸡胚。孵育6日后,统计分析新生血管数及血管面积/鸡胚面积比。结果种植细胞数为5×105/鸡胚时,5-8F、6-10B细胞部分鸡胚有移植瘤形成,CNE-2细胞鸡胚均未见成瘤。种植细胞数为2×106/鸡胚时,三种鼻咽癌细胞株均可100%成瘤,其新生血管数依次递减,分别为（38.7±2.50）、（33.5±4.43）、（29.7±2.71）,差异有统计学意义（P〈0.05）;血管面积/鸡胚面积比分别为（22.2±2.18）%、（18.7±2.45）%、（16.9±2.62）%,均高于对照组的（9.5±1.86）%,差异有统计学意义（P〈0.05）,且5-8F面积比高于其他两种细胞株（P〈0.05）。结论鼻咽癌5-8F、6-10B及CNE-2三种细胞株在鸡胚尿囊膜模型上血管生成能力依次减弱,实验研究可根据实际情况选用。     

The expression of leukocyte-endothelial adhesion molecules is increased in perennial allergic rhinitis.

Accumulating evidence supports the importance of leukocyte-endothelial cell adhesion molecule (CAM) expression as an initiating process in tissue inflammation. To investigate the relevance of CAM expression to allergic airways inflammation, nasal biopsies from patients with perennial allergic rhinitis (n = 8) and from nonatopic healthy volunteers (n = 8) were immunostained with monoclonal antibodies directed against the CAMs, intercellular adhesion molecule-1 (ICAM-1), endothelial cell adhesion molecule-1 (ELAM-1), and vascular cell adhesion molecule-1 (VCAM-1). The endothelial staining of these CAMs was related to the number of vessels within each biopsy, delineated by a monoclonal antibody against Ulex europaeus-1 lectin bound to endothelial cells, and to the number of tissue leukocytes staining for one of the ligands of ICAM-1, the beta 2 integrin, lymphocyte function-associated antigen (LFA-1). Expression of CAMs was related to the number of infiltrating neutrophils, eosinophils, and lymphocytes identified immunohistochemically within the biopsies. ICAM-1 was the most prominent CAM present on the endothelium of the normal nasal mucosa, with less expression of ELAM-1 and only minimal or absent expression of VCAM-1. In perennial rhinitis, both ICAM-1 (P less than 0.05) and VCAM-1 (P less than 0.01) expression on endothelial cells were increased and were positively correlated in their level of expression (P less than 0.002). The number of tissue LFA-1-positive cells was significantly greater (P less than 0.05) in the biopsies from the perennial rhinitics (median, 27.3/mm2) than from the healthy controls (median, 5.3 cells/mm2). LFA-1 expression significantly correlated with the number of ICAM-1-positive vessels (P less than 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of vascular endothelial cell growth factor gene transfer to enhance implantable sensor function in vivo

In the current study, we developed and validated a simple, rapid and safe in vivo model to test gene transfer and sensor function in vivo. Using the model, we tested the specific hypothesis that in vivo gene transfer of angiogenic factors at sites of biosensor implantation would induce neovascularization surrounding the sensor and thereby enhance biosensor function in vivo. As the in vivo site for testing of our gene transfer cell and biosensor function systems, the developing chorioallantoic membrane (CAM) of the embryo was utilized. Vascular endothelial cell growth factor (VEGF) was used as a prototype for angiogenic factor gene transfer. A helper-independent retroviral vector derived from Rous sarcoma virus (RSV), designated RCAS, was used for gene transfer of the murine VEGF (mVEGF) gene (mVEGF:RCAS) into the DF-1 chicken cell line (designated mVEGF:DF-1). Initially, the ability of VEGF:DF-1 cells to produce VEGF and RCAS viral vectors containing the mVEGF gene (mVEGF:RCAS) was validated in vitro and in vivo, as was the ability of the mVEGF:DF-1 cells to induce neovascularization in the ex ova CAM model. Using the system, we determined the ability of mVEGF:DF-1 cells to enhance acetaminophen sensor function in vivo, by inducing neovascularization at sites of sensor implantation in the ex ova CAM model. For these studies, acetaminophen sensors were placed on 8-day-old ex ova CAMs, followed by addition of media or cells (mVEGF:DF-1 cells or GFP:DF-1 cells) at the sites of biosensor implantation on the CAM. At 4 to 10 days after sensor placement, the biosensor function was determined by measuring sensor response to an intravenous injection of acetaminophen. Sensors implanted on CAMs with buffer or control cells (GFP:DF-1 cells) displayed no induced neovascularization around the sensor and had minimal/baseline sensor responses to intravenous acetaminophen injection (media, 133.33 +/- 27.64 nA; GFP:DF-1, 187.50 +/- 55.43 nA). Alternatively, the sensors implanted with mVEGF:DF-1 cells displayed massive neovascularization and equally massive sensor response to intravenous injection of acetaminophen (VEGF:DF-1, 1387.50 +/- 276.42 nA). These data clearly demonstrate that enhancing vessel density (i.e., neovascularization) around an implanted sensor dramatically enhances sensor function in vivo.     

Engineered endothelial cell adhesion via VCAM1 and E-selectin antibody-presenting alginate hydrogels

Materials that adhere to the endothelial cell (EC) lining of blood vessels may be useful for treating vascular injury. Cell adhesion molecules (CAMs), such as endothelial leukocyte adhesion molecule-1 (E-selectin) and vascular cell adhesion molecule-1 (VCAM1), modulate EC-leukocyte interactions. In this study, we mimicked cell-cell interactions by seeding cells on alginate hydrogels modified with antibodies against E-selectin and VCAM1, which are upregulated during inflammation. ECs were activated with interleukin-1α to increase CAM expression and subsequently seeded onto hydrogels. The strength of cell adhesion onto gels was assessed via a centrifugation assay. Strong, cooperative EC adhesion was observed on hydrogels presenting a 1:1 ratio of anti-VCAM1:anti-E-selectin. Cell adhesion was stronger on dual functionalized gels than on gels modified with anti-VCAM1, anti-E-selectin or the arginine-glycine-aspartic acid (RGD) peptide alone. Anti-VCAM1:anti-E-selectin-modified hydrogels may be engineered to adhere the endothelium cooperatively.     

Aquaporin-1 expression in the chick embryo chorioallantoic membrane

The chick embryo chorioallantoic membrane (CAM) is commonly used in vivo to study both angiogenesis and anti-angiogenesis. Rapid membrane water transport is mediated by a family of molecular water channels, called aquaporins (AQPs), which have been identified in the epithelial and endothelial cells of higher vertebrates. AQP1, expressed in adsorptive and secretory epithelia, is also expressed in endothelial cells of capillaries and arteries. Its mRNA has been found in vascular smooth muscle cells (VSMCs) of arteries and capillaries, as well as in a subset of VSMCs of human atherosclerotic plaques. This study investigated the developmental expression of AQP1 in the chick CAM by Western blot and immunohistochemistry. Western blot results show that a major nonglycosylated band was observed with electrophoretic mobility of approximately 28 kDa in the three developmental stages examined. Immunohistochemistry data demonstrate that AQP1 was clearly expressed in the ectodermal and endodermal epithelia, the vascular endothelium, and the VSMCs. Because little information is available on the behavior of microvessel AQP1 during angiogenesis in normal and pathological conditions, our data relative to the pattern of expression of AQP1 in CAM blood vessels in normal conditions may be considered a useful tool to further investigate its modifications in several experimental conditions implying a stimulation or an inhibition of angiogenesis in the CAM assay.     

Epoxides and soluble epoxide hydrolase in cardiovascular physiology

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites that importantly contribute to vascular and cardiac physiology. The contribution of EETs to vascular and cardiac function is further influenced by soluble epoxide hydrolase (sEH) that degrades EETs to diols. Vascular actions of EETs include dilation and angiogenesis. EETs also decrease inflammation and platelet aggregation and in general act to maintain vascular homeostasis. Myocyte contraction and increased coronary blood flow are the two primary EET actions in the heart. EET cell signaling mechanisms are tissue and organ specific and provide significant evidence for the existence of EET receptors. Additionally, pharmacological and genetic manipulations of EETs and sEH have demonstrated a contribution for this metabolic pathway to cardiovascular diseases. Given the impact of EETs to cardiovascular physiology, there is emerging evidence that development of EET-based therapeutics will be beneficial for cardiovascular diseases.     

Capillary, mast cell and connective tissue response to myxoviruses in the rat

Summary Myxoviruses injected intradermally into rats provoked activation of capillary endothelium, vasodilatation, increased capillary permeability, edema formation and severe disruption of the capillary wall without mastcell disruption, as visualized by the transparent skin techniques. Minute amounts of histamine and other stimuli, applied at the same site of the skin, further aggravated the vascular response to viruses and disrupted the perivascular mast cells. Neuraminidase, N-acetyl neuraminic acid, hyaluronidase and hyaluronic acid had a similar effect on the minute vessels, but there was no mast-cell disruption when histamine was injected at the same sites. Nasal discharge collected from a vasomotor rhinitis case, had no damaging effect on the capillaries. Nasal discharge from common cold cases, collected during the first three days of the onset provoked the same vascular damage as the myxoviruses. Samples collected at later periods did not effect the morphology of the capillaries. Vascular damage produced by the myxoviruses was not inhibited by levomepromazine, butazolidin or hydrocortisone.This study was partially supported by the Ministry of Health of the Province of Quebec (Federal-Provincial grants for public health research).     

Histologic analysis of an immune response in the lung parenchyma of mice. Angiopathy accompanies inflammatory cell influx.

To determine the histologic changes occurring during a pulmonary immune response, the lungs of antigen-primed C57BL/6 mice were examined on various days after intratracheal challenge with 10(8) sheep erythrocytes. The response was characterized by 1) dense perivascular aggregates composed largely of mononuclear cells; 2) endothelial cell hypertrophy and subendothelial inflammatory cell collections in vessels of a variety of sizes; 3) variable degrees of focal, reversible vascular injury (angiopathy) of both muscular arteries and small veins; and 4) increased cellularity of alveolar walls. Inflammatory cells appeared to emanate from small veins and venules and from minute thin-walled vessels adjacent to large arteries. The reaction peaked at 3 to 4 days and then gradually declined over a period of 6 weeks, never quite reaching baseline. We believe that this experimental model will be an important means of further defining both the mechanisms of lymphocyte entry to the lungs in response to antigen and the factors controlling the pathogenesis of related angiopathies.     

Vascular morphogenesis and differentiation after adoptive transfer of human endothelial cells to immunodeficient mice

To establish a model for adoptive transfer of endothelial cells, we transferred human umbilical vein endothelial cells (HUVECs) to immunodeficient mice (Rag 2(-/-)). HUVECs were suspended as single cells in Matrigel and injected subcutaneously in the abdominal midline. Within 10 days after injection, HUVECs expressed pseudopod-like extensions and began to accumulate in arrays. By day 20, we observed human vessels that contained erythrocytes, and on day 30 we observed perivascular cells that expressed smooth muscle actin, thus resembling mature vessels. Throughout the experimental period, HUVECs bound Ulex europaeus lectin and expressed CD31, VE-cadherin, von Willebrand factor, as well as ICAM-2. A fraction of the cells also expressed the proliferation marker Ki67. Moreover, the sialomucin CD34, which is rapidly down-regulated in cultured HUVECs, was reinduced in vivo. However, we found no reinduction of CD34 in cells cultured inside or on top of Matrigel in vitro. We also injected cells suspended in Matrigel around the catheter tip of implanted osmotic pumps. Delivery of recombinant human interferon-gamma by this route led to strong induction of MHC class II and ICAM-1 on the human vessels. In conclusion, isolated human endothelial cells can integrate with the murine vascular system to form functional capillaries and regain in vivo properties.     

Endogenous nitric oxide and epoxyeicosatrienoic acids modulate angiotensin II-induced constriction in the rabbit afferent arteriole

Nitric oxide (NO) and epoxyeicosatrienoic acids (EETs), cytochrome P450 epoxygenase metabolites of arachidonic acid, are released by the vascular endothelium and play important roles in the control of glomerular haemodynamics. We examined whether endogenous NO or EETs modulate angiotensin II- (AngII) induced constriction in isolated microperfused afferent arteriole (Af-Art) of the rabbit kidney. When Af-Arts were treated with NG-nitro-L-arginine methyl ester (L-NAME, an inhibitor of NO synthese; 10-4 mol L-1) or miconazole (an inhibitor of P450 epoxygenase; 10-6 mol L-1), basal diameter was decreased by 34.5 +/- 2.2 and 13.9 +/- 3.2%, respectively. AngII added to both the bath and lumen decreased the diameter of Af-Arts in a dose-dependent manner. Pretreatment with either L-NAME or miconazole also augmented the constrictor response to AngII. AngII at 10-8 mol L-1 decreased the diameter to 39.2 +/- 1.4, 32.9 +/- 3.6, and 12.7 +/- 4.6%, in control, L-NAME-, and miconazole-treated group, respectively. In order to study whether the AngII type2 (AT2) receptor modulates AngII action via NO or EETs, we repeated the experiments in the presence of PD123319 (an AT2 receptor antagonist; 10-7 mol L-1). In the presence of PD123319, L-NAME still augmented the constrictor response to AngII, however, miconazole had no effect. In the presence of PD123319, AngII at 10-8 mol L-1 decreased the diameter to 25.0 +/- 4.6, 9.4 +/- 4.0, and 26.0 +/- 3.3%, in control, L-NAME-, and miconazole-treated group, respectively. These results suggest that (1) tonic release of NO and EETs attenuates the vasoconstrictor response to AngII in Af-Arts and (2) AT2 receptor seems to be coupled to EETs rather than the NO pathway.