Proximal atherosclerotic lesion as a cause of very late stent thrombosis

Very late stent thrombosis is defined as in-stent thrombosis occurring after 1 year of an intra-coronary artery stent placement. Drug eluting stents have lately been criticized for increased reports of very late stent thrombosis. The exact cause of these very late stent thromboses is not clearly understood. Virchow's triad describes the three main factors of thrombus formation to be stasis of blood flow, endothelial injury and hypercoagulability. Based on Virchow's triad, we propose the cause of very late stent thrombosis to be formation of a de novo atherosclerotic lesion in the proximal segment of a stented artery. The de novo atherosclerotic lesion narrows the vessel lumen and causes stasis of blood flow in the distal stent. The de novo lesion can also cause myocardial ischemia creating a prothrombotic environment in the stented region. Stasis of blood flow and prothrombotic environment in the stented region can lead to the formation of very late stent thrombosis. Since atherosclerosis is a dynamic aging process in humans, we propose de novo proximal lesions in the coronary arteries can predispose to very late stent thrombosis.     

兔股动脉结扎诱导动脉生成过程中iNOS和eNOS的表达

目的:研究兔后肢动脉生成过程中诱导型一氧化氮合酶(iNOS)和内皮型一氧化氮合酶(eNOS)表达特征.方法:将兔一侧股动脉结扎,另一侧设为对照组.1周后动物被处死.应用免疫荧光组织化学技术检测侧支血管中iNOS及eNOS的表达.用Leica激光共聚焦显微镜观察并拍照.图片用silicongraphicsoctane进行处理.结果:在正常小动脉血管中iNOS的表达很低,在生长的侧支血管iNOS的表达显著上调,是正常小动脉血管的2.8倍.其表达在血管壁的各层可见.正常小动脉血管eNOS的表达呈现一定基础水平,在生长的侧支血管中eNOS的表达呈强阳性,集中在内皮细胞,是正常小动脉血管的2.2倍.结论:侧支血管发育过程中iNOS的表达上调,上调的iNOS和eNOS可能通过生成NO,调节内皮细胞的增殖、移动及炎症的形成,从而对侧支血管的生长发挥重要作用.     

Hemodynamic effect of Neuroform stent on intimal hyperplasia and thrombus formation in a carotid aneurysm

Stents play an important role in management of cerebral aneurysms. A stent reconstructs the parent artery, assists coil embolization, and decreases flow activity within an aneurysm. However, an in-stent stenosis often occurs within the stented artery and compromises the circulation at the parent artery. Hemodynamic basis of re-stenoses from aneurysm stenting is not fully understood. An 8mm cavernous carotid aneurysm with a wide neck was treated by a Neuroform stent first and by coils five weeks later. A comparison of the difference in morphology during this five-week period reveals the presence of intimal hyperplasia in the internal carotid artery, 1.3mm thick at the proximal end and 1mm at the distal end of the stent, and 1mm thick thrombus at the postero-inferior side of the aneurysm. Computational fluid dynamic analyses show that the site of intimal hyperplasia is exposed to low wall shear with high oscillatory shear index (OSI), and the location of thrombus is subject to high OSI. Intimal hyperplasia and thrombus occur at comparable rates, but at different hemodynamic conditions; however, both prefer regions with high OSIs.     

罗格列酮对改善糖尿病大鼠心肌微血管内皮细胞功能的影响

目的探讨罗格列酮对糖尿病大鼠心肌微血管内皮细胞功能的影响。方法腹腔注射小剂量链脲佐菌素结合高能量饲料制备2型糖尿病大鼠模型，随机分为未治疗组与治疗组：罗格列酮4mg／（kg·d）。16周后定量测定血管内皮损伤标志物：可溶性血管内皮细胞蛋白C受体（sEPCR）、可溶性血栓调节蛋白（sTM）、血管性血友病因子（VWF）和一氧化氮（NO）的血浆浓度。分光光度法及免疫组化法分别测定心肌组织内NO浓度、NO合酶（NOS）的活性及内皮型NO合酶（cNOS）在微血管内皮细胞的表达。结果罗格列酮治疗组血糖及血浆中sEPCR、sTM、vWF显著低于未治疗组，但高于非糖尿病对照组。与未治疗组比较，罗格列酮治疗组心肌组织中NO、结构型NOS（cNOS）含量增高，eNOS阳性反应产物量增多，而诱导型NOS（iNOS）含量降低。结论罗格列酮可以降低糖尿病大鼠血糖，减轻内皮细胞损伤与改善心肌微血管内皮细胞功能，有助于减少糖尿病时心肌微血管病的发生与发展。     

A Simplified Murine Intimal Hyperplasia Model Founded on a Focal Carotid Stenosis

Murine models offer a powerful tool for unraveling the mechanisms of intimal hyperplasia and vascular remodeling, although their technical complexity increases experimental variability and limits widespread application. We describe a simple and clinically relevant mouse model of arterial intimal hyperplasia and remodeling. Focal left carotid artery (LCA) stenosis was created by placing 9-0 nylon suture around the artery using an external 35-gauge mandrel needle (middle or distal location), which was then removed. The effect of adjunctive diet-induced obesity was defined. Flowmetry, wall strain analyses, biomicroscopy, and histology were completed. LCA blood flow sharply decreased by ∼85%, followed by a responsive right carotid artery increase of ∼71%. Circumferential strain decreased by ∼2.1% proximal to the stenosis in both dietary groups. At 28 days, morphologic adaptations included proximal LCA intimal hyperplasia, which was exacerbated by diet-induced obesity. The proximal and distal LCA underwent outward and negative inward remodeling, respectively, in the mid-focal stenosis (remodeling indexes, 1.10 and 0.53). A simple, defined common carotid focal stenosis yields reproducible murine intimal hyperplasia and substantial differentials in arterial wall adaptations. This model offers a tool for investigating mechanisms of hemodynamically driven intimal hyperplasia and arterial wall remodeling.Arterial occlusive disease remains a major health issue in developed nations, affecting approximately 35% of the adult population in the United States.^1–3 Various open and endovascular procedures are currently used to treat occlusive lesions; however, postprocedure intimal hyperplasia and vascular wall maladaptation in treated arteries continue to plague these interventions in as many as 50% of patients, leading to recurrent end-organ ischemia.^4–6To understand the mechanisms of intimal hyperplasia and arterial wall remodeling, many animal models (eg, pig, rabbit, rat, and mouse) have been generated using chemical, mechanical, and electrical approaches to induce arterial injury. The mouse species holds great appeal because of advanced genetic knowledge. Murine common carotid artery (CCA) blood flow cessation via complete ligation and flow restriction via outflow branch ligation models have been popular approaches in the investigation of neointimal hyperplasia and vascular remodeling.^7–9 In the complete ligation model, blood flow in the left carotid artery (LCA) is disrupted by a ligature near the bifurcation; at 4 weeks, this manipulation results in blood flow cessation, substantial luminal area reduction, extensive smooth muscle cell–dominant intimal hyperplasia, and matrix deposition.⁷ Though technically simple, this model lacks direct clinical relevance. In contrast, the outflow branch ligation model requires advanced microsurgical skills and yields only limited intimal hyperplasia.⁹Combining the simplicity of complete carotid artery ligation with the hemodynamic principles of outflow branch ligation, we hypothesized that flow perturbation via a high-grade focal stenosis would yield arterial intimal hyperplasia and arterial wall remodeling. Herein, we detail the technical aspects and the physiologic and biological outcomes of this model.     

Carotid endarterectomy for symptomatic complete occlusion of the internal carotid artery

We described 9 consecutive patients who underwent operative carotid artery exploration with attempted carotid endarterectomy (CEA) for symptomatic internal carotid artery (ICA) occlusion. Indications for this surgery based on vascular imaging included segmental occlusion of the proximal ICA and also extensive occlusion of the distal ICA in selected patients in whom color-flow duplex ultrasound showed a poorly echogenic or anechoic thrombus with a flow void, suggestive of an acute thrombus. CEA was performed successfully to restore blood flow in all 9 patients:CEA in 5 and CEA with Fogarty thrombectomy in 4. Postoperative magnetic resonance (MR) angiography confirmed that revascularization had been successful in all 9 patients, and MR imaging displayed improved perfusion in 4 patients. Despite the lack of a generalized efficacy of surgical revascularization for symptomatic ICA occlusion, our study demonstrated that preoperative vascular imaging allows the selection of patients who may benefit from CEA.     

Short- and long-term treatment with folic acid suppresses thrombus formation in atherogenic mice in vivo

In the present study, we examined the effects of short- and long-term treatment with folic acid (FA) on thrombus formation in vivo in atherogenic mice to explore a novel agent for the prevention of atherothrombotic disease. Apolipoprotein E and low-density lipoprotein receptor double deficient (ApoE^−/−LDLR^−/−) mice were orally administrated a single bolus of FA (20 mg/kg) or fed an atherogenic diet with or without FA (0.02, 0.5, and 1.5 mg/kg) for 12 weeks. Thrombus formation and endothelial function were assessed in vivo using the He–Ne laser-induced carotid artery thrombus formation test and the flow-mediated vasodilation method. Platelet reactivity was assessed ex vivo using haemostatometry. Short-term treatment with FA markedly increased plasma folate levels and significantly suppressed laser-induced thrombus formation in apoE^−/−LDLR^−/− mice. Short-term treatment with FA suppressed platelet reactivity in apoE^−/−LDLR^−/− mice, but FA treatment did not affect endothelial function or plasma homocysteine levels. Long-term treatment with FA increased plasma folate levels dose-dependently. Thrombus formation and endothelial dysfunction were suppressed by treatment with 0.5 and 1.5 mg/kg of FA, respectively, but not with 0.02 mg/kg of FA, whereas platelet reactivity was not altered by treatment with any dose of FA. Long-term treatment with all doses of FA decreased the plasma homocysteine levels in apoE^−/−LDLR^−/− mice, although this result was not consistent with its anti-thrombotic action. In conclusion, our data showed that short- and long-term treatment with FA could suppress in vivo thrombus formation in an atherogenic setting, independent of its hypohomocysteinemic action.     

Determination of nitric oxide synthase activity and apoptosis of germ cells in different obstruction models

We aimed to determine the changes of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) immunoreactivity and apoptosis after proximal and distal obstruction models on ipsilateral and contralateral testicular tissues. Male albino Wistar rats were randomly divided into three groups (n=30): a control group which underwent sham operations (n=10), a unilateral vasal ligation (n=10) and a unilateral epididymal ligation group (n=10). iNOS and eNOS distribution and apoptosis were studied in both ipsilateral and contralateral testes using quantitative immunohistochemistry. Nitric oxide synthase activity was significantly affected in ipsilateral and contralateral testes cells after vasal and epididymal ligation. eNOS immunoreactivity increased markedly after ipsilateral vasal ligation (ILVL). Degeneration-related changes were also associated with changes in apoptotic rate. Analysis using the terminal dUTP nick end-labeling TUNEL method revealed that apoptotic cell numbers significantly increased after ILVL. p53 and bcl-2 immunoreactivity increased in both experimental groups compared with the sham-operated group. Changes in iNOS and eNOS immunolocalisation were strongly associated with cell damage, because germ cell degeneration was more prominent in the ILVL group. Altered p53 immunolocalisation was also associated with cell degeneration, and a rise in bcl-2 immunoreactivity might be considered to reflect a protective mechanism in the testis. These cellular changes could enlighten understanding of the interaction between testicular functioning and damage.     

硫化氢对FeCl_3诱导的小鼠颈动脉血栓模型中凝血和纤溶活性的影响

目的:探讨外源性硫化氢(H_2S)对三氯化铁(FeCl_3)诱导的小鼠颈动脉血栓模型凝血和纤溶活性的影响。方法:本实验分为空白对照组、模型组、不同浓度(12.5、25和50μmol/kg)的硫氢化钠(NaHS,H_2S供体)处理组和30 mg/kg氯吡格雷(clopidogrel,阳性对照)。不同浓度NaHS腹腔注射及硫酸氢氯吡格雷灌胃给药处理3d后,采用10%FeCl_3诱导小鼠颈动脉血栓模型。取各组颈动脉制成冰冻切片后,HE染色观察血栓形成及血管病理变化;对形成的血栓重量进行测量,计算血栓形成抑制率;采用血凝仪检测血浆凝血酶原时间(PT)、活化部分凝血活酶时间(APTF)、纤维蛋白原(FIB)和纤维蛋白降解产物(FDP)的变化;ELISA法检测血浆中血栓烷素B_2(TXB_2)、6-酮-前列腺素F_(1α)(6-keto-PGF_(1α))和纤溶酶原活化抑制剂(PAI)的含量。结果:与模型组相比,NaHS剂量依赖性地抑制小鼠颈动脉血栓的形成;NaHS处理可延长血栓模型中PT和APTT,减少FIB含量,增加FDP含量;NaHS处理能够降低颈动脉血栓模型中TXB_2及PAI含量,恢复6-keto-PGF_(1α)含量,并且6-keto-PGF_(1α)/TXB_2与血栓重量呈负相关。结论:硫化氢通过抑制机体凝血功能并激活纤溶活性而抑制FeCl_3诱导的颈动脉血栓形成。     

一种颈总动脉部分结扎-内膜新生模型的制备

目的 探讨一种左颈总动脉部分结扎后诱导血管重构（内膜新生）的方法。方法 8周龄雄性C57BL/6 J小鼠40只,随机分为6组：正常组（WT）、假手术组（sham）、术后4周、8周、10周和12周组。手术结扎左颈总动脉（LCA）远心端4个分支中的颈内、颈外和枕动脉,保留甲状腺上动脉,造成左颈总动脉血管内膜新生和血管重构。术后观察小鼠体重和饲料的消耗情况;HE染色观察血管形态学变化;天狼猩红染色观察血管胶原纤维聚集情况。结果 除正常组小鼠外,其他组小鼠术后1～2 d体重和饮食量均降低,第3天开始上升,假手术组小鼠第2天开始上升;HE染色表明,假手术组和正常组小鼠LCA均未形成新生内膜,部分结扎手术后4、 8、10和12周组小鼠LCA均出现内膜和中膜层的增厚、管腔狭窄等。右侧未结扎的颈总动脉无内膜增厚,但出现了管腔增大;天狼猩红染色显示,内膜和中膜增生部分有胶原积聚。结论 左颈总动脉部分结扎可以建立小鼠动脉内皮损伤模型并伴有明显的内膜新生。     

The effect of myocardial bridging of the coronary artery on vasoactive agents and atherosclerosis localization

The relationship between alterations in the immunohistochemical expression of three vasoactive agents [endothelial nitric oxide synthase (eNOS), endothelin-1 (ET-1), and angiotensin-converting enzyme (ACE)] and the occurrence human atherosclerosis was investigated in relation to the myocardial bridge (MB) of the left anterior descending coronary artery (LAD), an anatomical site that experiences increased shear stress. Five millimetre cross-sections of LADs with MB from 22 autopsied cases were taken from the left coronary ostium to the cardiac apex and were immunohistochemically stained with antibodies against eNOS, ET-1, and ACE. The extent of atherosclerosis in each section was calculated using the atherosclerosis ratio (intimal cross-sectional area/medial cross-sectional area) determined by histomorphometry. The results were analysed according to their anatomical location relative to the MB, either proximal, beneath, or distal. The extent of atherosclerosis was significantly lower beneath the MB, compared with proximal and distal segments. The expression of eNOS, ET-1, and ACE was also significantly lower beneath the MB. The expression of these agents correlated significantly with the extent of atherosclerosis. Because nitric oxide, after its production by eNOS, is believed to be degraded by superoxide radicals, the effect of eNOS expression on atherosclerosis remains controversial. However, the present findings clearly indicate that the expression of ET-1 and ACE is directly related to the development of human coronary atherosclerosis in vivo through shear stress.     

Methionine diet-induced hyperhomocysteinemia accelerates cerebral aneurysm formation in rats

Background and purpose: The pathophysiology of cerebral aneurysms (CA) is linked to chronic inflammation. Endothelial damage is one of the first changes in CA walls resulted from inflammation. It has been shown that increase in plasma homocysteine (Hcy) impairs vascular endothelium and correlates with the development of atherosclerosis. However, the effect of hyperhomocysteinemia (HHcy) on the formation of cerebral aneurysm remains unknown. Methods: Male Sprague–Dawley rats examined for developing cerebral aneurysms after surgical induction in the presence and absence of hypercysteinemia induced by a high l-methionine diet (1 g/kg/d). Aneurysms developed at the anterior cerebral–olfactory artery bifurcation were classified as 4 stages from no abnormality to saccular aneurysm. Plasma homocysteine levels and expression of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), matrix metalloproteinase-2 (MMP-2), and MMP-9 in aneurysmal walls was examined and correlated with CA formation 3 months after surgery. Results: Methionine diet significantly increased plasma homocysteine levels, accelerates CA formation after ligation of the left common carotid artery. Expression of VEGF, iNOS, MMP-2, and MMP-9 in aneurysmal walls was also increased by methionine treatment. Conclusion: Hyperhomocysteinemia accelerates cerebral aneurysm formation, potentially through differential effects on expression of molecules critical for vascular wall modeling in a rat model.     

The hemodynamic effect of bilateral carotid artery ligation and the morphometry of the main communicating circuit in normotensive and spontaneously hypertensive rats

After reducing the number of patent conduit arteries to the brain by bilateral ligation of the carotid artery, the percentage decrease in blood pressure from the aorta to the internal carotid artery distal to the ligation was larger in spontaneously hypertensive rats than in normotensive rats. The pressure drop corresponded to the degree of hypertension as well as to morphometrically determined structural arterial alteration in the main communicating circuit, i.e. larger media to internal radius ratio and smaller internal radius in the posterior communicating arteries, the proximal part of the posterior cerebral arteries, the basilar artery and the vertebral arteries. The discrepancy between the sum of the luminal cross sectional areas of the communicating circuit and the luminal areas of the ligated conduit arteries was larger in the hypertensive than in the normotensive rats. It is to be expected that occlusion of conduit arteries to the brain will have a larger impact on the cerebral arterial perfusion pressure head in the presence of such hypertensive structural alterations known to increase flow resistance.     

高血压与低切应力对大鼠颈总动脉血管重建的影响

目的研究高血压与低切应力对血管重建的影响及其机制,这对于阐明血管疾病的发病机理以及提供诊断、治疗的一些基本原理都将有重要的理论和实际意义。方法通过腹主动脉缩窄,结扎左颈总动脉的部分分支建立高血压、左颈总动脉低切应力以及高血压伴有低切应力大鼠动物模型。几何形态学方法观测左颈总动脉的壁厚及壁厚/内径比的变化;金属蛋白酶谱法分析MMP-2活性;免疫印迹法检测信号通路分子p-Akt分子以及Rho GDIα的表达变化。结果高血压和低切应力均可诱导颈总动脉MMP-2活性和壁厚及壁厚/内径比显著增加;当高血压伴有低切应力时,两者的协同作用诱导颈总动脉MMP-2活性和壁厚及壁厚/内径比进一步增加,从而促进血管重建。低切应力可诱导颈总动脉p-Akt的表达水平,且与低切应力大小相关,切应力低,p-Akt的表达水平高。当高血压伴有低切应力时,两者的协同作用诱导p-Akt的表达水平进一步增加。高血压和低切应力可诱导颈总动脉RhoGDIα表达增加;当高血压伴有低切应力时,两者的协同作用诱导颈总动脉Rho GDIα表达进一步增加。结论高血压与切应力协同作用对血管重建的影响最为显著,Akt和Rho GDIα信号通路参与了高血压与低切应力诱导的血管重建过程。     

Craniofacial growth influenced by blood supply. 1. Collaterals and anastomoses following unilateral ligation of the common carotid artery

The influence of the blood supply on craniofacial growth was investigated in 396 albino rats (Rattus norvegicus Berkenhout). This was done by blocking the right common carotid artery of animals aged 42 d. The rats were slaughtered at previously specified intervals after the operation. The ligature led to dilation of the other major arteries of the neck. Numerous collaterals and anastomoses were also formed. These occurred on the ligature side in the flow region between the proximal and distal stumps of the right common carotid artery, between the flow regions of the right external carotid artery and the right vertebral artery and between the flow regions of the right subclavian artery and the right external carotid artery. Further anastomoses arose between the arteries of the other side, i.e. the rostral thyroid artery, lingual artery, submental artery and labial mandibular artery.     

A computational model based on fibrin accumulation for the prediction of stasis thrombosis following flow-diverting treatment in cerebral aneurysms

Flow diverters, the specially designed low porosity stents, have been used to redirect blood flow from entering aneurysm, which induces flow stasis in aneurysm and promote thrombosis for repairing aneurysm. However, it is not clear how thrombus develops following flow-diversion treatment. Our objective was to develop a computation model for the prediction of stasis-induced thrombosis following flow-diversion treatment in cerebral aneurysms. We proposed a hypothesis to initiate coagulation following flow-diversion treatment. An experimental model was used by ligating rat’s right common carotid artery (RCCA) to create flow-stasis environment. Thrombus formed in RCCA as a result of flow stasis. The fibrin distributions in different sections along the axial length of RCCA were measured. The fibrin distribution predicted by our computational model displayed a trend of increase from the proximal neck to the distal tip, consistent with the experimental results on rats. The model was applied on a saccular aneurysm treated with flow diverter to investigate thrombus development following flow diversion. Thrombus was predicted to form inside the sac, and the aneurysm was occluded with only a small remnant neck remained. Our model can serve as a tool to evaluate flow-diversion treatment outcome and optimize the design of flow diverters.     

Endothelial nitric oxide synthase is differently expressed in human endometrial vessels during the menstrual cycle

Endogenous nitric oxide (NO) can be synthesized by endothelial cells and can act as a potent vasodilator. We investigated endothelial nitric oxide synthase (eNOS), one of the three different enzymes responsible for the synthesis of NO by immunohistochemical methods throughout the menstrual cycle on 34 endometrial samples and compared its detection with the von Willebrand Factor (vWF) as a reliable marker molecule of the endothelium on serial sections. Immunoreactivity for eNOS was clearly localized in various types of arterial and venous endothelial cells as well as in capillaries. In addition, in some samples there was a positive staining in endometrial glandular epithelium. There was no staining in endometrial fibroblasts or in myometrial smooth muscle cells. Whereas the endothelium was constantly stained by the monoclonal antibody against vWF, eNOS was not always expressed in the endothelial lining of the vessels during the menstrual cycle. The number of vessels positively stained for eNOS increased gradually during the proliferation phase and most of the vessels were positive in the early secretory phase. These results suggest that its markedly increased expression during the early secretory phase of the menstrual cycle indicates a physiological significance.     

eNOS基因体外转染对大鼠颈总动脉球囊损伤后血管平滑肌细胞增殖规律的影响

目的将内皮型一氧化氮合酶(endothilialnitircoxidesynthase,eNOS)基因,通过基因工程技术转染至体外培养的大鼠颈总动脉球囊损伤后的血管平滑肌细胞中,观察其对血管平滑肌细胞增殖规律的影响。方法三月龄雄性Wastar大鼠20只,分为正常对照组、空白对照组、pcDNA3.1(-)转染组和pcDNA3.1-eNOS转染组,每组各5只。除正常对照组外15只Wastar大鼠,施行经皮冠状动脉腔内成形术(PTCA)。术后一月,处死所有20只大鼠,取出左侧颈总动脉,以组织块贴壁法培养损伤后血管平滑肌细胞,采用脂质体载体Fugene6介导真核表达载体pcDNA3.1-eNOS和空载体pcDNA3.1(-)分别转染eNOS转染组和pcDNA3.l(-)转染组的平滑肌细胞,以RT-PCR法、原位杂交法和免疫荧光法检测eNOS基因的表达;以MTT法检测细胞增殖程度。结果在转染pcDNA3.l-eNOS基因的损伤后血管平滑肌细胞中可以检测出eNOSmRNA和蛋白的表达,且其细胞增殖程度显著低于未转染eNOS基因者(P<0.05)。结论eNOS基因体外转染可以明显抑制大鼠颈总动脉球囊损伤后血管平滑肌细胞的增殖活性。     

Analysis of intrapulmonary vessels and epithelial-endothelial interactions in the human developing lung

The establishment of a sufficiently wide and functional blood-gas interface is of critical importance in lung development, but development of the intrapulmonary vascular system including alveolar capillary vessels still remains unclear. In this study, we first characterized the structural development of the vascular system in accordance with that of airways in human fetal lungs at the pseudoglandular phase (8, 13, and 16 weeks gestation) by examining the immunohistochemical distribution of CD34 and alpha-smooth muscle actin (SMA). Using double immunohistochemistry and 3-dimensional reconstruction techniques, endothelial cells in the developing lung could be classified into two different types according to the characteristics of their adjacent cells (presence or absence of SMA-positive cells) and their distribution (proximal or distal lung parenchyme). Endothelial cells without SMA-positive cells developed into a capillary network surrounding the budding components of distal airways during the mid-pseudoglandular phase before communicating with proximal vessels. We then examined the immunoreactivity of thrombomodulin and von Willebrand factor (vWF) in endothelial cells. Endothelial cells of the capillary network were mainly positive for vWF during the early gestational stages, but altered their phenotypes to those of mature lungs (vWF negative and thrombomodulin positive) during the terminal sac phase. We subsequently determined the immunohistochemical distribution of vascular endothelial growth factor (VEGF). Epithelial cells of the most distal airways were intensely positive for VEGF. These results suggest that VEGF present in airway epithelial cells is involved in the maturation as well as proliferation of capillary endothelial cells. Epithelial-endothelial interactions during lung development are considered very important in the establishment of the functional blood-gas interface.     

The estrogen effects on endothelial repair and mitogen-activated protein kinase activation are abolished in endothelial nitric-oxide (NO) synthase knockout mice, but not by NO synthase inhibition by N-nitro-L-arginine methyl ester

We have previously shown that estrogen exerts a vasoprotective effect by accelerating reendothelialization after perivascular artery injury through activation of the estrogen receptor alpha. Because 17beta-estradiol (E2) is known to increase the bioavailability of nitric oxide, in this study, we used the same perivascular model to characterize the role of the endothelial nitric oxide synthase (eNOS) pathway in reendothelialization. Surprisingly, we found that the stimulatory effect of E2 on reendothelialization was not altered following pharmacological inhibition of nitric-oxide synthase enzymatic activity by N-nitro-L-arginine methyl ester, whereas it was abolished in eNOS-deficient (eNOS-/-) mice. This discrepancy between eNOS gene inactivation and the pharmacological inhibition of eNOS was confirmed in a classical model of endovascular injury. When assessing the involvement of eNOS in short-term membrane-associated signaling events induced by E2, we found that E2 stimulated phosphorylation of extracellular signal-regulated kinase 1/2 in isolated perfused carotid arteries from wild-type mice in the absence or presence of N-nitro-l-arginine methyl ester, whereas this stimulation was abolished in carotid arteries from eNOS-/- mice. Similar results were obtained in primary cultures of mouse aortic endothelial cells. These data reveal an original and unexpected role of eNOS, in which its presence but not its enzymatic activity appears to be a determinant for estrogen signaling in the endothelium. The consequences of this novel function of eNOS with respect to vascular diseases should be explored.