Bingwei Li

Melatonin inhibits IL-1β-induced monolayer permeability of human umbilical vein endothelial cells via Rac activation

Abstract:  Melatonin is a key factor in the coordination of circadian rhythms and seasonal reproduction. Melatonin and its metabolites directly scavenge free radicals, increase the expression of antioxidant enzymes, and play a role in the anti‐inflammatory phase of defense responses. At present, there are no direct data available as to melatonin’s possible influence on endothelial cell monolayer permeability, which is a major biological process responsible for vascular diseases. The aim of this study was to investigate the effect of melatonin on IL‐1β‐induced human umbilical vein endothelial cells (HUVECs) monolayer permeability and then to test the involvement of small GTPase Rac in the melatonin‐induced endothelial barrier‐protective effects as well as cell contact reorganization. It was found that IL‐1β treatment increased the permeability of HUVECs monolayer, disrupted adherens junctions, and down‐regulated the expression of VE‐cadherin which is the main functional protein of adherens junctions. Melatonin, however, decreased dextran permeability and increased intercellular adherens junction areas reflecting an endothelial cell barrier‐protective response. Furthermore, melatonin dramatically improved IL‐1β‐induced Rac inactivation. Our results show that the barrier‐protective effects of melatonin on endothelial cells are mediated by Rac activation and leads to enhancement of adherens junctions.

Enhanced matrix metalloproteinases-2 activates aortic endothelial hypermeability, apoptosis and vascular rarefaction in spontaneously hypertensive rat.

Microvascular rarefaction with endothelial cells apoptosis is a common characteristic of various microvascular complications in the spontaneously hypertensive rat (SHR). Elevated levels of proteolytic (e.g. matrix metalloproteinase, MMPs) activity and apoptosis in aortic endothelial cells of SHR were found when compared to its normotensive control. However, the exact mechanisms of microvascular rarefaction and the role of MMPs in this process remain poorly understood. Besides cleavage of VEGFR2 via unbalanced MMPs, we hypothesize that selected cleavage of Beta-Catenin and VE-cadherin by MMPs could induce apoptosis of rat aortic endothelial cells (RAECs) and rarefaction. Primary RAECs were isolated, identified and used in a in-vitro model. Transwell system was used to analyze the permeability of Wistar RAECs, SHR RAECs and SHR RAECs with pretreatment by doxycycline. Qualitative and semi-quantitative analysis of major endothelial adhesion molecules were detected by immunofluorescence technique and Western blot, respectively. MMP-2 activity of SHR RAECs was increased significantly and doxycycline (50 μM) effectively reduced the level of MMP-2 and hyper-permeability in SHR RAECs. SHR RAECs showed enhanced cleavage of VEGFR2, VE-cadherin and B-catenin, which could be prevented by doxycycline (50 μM). Doxycycline (50 μM) attenuated hyper-permeability via decreased MMP-2 by protecting VEGFR2, VE-cadherin, Beta-catenin from cleavage and inhibited the reduction of mitochondrial transmembrane potential (MTP), thus prevented mitochondria-mediated apoptotic signaling and capillary rarefaction in the SHR. It might be a novel insight into the mechanisms of SHR microvascular rarefaction that is independent of pressure but relevant to MMP-2.

Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2.

Matrix metalloproteinase-2 (MMP-2), also known as gelatinase A, is involved in vascular calcification. Another member of gelatinases is MMP-9 (gelatinase B). However, the role of gelatinases in the pathogenesis of vascular calcification is not well understood. The current study aims to clarify the relationship between gelatinases and vascular calcification and to elucidate the underlying mechanism. Beta-glycerophosphate (β-GP) was used to induce calcification of vascular smooth muscle cells (VSMCs) with or without 2-[[(4-Phenoxyphenyl)sulfonyl]methyl]-thiirane (SB-3CT), a specific gelatinases inhibitor. Levels of calcification were determined by assessing calcium content and calcification area of VSMCs. Phenotype transition of VSMCs was observed by assessing expressions of alkaline phosphatase (ALP), smooth muscle α-actin (SM-α-actin) and desmin. Gelatin zymography was applied to determine the activities of gelatinases, and western blot was applied to determine expressions of gelatinases, bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (RUNX2) and msh homeobox homolog 2 (Msx-2). Gelatinases inhibition by SB-3CT alleviated calcification and phenotype transition of VSMCs induced by β-GP. Increased gelatinases expression and active MMP-2 were observed in calcifying VSMCs. Gelatinases inhibition reduced expression of RUNX2, Msx-2 and BMP-2. BMP-2 treatment increased expressions of RUNX2 and Msx-2, while noggin, an antagonist of BMP-2, decreased expressions of RUNX2 and Msx-2. Gelatinases promote vascular calcification by upregulating BMP-2 which induces expression of RUNX2 and Msx-2, two proteins associated with phenotype transition of VSMCs in vascular calcification. Interventions targeting gelatinases inhibition might be a proper candidate for ameliorating vascular calcification.

The distinct abilities of tube-formation and migration between brain and spinal cord microvascular pericytes in rats

Pericytes are contractile cells that wrap around the endothelial cells of capillaries throughout the body. They play an important role in regulating the blood brain barrier (BBB) and blood spinal cord barrier (BSCB). The differences between brain and spinal cord microvascular endothelial cells have been investigated. However, no report has elucidated the similarities and differences between brain microvascular pericytes (BMPs) and spinal cord microvascular pericytes (SCMPs) in vitro. The similarities were found between the two types of pericytes not only in the proliferation ability but also in the expression of toll like receptor 4. On the other hand, BMPs showed more than 2 fold in tubular length formation compared with SCMPs. The number of migratory SCMPs was larger than that of migratory BMPs. The expressions of connexin 43 and vascular endothelial growth factor (VEGF) in BMPs were increased compared with those in SCMPs, while SCMPs expressed more desmin and N-cadherin than BMPs. The abilities of tube-formation and migration between BMPs and SCMPs were markedly different, which might be mediated by VEGF, connexin 43, N-cadherin and desmin. These distinguishing features may reflect the more widespread differences between the BBB and BSCB which directly impact pathophysiological processes in various major diseases.

Insulin treatment restores islet microvascular vasomotion function in diabetic mice

The microcirculation plays an important role in the pathogenesis of diabetes and its complications. We hypothesized that pancreatic islet microvascular (PIM) vasomotion, as a parameter of pancreatic islet microcirculation function, is abnormal in diabetic mice and that insulin treatment may reverse this dysfunction.

Functional status of microvascular vasomotion is impaired in spontaneously hypertensive rat

Accumulating evidence demonstrates that microcirculation plays a role in the pathogenesis of hypertension. In the current study, we demonstrated that pancreatic islet microvascular vasomotion of spontaneously hypertensive rats (SHRs) lost the ability to regulate blood flow perfusion and exhibited a lower microvascular blood perfusion pattern which was negative correlated with blood glucose level. SHRs administrated with insulin revealed an improvement of pancreatic islet microvascular vasomotion and blood perfusion pattern. In vitro, the expressions of endothelial nitric oxide synthase (eNOS) and phospho-eNOSser1177 (p-eNOSser1177) were significantly decreased in high glucose exposed islet endothelial cells (iECs), accompanied with a higher ratio of eNOS monomer to eNOS dimer and a significantly increased malondialdehyde and nitrite levels. Meanwhile, barrier function, tube formation and migration capacities of high glucose exposed iECs were significantly inhibited. In contrast, iECs dysfunction induced by glucose toxicity and oxidative stress was attenuated or improved by supplement with insulin, L-arginine and β-mercaptoethanol. In summary, our findings suggest that functional status of pancreatic islet microvascular vasomotion is impaired in SHRs and provide evidence that treatment with insulin, L-arginine and β-mercaptoethanol improves endothelium-dependent microvascular vasomotion and meliorates iECs function due to anti-hyperglycemic and anti-oxidative effects, partly through mechanism involving regulation of eNOS and p-eNOSser1177.

Effects of melatonin on spinal cord injury-induced oxidative damage in mice testis.

This study evaluated the effects of melatonin on spinal cord injury (SCI)‐induced oxidative damage in testes. Adult male C57BL/6 mice were randomly divided into sham‐, SCI‐ or melatonin (10 mg/kg, i.p.)‐treated SCI groups. To induce SCI, a standard weight‐drop method that induced a contusion injury at T10 was used. After 1 week, testicular blood flow velocity was measured using the Laser Doppler Line Scanner. Malondialdehyde (MDA), glutathione (GSH), oxidised glutathione (GSSG) and myeloperoxidase (MPO) were measured in testis homogenates. Microvascular permeability of the testes to Evans Blue was examined by spectrophotometric and fluorescence microscopic quantitation. The tight junction protein zonula occludens‐1 (ZO‐1) and occludin in testes were assessed by immunoblot analysis. Melatonin increased the reduced blood flow and decreased SCI‐induced permeability of capillaries. MDA levels and MPO activity were elevated in the SCI group compared with shams, which was reversed by melatonin. In contrast, SCI‐induced reductions in GSH/GSSG ratio were restored by melatonin. Decreased expression of ZO‐1 and occludin was observed, which was attenuated by melatonin. Overall, melatonin treatment protects the testes against oxidative stress damage caused by SCI.

Absolute circulating pericyte progenitor cell counts in mice by flow cytometry: comparison of 2 single-platform technologies.

Background Accumulating evidence indicates that circulating pericyte progenitor cells (CPPCs) may be angiogenic biomarkers in cancer and diabetes. Their validity as biomarkers depends on the accuracy of techniques used for enumeration. In this report, absolute CPPC counts were performed by 2 single-platform technologies. The reliability of the 2 methods, including retest reliability and intraobserver and interobserver variability, was assessed according to the intraclass correlation coefficient (ICC). The linear correlation and agreement among both methods were assessed, and the stability of CPPC numbers in blood samples was analyzed. Methods The blood samples were obtained from ICR mice. The samples were processed through a no-lyse, 1-wash procedure, and Syto16+CD45-CD31-CD140b+ CPPCs were analyzed by exclusion of dead cells and by fluorescence-minus-one control. CPPCs were enumerated by 2 methods: bead-based 123count eBeads count (eBioscience) and direct volume–based Accuri C6 Flow Cytometer count (BD). The cells were measured immediately and after storage of blood samples for 24 and 48 hours. Results There were excellent retest correlations and intraobserver and interobserver agreement in both methods. The 2 methods showed a high linear correlation (R2 = 0.923) and with a high level of agreement (0.986). It was demonstrated that CPPCs are unstable in blood samples. Conclusions In this study, 2 reproducible protocols for CPPC quantification were established. These protocols should facilitate future studies to further define the role of CPPCs as cellular biomarkers.

Gene expression profiles of glucose toxicity-exposed islet microvascular endothelial cells

Islet microcirculation is mainly composed by IMECs. The aim of the study was to investigate the differences in gene expression profiles of IMECs upon glucose toxicity exposure and insulin treatment.

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon dioxide and waste products. The impairment of microvascular vasomotion might be a crucial step in the development of microcirculation-related diseases. In addition, the highly vascularized pancreatic islet is adapted to support endocrine function. In this respect, it seems possible to infer that the functional status of pancreatic islet microvascular vasomotion might affect pancreatic islet function. Analyzing the pathological changes of the functional status of pancreatic islet microvascular vasomotion may be a feasible strategy to determine contributions that pancreatic islet microcirculation makes to related diseases, such as diabetes mellitus, pancreatitis, etc. Therefore, this protocol describes using a laser Doppler blood flow monitor to determine the functional status of pancreatic islet microvascular vasomotion, and to establish parameters (including average blood perfusion, amplitude, frequency, and relative velocity of pancreatic islet microvascular vasomotion) for evaluation of the microcirculatory functional status. In a streptozotocin-induced diabetic mouse model, we observed an impaired functional status of pancreatic islet microvascular vasomotion. In conclusion, this approach for assessing pancreatic islet microvascular vasomotion in vivo may reveal mechanisms relating to pancreatic islet diseases.