Endothelium : journal of endothelial cell research最新文献

Previous studies have demonstrated that endothelial cells exposed to laminar shear stress are protected from apoptotic stimuli such as tumor necrosis factor (TNF)-alpha. The authors investigated the role of phosphatidylserine (PS) in this phenomenon. Western blot analysis of cleaved caspase 3 was used as an indicator of apoptosis and revealed that in the absence of serine, endothelial cells exposed to laminar shear stress were unable to protect against TNF-alpha-induced apoptosis, in contrast to sheared cells grown in regular medium. It was also found that shear-induced activation of the Akt pathway was significantly decreased in cells grown without serine. In addition, quantitation of PS using a novel isotopic labeling technique involving the use of formalin revealed that stearoyl-oleic PS (18:0/18:1) did not increase during shear treatment. These findings suggest that basal levels of PS are required to activate survival pathways in endothelial cells and thereby contribute to the overall protective mechanism initiated by shear stress.

Using cationic liposomes to deliver cytotoxic molecules to the tumor microvasculature is currently being developed for the treatment of cancer and other angiogenesis-related diseases. To improve on their beneficial properties, the authors have examined whether the particular cationic lipid type and lipid content employed are important factors influencing cellular interactions and formulation effects. The authors prepared different PEG (polyethylene glycol)-modified cationic liposomes (PCLs) with varying percent cationic lipid content and lipid type, and evaluated liposome size, surface charge (zeta) potential, and cellular properties in vitro. The cell lines used were human umbilical vein (HUVEC), lung microvascular (HMVEC-L and HPVE-26), coronary microvascular (HMVEC-C), dermal microvascular (HMVEC-D), and immortalized dermal microvascular (HMEC-1) endothelial cells. In vitro experiments consisted of cellular uptake and cytotoxicity studies, fluorescence-activated cell sorting (FACS) analysis, fluorescence, and transmission electron microscopic analysis. Liposome size and zeta potential analysis of five different PCLs revealed significant differences in their physicochemical properties. Some cationic lipids formed relatively toxic liposomes compared to others. The efficiency of loading chemotherapeutic drugs (doxorubicin hydrochloride, etoposide), affinity of PCLs for endothelial cells, and formulation effects varied according to cationic lipid content and the lipid type. Cellular uptake was observed in lung, dermal, and coronary endothelial cells. Heparan sulfate proteoglycans were found present on HMEC-1 cells, which may have enabled PCL uptake. In conclusion, physicochemical properties of cationic liposomes and their ability to interact with endothelial cells are important factors to consider during the early stages of formulation development for the treatment of cancer and other angiogenesis-dependent diseases.

The data reported in literature revealed a novel function for matrix metalloproteinases (MMPs) as modulators of adipogenesis. However, their expression profile and role in the cellular microenvironment during obesity-mediated adipose tissue development remain poorly defined. The authors hypothesized that MMP-2 and MMP-9 levels might be abnormal in obesity, reflecting alterations in extracellular matrix (ECM) turnover. One hundred and sixty three obese patients and 165 controls were enrolled. The following were measured: body mass index (BMI), waist circumference (WC), fasting plasma glucose (FPG), fasting plasma insulin (FPI), homeostasis model assessment (HOMA) index, systolic blood pressure (SBP), diastolic blood pressure (DBP), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (Tg), lipoprotein(a) (Lp(a)), and plasma levels of MMP-2 and MMP-9. A significant increase of BMI and WC (p< .0001) was observed in obese patients. No FPG change was present in obese group, whereas FPI and HOMA index increases (p< .0001) were obtained in obese patients compared to control subjects. No SBP and DBP variations were observed in obese group. Significant TC and LDL-C increases (p< .0001) were present in obese patients, whereas no HDL-C, Tg, and Lp(a) changes were obtained in both groups. MMP-2 and MMP-9 levels were significantly higher in obese group (p< .0001). Plasma levels of MMP-2 and MMP-9 are increased in obese patients which may reflect abnormal ECM metabolism.

Human endothelial cells (ECs) are heterogeneous, although little is known regarding regional variations in their regulation of vascular tone. This study compares activation of the key enzyme phospholipase D (PLD) by the vasoconstrictors angiotensin II (AII) and lysophosphatidylcholine (lysoPC), and the vasodilator insulin, in primary human microvascular endothelial cells (HMVECs) and human umbilical vein endothelial cells (HUVECs). PLD activity was measured by [(3)H]phosphatidylethanol production in cells labeled with [(3)H]myristic acid. AII maximally activated PLD in both cell types at 1 nmol/L. AII also significantly activated PLD at 100 pmol/L in HUVECS but not in HMVECs. LysoPC dose dependently activated PLD in both cell types, although HUVECs were more sensitive to the agonist; being significantly activated by 10 micromol/L lysoPC and displaying an approximately sevenfold greater PLD activity with 20 micromol/L lysoPC compared to HMVECs. Insulin significantly increased PLD activity in both cell types with maximum activation at 1 nmol/L. Again differential sensitivity was observed; 10 nmol/L insulin significantly stimulated PLD in HUVECs but not HMVECs. Differential sensitivity of PLD activation in human endothelial cells from different vascular beds in response to vasoactive agents was observed, with the HUVECs displaying greater sensitivity to vasoconstricting agents than HMVECs.

Unlabelled: Liver sinusoidal endothelial cells (LSECs) constitute an attractive target for gene therapy of several liver and systemic diseases. However, there are few reports showing an efficient plasmid-based or viral methodology to deliver recombinant genes into these cells. In the present study, the authors evaluated in vitro gene transfer efficiency of standard plasmid-based techniques (i.e., electroporation, lipofection, and calcium phosphate) and lentiviral-mediated gene transduction into primary murine LSECs, using reporter genes. The results show that electroporation is the most effective in vitro plasmid-gene transfer method to deliver GFP into LSECs (31%), as compared with lipofection and calcium phosphate transfection (6% and 4%, respectively). However, lentiviral transduction resulted in higher, efficient, and stable gene transfer (70%) as compared with plasmid-based techniques.

Conclusions: The highly efficient gene expression obtained by lentiviral transduction and electroporation shows that these methodologies are highly reliable systems for gene transfer into LSECs.

By the time the clinical findings of atherosclerotic disease appear, involvement is usually at an advanced stage and procedures after this stage are usually palliative or aimed at secondary protection. On the other hand, prevention can be achieved by the detection and treatment of endothelial dysfunction, which is one of the most important changes in the early subclinical stage of atherosclerotic disease. When the systemic involvement of endothelial dysfunction is taken into consideration, checking from the peripheral arteries with noninvasive methods gives one-to-one correct information. Currently, endothelial dysfunction can be detected using simple, inexpensive, and noninterventional methods. Particularly, easily accessible localization of the brachial artery is ideal for the evaluation of endothelial dysfunction. Flow-mediated dilation method (FMD; endothelial-dependent vasodilation), which can be carried out noninvasively with ultrasonography on the brachial artery, is a frequently used method for the assessment of endothelial dysfunction. A sphygmomanometer is placed on the forearm to create a flow stimulation in the brachial artery. The sphygmomanometer is inflated until the systolic pressure is above 50 mm Hg, thus stopping the antegrade blood flow and creating ischemia. Consequently, vasodilation occurs at the resistance arteries distal to where the flow is blocked. When the sphygmomanometer is deflated, a reactive hyperemia occurs in the brachial artery. The % difference between the diameter measured after reactive hyperemia and the basal diameter is taken as FMD. The effects of the treatments on endothelial dysfunction can be monitored with this method. Studies have shown that angiotensin-converting enzyme (ACE) inhibitors, angiotensin 1 (AT1) receptor blockers, latest-generation beta blockers such as nebivolol and carvediol, statins, estrogen treatment, diet, and exercise increase FMD. Before this method becomes a part of routine clinical evaluation of cardiovascular disease (CVD) risk, measurement technique and FMD values need to be standardized.

Protein-zero related (PZR) is an immunoglobulin V (IgV)-type immunoreceptor with two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). PZR interacts with Src homology 2 domain-containing tyrosine phosphatase (SHP-2) via its tyrosine-phosphorylated ITIMs, for which c-Src is a putative kinase. Towards elucidating PZR function in endothelial cells (ECs), the authors cloned PZR from bovine aortic endothelial cells (BAECs) and characterized it. Mature bovine PZR had 94.8% and 92.7% sequence identity with canine and human proteins, respectively, and the two ITIM sequences were conserved among higher vertebrates. PZR was expressed in many cell types and was localized to cell contacts and intracellular granules in BAECs and mesothelioma (REN) cells. Coimmunoprecipitation revealed that PZR, Grb-2-associated binder-1 (Gab1), and platelet endothelial cell adhesion molecule-1 (PECAM-1) were three major SHP-2-binding proteins in BAECs. H(2)O(2) enhanced PZR tyrosine phosphorylation and PZR/SHP-2 interaction in ECs in a dose-and time-dependent manner. To see if tyrosine kinases other than Src are also capable of phosphorylating PZR, the authors cotransfected HEK293 cells with PZR and one of several tyrosine kinases and found that c-Src, c-Fyn, c-Lyn, Csk, and c-Abl, but not c-Fes, phosphorylated PZR and increased PZR/SHP-2 interaction. These results suggest that PZR is a cell adhesion protein that may be involved in SHP-2-dependent signaling at interendothelial cell contacts.

The objective of the present study was to compare the early effects of treatment with nebivolol and quinapril on the endothelial function in hypertensive patients. A total of 54 hypertensive patients was enrolled in the present study. One of the groups (n = 27) received quinapril 20 mg/day, and the other group (n = 27) received nebivolol 5 mg/day for a period of 4 weeks. The endothelial dysfunction was assessed using FMD (flow-mediated vasodilation) of the brachial arteries. The baseline characteristics of both groups were similar in age, gender, left venticular ejection fraction, left ventricular mass index, and body mass index. No significant difference was also found between the groups in the distribution of atherosclerotic risk factors as well as other echocardiographic, demographic, and biochemical measurements. Although the reduction of diastolic blood pressure was more pronounced in the nebivolol group after a 4-week treatment, the change in the systolic blood pressure was found to be similar in both treatment arms. Although a statistically nonsignificant increase was observed in flow-mediated vasodilation in the quinapril group (4.77% +/- 3.92%, 5.60% +/- 6.18%; p = .587), the increase in the post-treatment FMD was statistically significant in the nebivolol group (3.78% +/- 4.25%, 8.56% +/- 6.39%; p = .002). A significant change was observed in the resistive index value following flow-mediated vasodilation for both groups after treatment (p = .043; p = .027), whereas the change in the value of flow volume was significant only in the nebivolol group (p = .019).

Thrombomodulin (TM) is a cell surface anticoagulant glycoprotein that plays a key role in the protein C pathway. TM expression in endothelial cells may be modulated by a variety of extracellular signals. Most notably, TM has been shown to be downregulated by inflammatory mediators, such as tumor necrosis factor-alpha and lipopolysaccharide. The objective of this study was to determine the effect of thrombin on TM expression and activity. Thrombin resulted in reduced TM in primary cultures of human endothelial cells by approximately 40% at the level of mRNA, protein, and activity. These effects were blocked by the thrombin inhibitor hirudin. These results suggest that activation of the coagulation cascade may result in a positive-feedback loop consisting of thrombin-mediated repression of TM-dependent protein C activation.

Sphingolipids, in particular sphingosine kinase (SphK) and its product sphingosine-1-phosphate (S1P), are now recognized to play an important role in regulating many critical processes in endothelial cells. Activation of SphK1 is essential in mediating the endothelial proinflammatory effects of inflammatory cytokines such as tumor necrosis factor (TNF). In addition, S1P regulates the survival and proliferation of endothelial cells, as well as their ability to undergo cell migration, all essential components of angiogenesis. Thus the inflammatory and angiogenic potential of the endothelium is in part regulated by intracellular components including the activity of SphK1 and levels of S1P. Herein a review of the sphingomyelin pathway with a particular focus on its relevance to endothelial cell biology is presented.