Spatial and temporal dynamics of the endothelium

The endothelium is a highly metabolically active organ that is involved in many physiological processes, including the control of vasomotor tone, barrier function, leukocyte adhesion and trafficking, inflammation, and hemostasis. Endothelial cell phenotypes are differentially regulated in space and time. Endothelial cell heterogeneity has important implications for developing strategies in basic research, diagnostics and therapeutics. The goals of this review are to: (i) consider mechanisms of endothelial cell heterogeneity; (ii) discuss the bench-to-bedside gap in endothelial biomedicine; (iii) revisit definitions for endothelial cell activation and dysfunction; and (iv) propose new goals in diagnosis and therapy. Finally, these themes will be applied to an understanding of vascular bed-specific hemostasis.     

Vascular complications in diabetes mellitus: the role of endothelial dysfunction

The endothelium is a complex organ with a multitude of properties essential for control of vascular functions. Dysfunction of the vascular endothelium is regarded as an important factor in the pathogenesis of diabetic micro- and macro-angiopathy. Endothelial dysfunction in Type I and II diabetes complicated by micro- or macro-albuminuria is generalized in that it affects many aspects of endothelial function and occurs not only in the kidney. The close linkage between microalbuminuria and endothelial dysfunction in diabetes is an attractive explanation for the fact that microalbuminuria is a risk marker for atherothrombosis. In Type I diabetes, endothelial dysfunction precedes and may cause diabetic microangiopathy, but it is not clear whether endothelial dysfunction is a feature of the diabetic state itself. In Type II diabetes, endothelial function is impaired from the onset of the disease and is strongly related to adverse outcomes. It is not clear whether impaired endothelial function is caused by hyperglycaemia or by other factors. Impaired endothelial function is closely associated with and may contribute to insulin resistance regardless of the presence of diabetes. Endothelial dysfunction in diabetes originates from three main sources. Hyperglycaemia and its immediate biochemical sequelae directly alter endothelial function or influence endothelial cell functioning indirectly by the synthesis of growth factors, cytokines and vasoactive agents in other cells. Finally, the components of the metabolic syndrome can impair endothelial function.     

Protein composition of plasminogen activator inhibitor type 1-derived endothelial microparticles

Endothelial microparticles (EMPs) are small vesicles released from the plasma membrane of endothelial cells in response to cell injury, apoptosis, or activation. Low levels of MPs are shed into the blood from the endothelium, but in some pathologic states, the number of EMPs is elevated. The mechanism of MP formation and the wide-ranging effects of elevated EMPs are poorly understood. Here, we report the protein composition of EMPs derived from human umbilical cord endothelial cells stimulated with plasminogen activator inhibitor type 1 (PAI-1). Two-dimensional gel electrophoresis followed by mass spectrometry identified 58 proteins, of which some were verified by Western blot analysis. Gene Ontology database searches revealed that proteins identified on PAI-1-derived EMPs are highly diverse. Endothelial microparticles are composed of proteins from different cellular components that exhibit multiple molecular functions and are involved in a variety of biological processes. Important insight is provided into the generation and protein composition of PAI-1-derived EMPs.     

角膜内皮损伤的相关因素分析

角膜保持透明性的重要条件是内皮细胞的生理功能正常和解剖完整，内皮损伤的影响因素取决于年龄，外伤，遗传，代谢遗传，可在相关术后被破坏其组织结构，眼病相关及全身疾病都会影响角膜内皮的生理特性，成人内皮损伤后不能再生，只能靠邻近细胞迁移及扩展，近年来专家和学者对角膜内皮相关研究越发关注，本文就引起角膜内皮损伤的个性因素及相关原理作一综述。     

Endothélium et microcirculation au cours des états critiques. Actes du séminaire de recherche translationnelle de la Société de réanimation de langue française (1<Superscript>er</Superscript> décembre 2015)

Under physiological conditions, the endothelium regulates vasomotor tone, the coagulation cascade, platelet aggregation, and vascular permeability. Endothelial cells have common properties, but these vascular cells also have organ-related characteristics, defining the endothelial heterogeneity. Endothelial dysfunction has been associated with many pathophysiological processes such as inflammation and oxidative stress. Impaired endothelial function leads to phenotypic changes and is involved in the pathophysiology of organ failure during critical conditions. The development of accurate tools for the early detection of endothelial dysfunction and microcirculatory hypoperfusion could be of great interest in critically ill patients.     

内皮祖细胞内皮修复的影响因素

内皮祖细胞（EPC）来源于骨髓,能够定向分化为成熟有功能的血管内皮细胞,是血管内皮细胞的前体细胞,又称血管母细胞。内皮祖细胞在组织缺血和血管损伤时动员入血,促进微血管的生成和血管内皮的修复,不仅参与胚胎血管生成,也参与出生后的血管发生过程,具有保护血管内皮、减少心血管疾病的发生率和死亡率的作用。许多试验证实冠状动脉粥样硬化性心脏病（冠心病）和心力衰竭患者EPC数量减少,外周循环内皮细胞功能受损。研究发现,EPC的生物学特性受到诸多冠心病危险因素的影响,致使其功能异常。     

Metabolism of vasoactive peptides by human endothelial cells in culture. Angiotensin I converting enzyme (kininase II) and angiotensinase.

Cultured endothelial cells provide a model for the study of interactions of vasoactive peptides with endothelium. Endothelial cell cultured from veins of human umbilical cords contain both angiotensin I converting enzyme (kininase II) and angiotensinase activities. Intact monolayers of cells can both activate angiotensin I and inactivate bradykinin when the peptides are added to culture flasks in protein-free medium. Intact suspended cells or lysed cells convert angiotensin I to angiotensin II, inactivate bradykinin, and hydrolyze hippuryldiglycine to hippuric acid and diglycine. These actions are inhibited by SQ 20881, the specific inhibitor of converting enzyme. The kininase activity of endothelial cells was partially inhibited by antibody to human lung converting enzyme. Endothelial cells also inactivate longer analogs of bradykinin, such as kallidin, methionyl-lysyl bradykinin, and bradykinin coupled covalently to 500,000 mol wt dextran. The endothelial cells retained converting enzyme activity through four successive subcultures, indicating that the enzyme is synthesized by the cells surface, and it is apparently a marker for endothelial cells, since cultured human fibroblasts, smooth muscle cells, and baby hamster kidney cells do not have it. Endothelial cells also contain an aminopheptidase which hydrolyzes both angiotensin II and the synthetic substrate, alpha-L-aspartyl beta-naphthylamide. The angiotensinase activity increased when the cells were lysed, which suggests that the enzyme is localized within the cells, Hydrolysis of both alpha-L-aspartyl beta-naphthylamide and angiotensin II was inhibited by omicron-phenanthroline, indicating that the enzyme is an A-tipe anigotensinase.     

Sickle erythrocyte adherence to large vessel and microvascular endothelium under physiologic flow is qualitatively different.

Complications in sickle syndromes are thought to result from regional disturbances of normal blood flow with subsequent ischemic damage. Adherence of sickle erythrocytes has been implicated in the pathophysiology of occlusive complications. Most previous studies have explored adherence of sickle erythrocytes to endothelial cells from large vessels, even though the majority of the pathophysiologic models implicate the microvascular system. To explore potential variation in endothelial interactions at low shear rates, adherence of sickle erythrocytes to large vessel umbilical vein endothelium and microvascular endothelium was compared under flow conditions in a parallel-plate flow chamber at a shear stress of 1.0 dyne/cm2. Autologous plasma promotes high levels of sickle red cell adherence to microvascular endothelial cells, but only low levels of adherence to human umbilical vein endothelium. On average, autologous plasma promotes sixfold more sickle cell red adherence to microvascular endothelial cells. In contrast to umbilical vein endothelium, high molecular von Willebrand factor does not elevate sickle cell adherence to microvascular endothelial cells, and the integrin receptor agonist peptide, RGD, does not inhibit adherence to microvascular endothelial cells. These results demonstrate that sickle erythrocyte adherence to large vessel and microvascular endothelium is quantitatively and qualitatively different and that plasma factors may have significant impact on sickle erythrocyte adherence to endothelium in the microvessels. Since microvascular occlusion has been suggested as an antecedent of ischemic damage in sickle syndromes, plasma enhanced adherence to microvascular endothelium may contribute to the pathophysiology of episodic occlusion in sickle cell anemia.     

Biochemical and therapeutic effects of Omega-3 fatty acids in sickle cell disease

Sickle cell disease (SCD) is a hematologic disorder with complex pathophysiology that includes chronic hemolysis, vaso-occlusion and inflammation. Increased leukocyte-erythrocyte-endothelial interactions, due to upregulated expression of adhesion molecules and activated endothelium, are thought to play a primary role in initiation and progression of SCD vaso-occlusive crisis and end-organ damage. Several new pathophysiology-based therapeutic options for SCD are being developed, chiefly targeting the inflammatory pathways.Omega−3 fatty acids are polyunsaturated fatty acids that are known to have effects on diverse physiological processes. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are the principal biologically active omega-3 fatty acids. The therapeutic effects of DHA and EPA on chronic inflammatory disorders and cardiovascular diseases are well recognized. The therapeutic effects of omega-3 fatty acids are attributed to their anti-inflammatory and anti-thrombotic eicosanoids, and the novel class of EPA and DHA derived lipid mediators: resolvins, protectins and maresins.Blood cell membranes of patients with SCD have abnormal fatty acids composition characterized by high ratio of pro-inflammatory arachidonic acid (AA) to anti-inflammatory DHA and EPA (high omega-6/omega-3 ratio). In addition, experimental and clinical studies provide evidence that treatment with DHA does confer improvement in rheological properties of sickle RBC, inflammation and hemolysis. The clinical studies have shown improvements in VOC rate, markers of inflammation, adhesion, and hemolysis. In toto, the results of studies on the therapeutic effects of omega-3 fatty acids in SCD provide good body of evidence that omega-3 fatty acids could be a safe and effective treatment for SCD.     

Functions of the endothelium and its role in hematopoietic cell transplantation

The endothelium is a single-layered structure that responds to physical and chemical signals with various factors it synthesizes. In the early days of its discovery, as the inner wall of the vessels, the endothelium was thought to be a simple barrier that lays on the surface. Over time it is discovered that endothelium maintains body homeostasis with the molecules it synthesizes, despite its simple single-layer structure. It has been accepted as an important organ that contributes to the maintenance of vascular tone, cell adhesion, inflammation, vascular permeability and coagulation. Any imbalance in these physiological and pathological events causes endothelial dysfunction. This can cause many diseases such as atherosclerosis, hypertension, diabetes, or it can occur because of these. Endothelial related disorders may also complicate hematopoietic stem cell transplantation (HSCT), which is used to treat various hematologic and neoplastic diseases. These life-threatening complications include graft-versus-host disease, hepatic veno-occlussive disease, transplant-associated thrombotic microangiopathy and diffuse alveolar hemorrhage. They share a similar pathophysiology involving endothelial cells with different clinical presentations. Therefore, current researche on the issue is putting the endothelium under the spotlight for novel markers and treatment options that should be used to monitor or treat at least some of these complications following HSCT.     

Iron enhances endothelial cell activation in response to Cytomegalovirus or Chlamydia pneumoniae infection

BACKGROUND: Chronic inflammation has been implemented in the pathogenesis of inflammatory diseases like atherosclerosis. Several pathogens like Chlamydia pneumoniae (Cp) and cytomegalovirus (CMV) result in inflammation and thereby are potentially artherogenic. Those infections could trigger endothelial activation, the starting point of the atherogenic inflammatory cascade. Considering the role of iron in a wide range of infection processes, the presence of iron may complicate infection-mediated endothelial activation. MATERIALS AND METHODS: Endothelial intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelial selectin (E-selectin) expression were measured using flow cytometry, as an indication of endothelial activation. Cytotoxicity was monitored using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Immunostaining was applied to measure Cp and CMV infectivity to endothelial cells. RESULTS: An increased number of infected endothelial cells in a monolayer population leads to a raised expression of adhesion molecules of the whole cell population, suggesting paracrine interactions. Iron additively up-regulated Cp-induced VCAM-1 expression, whereas synergistically potentiated Cp-induced ICAM-1 expression. Together with CMV, iron also enhanced ICAM-1 and VCAM-1 expression. These iron effects were observed without modulation of the initial infectivity of both microorganisms. Moreover, the effects of iron could be reversed by intracellular iron chelation or radical scavenging, conforming modulating effects of iron on endothelial activation after infections. CONCLUSIONS: Endothelial response towards chronic infections depends on intracellular iron levels. Iron status in populations positive for Cp or CMV infections should be considered as a potential determinant for the development of atherosclerosis.     

In vitro modeling of the microvascular occlusion and thrombosis that occur in hematologic diseases using microfluidic technology

In hematologic diseases, such as sickle cell disease (SCD) and hemolytic uremic syndrome (HUS), pathological biophysical interactions among blood cells, endothelial cells, and soluble factors lead to microvascular occlusion and thrombosis. Here, we report an in vitro "endothelialized" microfluidic microvasculature model that recapitulates and integrates this ensemble of pathophysiological processes. Under controlled flow conditions, the model enabled quantitative investigation of how biophysical alterations in hematologic disease collectively lead to microvascular occlusion and thrombosis. Using blood samples from patients with SCD, we investigated how the drug hydroxyurea quantitatively affects microvascular obstruction in SCD, an unresolved issue pivotal to understanding its clinical efficacy in such patients. In addition, we demonstrated that our microsystem can function as an in vitro model of HUS and showed that shear stress influences microvascular thrombosis/obstruction and the efficacy of the drug eptifibatide, which decreases platelet aggregation, in the context of HUS. These experiments establish the versatility and clinical relevance of our microvasculature-on-a-chip model as a biophysical assay of hematologic pathophysiology as well as a drug discovery platform.     

Role of the vascular wall in the pathophysiology of the acute coronary syndrome

The endothelium is of important significance in the development of the acute coronary syndrome. As an endo-/paracrine organ, the endothelium plays a key role in the regulation of the vascular homeostasis. The endothelial integrity and above all the bioavailability of nitric oxide (NO) are essential for the correct function of the endothelium. Cardiac risk factors may lead to an endothelial dysfunction with a consecutive imbalance of the vascular homeostasis. In an inflammatory or prothrombotic state the endothelium shows a number of abnormalities such as oxidative stress, expression of cell adhesion molecules, activation of cell signal-systems (renin-angiotensin-system, CD40/CD40L-system) and especially the loss of NO. The inflammatory cascades lead to coronary atherosclerosis over years or, more instantly, to the acute coronary syndrome caused by endothelial erosion or the rupture of an instable plaque. The knowledge of the pathophysiological processes in the arterial wall during the acute coronary syndrome may lead to the identification of high risk patients and the development of more targeted therapies.     

Characterization of Bioeffects on Endothelial Cells under Acoustic Droplet Vaporization

Gas embolotherapy is achieved by locally vaporizing microdroplets through acoustic droplet vaporization, which results in bubbles that are large enough to occlude blood flow directed to tumors. Endothelial cells, lining blood vessels, can be affected by these vaporization events, resulting in cell injury and cell death. An idealized monolayer of endothelial cells was subjected to acoustic droplet vaporization using a 3.5-MHz transducer and dodecafluoropentane droplets. Treatments included insonation pressures that varied from 2 to 8 MPa (rarefactional) and pulse lengths that varied from 4 to 16 input cycles. The bubble cloud generated was directly dependent on pressure, but not on pulse length. Cellular damage increased with increasing bubble cloud size, but was limited to the bubble cloud area. These results suggest that vaporization near the endothelium may impact the vessel wall, an effect that could be either deleterious or beneficial depending on the intended overall therapeutic application.     

Circulating markers of endothelial function in cardiovascular disease

Endothelial dysfunction is a key event in cardiovascular disease. Measurement of endothelial dysfunction in vivo presents a major challenge, but has important implications since it may identify the clinical need for therapeutic intervention, specifically in primary prevention. Several biological markers have been used as indicators of endothelial dysfunction. The soluble adhesion molecules sICAM-1 and sVCAM-1 lack specificity and are increased in inflammatory processes. Both markers are increased in coronary artery disease. sICAM-1 level predicts the risk for cardiovascular disease or diabetes mellitus in healthy individuals. sE-selectin is specific for the endothelium and is increased in coronary artery disease and diabetes mellitus. sE-selectin is also associated with diabetic risk. The endothelium-specific marker, soluble thrombomodulin, is associated with severity of coronary artery disease, stroke or peripheral occlusive arterial disease and is not increased in healthy or asymptomatic subjects. Interestingly, thrombomodulin decreases during treatment of hypercholesterolemia or hyperhomocysteinemia. In contrast, von Willebrand factor is the best endothelial biomarker and predicts risk for ischemic heart disease or stroke.     

A fish oil emulsion used for parenteral nutrition attenuates monocyte-endothelial interactions under flow

Monocyte adhesion contributes to perfusion abnormalities, tissue damage, and activation of the coagulation system seen during trauma, shock, or overwhelming inflammation. This study was performed to determine whether an intravenous fish oil emulsion used for parenteral nutrition attenuates monocyte-endothelial interactions under flow and reduces procoagulant activity, measured as tissue factor (TF) expression on adherent monocytes in vitro. Endothelial cell monolayers were incubated with either an intravenous fish oil emulsion or a conventional omega-6 lipid emulsion at 0.05 to 1 mg/ml for 24 h. Six hours following activation with TNFalpha (25 ng/ml), expression of endothelial cell adhesion molecules was measured by flow cytometry. Adhesion of isolated monocytes to pretreated endothelium was examined in a parallel plate flow chamber at a shear stress of 1.5 dynes/cm2. Following perfusion, the cells were cocultured for an additional 4 h and TF expression on monocytes was determined by flow cytometry. In contrast to omega-6 lipids, fish oil down-regulated E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in a dose-dependent manner. P-selectin, however, remained unchanged. In addition, firm adhesion was reduced to 54%, whereas rolling interactions remained unchanged. Fish oil exhibited no effect on the TF expression on cocultured monocytes. We conclude that intravenous fish oil emulsions reduce both endothelial cell adhesion molecule expression and monocyte adhesion. However, under postcapillary flow conditions, rolling interactions via P-selectin remain unaltered. The functional importance of this effect is illustrated by the corresponding upregulation of TF in response to residual monocyte-endothelial interactions.     

Blood-derived angioblasts accelerate blood-flow restoration in diabetic mice

Endothelial cell progenitors, angioblasts, have been detected in the peripheral blood of adult humans, mice, and rabbits. These cells have been shown to incorporate into the endothelium of newly forming blood vessels in pathological and nonpathological conditions. Here we investigated the possibility that the CD34-expressing leukocytes (CD34(+) cells) that appear to be enriched for angioblasts could be used to accelerate the rate of blood-flow restoration in nondiabetic and diabetic mice undergoing neovascularization due to hindlimb ischemia. CD34(+) cells did not accelerate the restoration of flow in nondiabetic mice, but dramatically increased it in diabetic mice. Furthermore, CD34(+) cells derived from type 1 diabetics produced fewer differentiated endothelial cells in culture than did their type 2 diabetic- or nondiabetic-derived counterparts. In vitro experiments suggest that hyperglycemia per se does not alter the ability of angioblasts to differentiate or of angioblast-derived endothelial cells to proliferate. In contrast, hyperinsulinemia may enhance angioblast differentiation but impair angioblast-derived endothelial cell survival or proliferation. Our findings suggest that CD34(+) cells may be a useful tool for therapeutic angiogenesis in diabetics.     

Effects of tobacco and non-tobacco cigarette smoking on endothelium and platelets

Endothelial damage and platelet activation may mediate increased cardiovascular morbidity and mortality in tobacco cigarette smokers. Our study was designed to determine whether acute effects of tobacco smoking on endothelium and platelets could be avoided by the substitution of non-tobacco cigarettes. Twenty healthy nonsmokers smoked two tobacco cigarettes in 20 minutes and on another occasion (separated by 1 week) smoked two cigarettes made from wheat, cocoa, and citrus plants. Mean endothelial cell counts from venous blood before and after smoking tobacco cigarettes were 2.3 and 4.8 and before and after smoking non-tobacco cigarettes counts were 2.5 and 3.0. Mean platelet aggregate ratios before and after smoking tobacco cigarettes were 0.80 and 0.65 and before and after smoking non-tobacco cigarettes they were 0.81 and 0.78. Much greater effects of tobacco smoking on endothelial cell counts and platelet aggregate ratios suggest the possibility that non-tobacco cigarette smoking may be less harmful to the cardiovascular system than is tobacco cigarette smoking.     

Role of endothelial cells in restenosis after coronary angioplasty

Summary— Percutaneous transluminal coronary angioplasty (PTCA) is today a procedure of choice in many patients with atherosclerotic coronary artery disease. Despite high rates of initial success, restenosis, occurring in 30 to 40 percent of patients within the first six months, remains the major problem limiting the long-term efficacy of the procedure. Animal models have enhanced our knowledge in the understanding of the mechanisms involved in the restenotic process after experimental angioplasty. In fact, the two known determinants of restenosis are the proliferative and migrative response of underlying smooth muscle cells with production of extracellular matrix and the recently highlighted vascular remodeling. Endothelium, which regenerates from the leading edge of the de-endothelialized area within the weeks following arterial injury, is of particular interest in the modulation of the healing process after the procedure. Endothelial dysfunction, as an imbalance between relaxing and contracting factors, between anti- and procoagulant mediators or growth-inhibiting and growth-promoting factors, occurs at sites of regenerating endothelium. Experimental studies, using drugs that enhance endothelium-derived relaxing factors release or drugs that diminish endothelium-derived contracting factors production, have often been shown to be effective in the restenosis prevention. Thus, impairment in endothelial cell function may be considered as one of the major regulatory element in the restenotic process. This review discusses the interactions between endothelial and smooth muscle cells and has for aim to point out the major role of endothelial cells in the development of neointimal thickening and arterial remodeling.