Rat Microvascular Endothelial Cells Research Articles

Algorithm for biological second messenger analysis with dynamic regions of interest.

Physiological function is regulated through cellular communication that is facilitated by multiple signaling molecules such as second messengers. Analysis of signal dynamics obtained from cell and tissue imaging is difficult because of intricate spatially and temporally distinct signals. Signal analysis tools based on static region of interest analysis may under- or overestimate signals in relation to region of interest size and location. Therefore, we developed an algorithm for biological signal detection and analysis based on dynamic regions of interest, where time-dependent polygonal regions of interest are automatically assigned to the changing perimeter of detected and segmented signals. This approach allows signal profiles to be rigorously and precisely tracked over time, eliminating the signal distortion observed with static methods. Integration of our approach with state-of-the-art image processing and particle tracking pipelines enabled the isolation of dynamic cellular signaling events and characterization of biological signaling patterns with distinct combinations of parameters including amplitude, duration, and spatial spread. Our algorithm was validated using synthetically generated datasets and compared with other available methods. Application of the algorithm to volumetric time-lapse hyperspectral images of cyclic adenosine monophosphate measurements in rat microvascular endothelial cells revealed distinct signal heterogeneity with respect to cell depth, confirming the utility of our approach for analysis of 5-dimensional data. In human tibial arteries, our approach allowed the identification of distinct calcium signal patterns associated with atherosclerosis. Our algorithm for automated detection and analysis of second messenger signals enables the decoding of signaling patterns in diverse tissues and identification of pathologic cellular responses.

NMMHC IIA triggered lipid metabolize reprogramming resulting in vascular endothelial cellular tight junction injury.

Nonmuscle myosin heavy chain IIA, played an essential role in the promotion of tight junction injury in vascular endothelial cells under oxygen glucose deprivation condition. Rat microvascular endothelial cells had been confirmed to have the susceptibility to ox-LDL stimulation under OGD condition. We proposed the hypothesis that lipid metabolic reprogramming might be the root cause for damage to RBMCs tight junction. Untargeted shotgun and targeted lipid metabolomics mass spectrometry approaches combined with principal component analysis was applied to better define the lipids contributing to the variance observed between control and different OGD time. The protein expression of tight junction of RBMCs: occludin, claudin-5, and ZO-1 were detected with immunofluorescence staining and western blot. The proof of the interaction between NMMHC IIA and SREBP1 was investigated via GST-pull down, while their specific binding fragments were also confirmed. The regulation mechanism of NMMHC IIA on SREBP1 was investigated to explore downstream regulatory signaling pathways. Untargeted and targeted shotgun lipidomics data revealed that OGD might be the conditional factor in reshaping lipid components. Mechanistic studies showed that with the increase of OGD time, PCA analysis of lipidomics obtained from RBMCs indicated their specificity in reshaping lipid components, while ≥80% major lipid components phospholipids and sphingolipids transferred from phospholipids, sphingolipids, and neutral lipids, of which neutrallipids taken the largest proportion with OGD time course. Perturbing reprogramming of lipid composition was less susceptible to OGD condition via knockdown of NMMHC IIA of vascular endothelial cells. Knockdown of NMMHC IIA could promote tight junction defense to OGD condition. NMMHC IIA could directly bind with SREBP1, then could affect sterol regulatory element binding protein-1 to adjust lipid metabolize reprogramming of RBMCs. Mechanistic studies showed that perturbing reprogramming of lipid composition could enhance tight junction damage, which was mediated by the opposing effects of NMMHC IIA.

Quantitative analysis of effects of salvianic acid a combined with hydroxy safflower yellow a on rat endothelial cells after hypoxic injury using the combination index method

Cerebrovascular disease is the second most serious disease in the world. It has the features of high morbidity, high mortality and recurrence rate. Numerous research on the compatibility of Chinese medicine with effective ingredients of cerebral ischemia has been made during the past decades. The purpose of this study is to quantitatively analyze the combined pharmacological effect of effective ingredients in Danshen and Honghua (Dan Hong) on rat microvascular endothelial cells after gradually oxygen-glucose deprivation. The experimental concentration range for the compatibility of two effective ingredients were determined in the preliminary experiments by Cell Counting kit-8 (CCK-8) method. Drugs were added to rat brain microvascular endothelial cells at a non-toxic dose level. After that, the cells were cultured for 12 h, and placed in a hypoxic environment. Finally, the cell survival rate was used as a measure of drug effect. In order to determine synergism or antagonism, the combination index (CI)-isobologram method was performed to analyze the data from the experiments. Based on this theory, the potencies of each drug and the shapes of their does-effect curves are both taken into account. The results show that the synergism or the antagonism between two effective ingredients compatibility change with different proportion and dosage. Furthermore, it can be seen from the results of these experiments that when these drugs are used in combination, the dosage required to achieve the same therapeutic effects is greatly reduced compared with the case of single one. It is worth mentioning that our experiments also prove that the median-effect equation and the CI method can be applied in the field of traditional Chinese medicine.

The Short-Chain Fatty Acid Butyrate Attenuates Pulmonary Vascular Remodeling and Inflammation in Hypoxia-Induced Pulmonary Hypertension.

Pulmonary hypertension (PH) is a progressive cardiovascular disorder in which local vascular inflammation leads to increased pulmonary vascular remodeling and ultimately to right heart failure. The HDAC inhibitor butyrate, a product of microbial fermentation, is protective in inflammatory intestinal diseases, but little is known regarding its effect on extraintestinal diseases, such as PH. In this study, we tested the hypothesis that butyrate is protective in a Sprague–Dawley (SD) rat model of hypoxic PH. Treatment with butyrate (220 mg/kg intake) prevented hypoxia-induced right ventricular hypertrophy (RVH), hypoxia-induced increases in right ventricular systolic pressure (RVSP), pulmonary vascular remodeling, and permeability. A reversal effect of butyrate (2200 mg/kg intake) was observed on elevated RVH. Butyrate treatment also increased the acetylation of histone H3, 25–34 kDa, and 34–50 kDa proteins in the total lung lysates of butyrate-treated animals. In addition, butyrate decreased hypoxia-induced accumulation of alveolar (mostly CD68+) and interstitial (CD68+ and CD163+) lung macrophages. Analysis of cytokine profiles in lung tissue lysates showed a hypoxia-induced upregulation of TIMP-1, CINC-1, and Fractalkine and downregulation of soluble ICAM (sICAM). The expression of Fractalkine and VEGFα, but not CINC-1, TIMP-1, and sICAM was downregulated by butyrate. In rat microvascular endothelial cells (RMVEC), butyrate (1 mM, 2 and 24 h) exhibited a protective effect against TNFα- and LPS-induced barrier disruption. Butyrate (1 mM, 24 h) also upregulated tight junctional proteins (occludin, cingulin, claudin-1) and increased the acetylation of histone H3 but not α-tubulin. These findings provide evidence of the protective effect of butyrate on hypoxic PH and suggest its potential use as a complementary treatment for PH and other cardiovascular diseases.

Mechanism of Microvascular Endothelial Cells Protected by Pulsatillae Decoction Activated Neutrophils to Kill Bacteria

Abstract Neutrophils play critical a role in bacterial infection and tumor immunity. The migration of neutrophils across microvascular endothelial cells has been a focus of increased interest within the past decade. A traditional Chinese herbal formula, Pulsatillae Decoction (PD), has the effects of improving the permeability of MVECs, inhibiting apoptosis and enhancing cell activity. But there are very few studies linking the three, and the effect of PD on the bactericidal function of PMNs transendothelial migration is unclear. To investigate the protective effect of PD on the damage of Rat microvascular endothelial cells (RIMVECs) induced by LPS and it’s mechanism of activating neutrophils (PMNs) to kill bacteria by protecting RIMVECs, we established a model of LPS-induced RIMVECs injury and to evaluate the protection of PD against LPS-induced RIMVECs injury. We observed that LPS-injured RIMVECs inhibited the extracellular and intracellular bactericidal activity of transendothelial neutrophils, PD-protected RIMVECs promoted the extracellular and intracellular killing activity of transendothelial neutrophils and significantly increased NE and CG concentration in transendothelial neutrophils. In addition, SWATH/DIA proteome results showed that 11 consensus proteins were down-regulated in the model group and up-regulated in the treatment group, 23 consensus proteins were up-regulated in the model group and down-regulated in the treatment group. PD was likely to protect RIMVECs and activate the sterilization of PMNs by regulating these differential proteins.

Integrative Glycoproteomic and Phosphoproteomic Analyses to reveal Vascular Alterations in Response to a Hyperglycemic Microenvironment

Hyperglycemia‐induced vascular complications are preeminent factors for a decreased life expectancy in patients with diabetes. At a molecular level, increased concentrations of blood glucose is known to accelerate endothelial dysfunction through several negative intracellular signaling events. We hypothesize that these changes are not independent of spontaneous cell surface glycosylation. The cytosolic phosphoproteome and cell surface glycoproteome (both N‐and O‐glycoproteome) were isolated from rat microvascular endothelial cells (RMVECs) cultured under both normal (NG; 4.5 mM) and high glucose (HG; 25 mM) conditions for two weeks. This was followed by tandem mass spectrometry (MS/MS) and integrative bioinformatics analysis. Functional assays, namely tube formation, ROS production, and proliferation assays were performed on RMVECs in the presence or absence of HG conditions. The deleterious impact of hyperglycemic conditions on RMVEC function was confirmed by their inability to form robust tubes in vitro (n=3, p<0.05), and a diminished response to acetylcholine ex vivo (n=3, p<0.05). Hyperglycemic states resulted in differential glycosylation of several receptors, ion channels and cell surface antigens that are critical for endothelial functioning. Tandem MS/MS analysis revealed 87 proteins significantly increased in N‐glycosylation (65 unique) and 143 proteins significantly increased in O‐glycosylation (78 unique) in HG treated versus NG RMVECs (p<0.05). Targets included AT1R (N‐glycosylation: Unique HG, p<0.05), JAM‐A (O‐glycosylation: 2.05 fold, p<0.05) and CD59 (N‐glycosylation: 2.02 fold, p<0.05 and O‐glycosylation: Unique HG, p<0.05). Phosphopeptide pathway enrichment analysis revealed a 25% decrease in phosphorylation status, when filtered for scans (≥ 5) and p‐value (<0.05), in HG treated versus NG RMVECs. Pronounced alterations were observed in several components of insulin (INSR: 1.1 fold, p<0.05 and PKCβ: 2.3 fold, p<0.05), angiogenic (S1PR2: unique HG, p<0.05), and apoptotic (BCL2: −3.75 fold, p<0.05) signaling pathways. Furthermore, elimination of glucose moieties by the treating HG RMVECs with glycosidases, resulted in a normalization of several endothelial functional phenotypes. This study identified a multitude of changes in the glycosylation and phosphorylation status of several surface receptors and cytosolic proteins that are integral to vascular homeostasis. Since glycosidases reversed several of the impairments brought about by a hyperglycemic insult on the endothelium, reversion of dysfunctional endothelial cells to a healthy phenotype by negating a build‐up of glucose moieties on cell surface proteins may be a potential new avenue for the treatment of progressive cardiovascular complications in diabetes.Support or Funding InformationSupport for this project has been provided by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (K01‐DK105043 to BRH) and the Department of Biomedical Engineering at the Medical College of Wisconsin and Marquette University, Milwaukee, WI, USA.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Influence of Sugar and Artificial Sweeteners on Vascular Health during the Onset and Progression of Diabetes

As diabetes and obesity become a rising worldwide heath concern there has been an increased awareness of environmental factors, such as diet, that are contributing to the problem. The negative implications of consuming high amounts of dietary sugar on overall health have long been linked to diabetes, obesity, cardiovascular disease, and other systemic health problems. However, it was not until recently that the negative impact of consuming non‐caloric artificial sweeteners in the place of sugar had been increasingly recognized as a potential contributor to the dramatic increase in diabetes and obesity, along with the associated complications. This study tested the response of the vascular endothelium in vitro and the in vivo response of a diabetes susceptible BB‐DR rat model to glucose, aspartame, and acesulfame potassium supplementation through the use of high‐throughput ‘omics' analyses coupled with functional testing. We have demonstrated that an acute in vitro treatment of rat microvascular endothelial cells with high glucose (25 mM) results in impairment of through glycosylation as exhibited by a restored function with PNGaseF enzymatic removal of N‐glycosylations (p<0.05). Conversely, artificial sweetener treatments also exhibited endothelial impairment in an in vitro tube formation assay (p<0.05); however, gene expression analyses suggested the mechanisms of dysfunction were through different mechanisms than the glucose treatment. Furthermore, through a site‐specific proteomic analysis we have identified 87 proteins significantly increased in N‐glycosylation and 143 in O‐glycosylation (p<0.05) on the endothelial cell surface following high glucose treatment as compared to normal glucose (4.5 mM); many targets related to vascular homeostasis. Those experiments have been supplemented with a comprehensive metabolomics analysis of plasma from an acute three week study on diabetes susceptible BB‐DR rats fed a high glucose, aspartame, or acesulfame potassium diet. Through this set of experiments we have identified unique signatures of alterations in lipid metabolism, among others, following artificial sweetener consumption. Overall, results of this study suggests that exposure to high glucose and artificial sweetener administration lead to unique mechanisms of vascular impairment and homeostatic alterations that may be important during the onset and progression of diabetes and obesity.Support or Funding InformationSupport for this project has been provided by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (K01‐DK105043 to BRH) and the Department of Biomedical Engineering at the Medical College of Wisconsin and Marquette University, Milwaukee, WI, USA.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential response of GK and WKY rat microvascular endothelial cells to a hyperglycemic environment

BackgroundHyperglycemia, a characteristic feature of Type 2 diabetes mellitus (T2DM), is a well‐known causative factor for endothelial cell dysfunction. Both endothelial dysfunction and diabetes are significant contributors to a myriad of cardiovascular abnormalities. Previous experiments in our laboratory revealed significant impairments in the ability of endothelial cells from Goto‐Kakizaki (GK), a well‐established polygenic model of T2DM, to form tubes when cultured under both normal and a two‐week high glucose state, mimicking normal glycemic and hyperglycemic conditions.MethodsIn order to understand the causes and consequences of endothelial dysfunction in diabetes, we characterized the proteome of endothelial cells in both GK rats, and from control, Wistar Kyoto (WKY) rats, under normal and hyperglycemic conditions by tandem mass spectrometry analysis. This was followed by pathway exploration using database searching and protein enrichment analysis softwares. Additionally, functional experiments were performed to assess alterations in the GK rat endothelium under baseline and/or hyperglycemic conditions in relation to our dataset.ResultsSeveral proteins involved in energy regulation, metabolic and cardiovascular functions, were significantly altered in GK rats when compared to WKY rats under normal glycemic conditions. Hyperglycemic conditions significantly altered the levels of proteins involved in cardioprotective and angiogenic functionality to a greater degree in GK endothelial cells when compared to WKY endothelial cells. Mitochondrial protein complexes that specialize in oxidative phosphorylation and electron transport, were significantly altered in GK when compared to WKY endothelial cells, both under baseline, as well as under hyperglycemic states. Mitochondrial respiration assays further confirmed alterations in mitochondrial functioning, under both normal and hyperglycemic conditions, in GK and WKY endothelial cells.ConclusionBy utilizing high‐throughput proteomic analysis, in combination with in vitro functional assays, we have identified discrepancies in the endothelial angiogenic ability and mitochondrial bioenergetics in a model of T2DM. This study not only represents a comprehensive resource for the identification, characterization and functional enrichment of proteins in endothelial cells from GK and WKY rats, but also provides considerable insight into the phenotypic differences and mechanistic drivers of endothelial dysfunction in diabetes.Support or Funding Information: Support for this project has been provided by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (K01‐DK105043 to BRH) and the Department of Biomedical Engineering at the Medical College of Wisconsin and Marquette University, Milwaukee, WI, USA.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanisms of Mas1 Receptor-Mediated Signaling in the Vascular Endothelium.

Angiotensin II (AngII) has been shown to regulate angiogenesis and at high pathophysiological doses to cause vasoconstriction through the AngII receptor type 1. Angiotensin 1 to 7 (Ang-(1-7)) acting through the Mas1 receptor can act antagonistically to high pathophysiological levels of AngII by inducing vasodilation, whereas the effects of Ang-(1-7) signaling on angiogenesis are less defined. To complicate the matter, there is growing evidence that a subpressor dose of AngII produces phenotypes similar to Ang-(1-7). This study shows that low-dose Ang-(1-7), acting through the Mas1 receptor, promotes angiogenesis and vasodilation similar to a low, subpressor dose of AngII acting through AngII receptor type 1. In addition, we show through in vitro tube formation that Ang-(1-7) augments the angiogenic response in rat microvascular endothelial cells. Using proteomic and genomic analyses, downstream components of Mas1 receptor signaling were identified, including Rho family of GTPases, phosphatidylinositol 3-kinase, protein kinase D1, mitogen-activated protein kinase, and extracellular signal-related kinase signaling. Further experimental antagonism of extracellular signal-related kinases 1/2 and p38 mitogen-activated protein kinase signaling inhibited endothelial tube formation and vasodilation when stimulated with equimolar, low doses of either AngII or Ang-(1-7). These results significantly expand the known Ang-(1-7)/Mas1 receptor signaling pathway and demonstrate an important distinction between the pathological effects of elevated and suppressed AngII compared with the beneficial effects of AngII normalization and Ang-(1-7) administration. The observed convergence of Ang-(1-7)/Mas1 and AngII/AngII receptor type 1 signaling at low ligand concentrations suggests a nuanced regulation in vasculature. These data also reinforce the importance of mitogen-activated protein kinase/extracellular signal-related kinase signaling in maintaining vascular function.

Fermented Chinese formula Shuan-Tong-Ling attenuates ischemic stroke by inhibiting inflammation and apoptosis.

The fermented Chinese formula Shuan-Tong-Ling is composed of radix puerariae (Gegen), salvia miltiorrhiza (Danshen), radix curcuma (Jianghuang), hawthorn (Shanzha), salvia chinensis (Shijianchuan), sinapis alba (Baijiezi), astragalus (Huangqi), panax japonicas (Zhujieshen), atractylodes macrocephala koidz (Baizhu), radix paeoniae alba (Baishao), bupleurum (Chaihu), chrysanthemum (Juhua), rhizoma cyperi (Xiangfu) and gastrodin (Tianma), whose aqueous extract was fermented with lactobacillus, bacillus aceticus and saccharomycetes. Shuan-Tong-Ling is a formula used to treat brain diseases including ischemic stroke, migraine, and vascular dementia. Shuan-Tong-Ling attenuated H2O2-induced oxidative stress in rat microvascular endothelial cells. However, the potential mechanism involved in these effects is poorly understood. Rats were intragastrically treated with 5.7 or 17.2 mL/kg Shuan-Tong-Ling for 7 days before middle cerebral artery occlusion was induced. The results indicated Shuan-Tong-Ling had a cerebral protective effect by reducing infarct volume and increasing neurological scores. Shuan-Tong-Ling also decreased tumor necrosis factor-α and interleukin-1β levels in the hippocampus on the ischemic side. In addition, Shuan-Tong-Ling upregulated the expression of SIRT1 and Bcl-2 and downregulated the expression of acetylated-protein 53 and Bax. Injection of 5 mg/kg silent information regulator 1 (SIRT1) inhibitor EX527 into the subarachnoid space once every 2 days, four times, reversed the above changes. These results demonstrate that Shuan-Tong-Ling might benefit cerebral ischemia/reperfusion injury by reducing inflammation and apoptosis through activation of the SIRT1 signaling pathway.

Gene expression comparison reveals distinct basal expression of HOX members and differential TNF-induced response between brain- and spinal cord-derived microvascular endothelial cells.

BackgroundThe heterogeneity of endothelial cell types underlies their remarkable ability to sub-specialize and provide specific requirements for a given vascular bed. Here, we compared rat microvascular endothelial cells (MECs) derived from the brain and spinal cord in both basal and inflammatory conditions.MethodsWe used whole rat genome microarrays to compare, at different time points, basal and TNF-α-induced gene expression of rat MECs from in vitro models of the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB). Validation at both messenger RNA (mRNA) and protein levels was performed on freshly extracted microvessels (MVs) from the brain and spinal cord (BMVs and SCMVs, respectively), as these were considered the closest in vivo tissues to cultured MECs.ResultsMost of the genes encoding adhesion/tight junction molecules and known endothelial markers were similarly expressed in brain and spinal cord MECs (BMECs and SCMECs, respectively). However, one striking finding was the higher expression of several Hox genes, which encode transcription factors involved in positional identity. The differential expression of Hoxa9 and Hoxb7 at the mRNA levels as well as protein levels was confirmed in BMVs and SCMVs. Although the TNF-α response was in general higher in BMECs than in SCMECs at 12 h, the opposite was observed at 48 h. Furthermore, we found that expression of Tnfrsf1a and Tnfrsf1b encoding the TNF receptor super-family member 1a/TNFR1 and 1b/TNFR2, respectively, were constitutively higher in BMVs compared to SCMVs. However, only Tnfrsf1b was induced in SCMECs in response to TNF-α at 24 and 48 h.ConclusionsOur results support a role for HOX members in defining the positional identities of MECs in vivo. Our data also suggest that the delayed transcriptional activation upon TNF-α treatment in SCMECs results from the requirement of the TNF-induced expression of Tnfrsf1b. In contrast, its high basal expression in BMECs might be sufficient to confer an immediate and efficient TNF-α response.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0749-6) contains supplementary material, which is available to authorized users.

Angiotensin‐(1–7) Requires both Mas1 and AT1 Receptors to Restore Angiogenesis

The Renin‐Angiotensin System plays an important role in many physiological processes such as sodium homeostatis, regulating vascular tone and angiogenesis. The octapeptide Angiotensin II (AngII) acting through the AT1 receptor has long been known to promote vasoconstriction, fibrosis, cell proliferation and skeletal muscle angiogenesis in response to exercise or electrical stimulation. A second vasoactive peptide of the Renin‐Angiotensin System, Angiotensin‐1–7 (Ang‐(1–7)), acts through the Mas1 receptor and is generally thought to have many physiological actions that are antagonistic to the actions of the AngII‐AT1 axis, such as vasodilation, anti‐fibrotic and anti‐proliferative effects. We hypothesized Ang‐(1–7) acting through the Mas1 receptor could restore skeletal muscle angiogenesis in response to electrical stimulation. Little is known about the signaling events initiated by Mas1 receptor activation by Ang‐(1–7) relative to AngII‐AT1 activity. Here we show in Sprague Dawley rats with Renin suppressed through high dietary salt intake, which suppresses angiogenesis, that infused Ang‐(1–7) acting through the Mas1 receptor restores angiogenesis in vivo in skeletal muscle in response to electrical stimulation and in vitro in a tube formation assay using rat microvascular endothelial cells, in both cases independent of AngII. Using advanced proteomic methodology to characterize the components of Mas1 receptor signaling we identified signaling pathways that may play a role in the observed angiogenic response, such as nascent protein synthesis, exocytosis, cellular reorganization and regulators such as NO, NF‐kappaB and ERK1/2 signaling. Inhibition of ERK1/2 signaling inhibited tube formation by endothelial cells stimulated by AngII or Ang‐(1–7). We further assessed angiogenesis in response to electrical stimulation promoted by Ang‐(1–7) in SS‐Mas1em1Mcwi (Mas1 mutant knockout) and SS‐Agtr1aem5Mcwi (AT1 mutant knock out) strains, and compared to wild‐type SS/Mcwi. We found reduced angiogenesis to electrical stimulation after Ang‐(1–7) or orally active Ang‐(1–7) analog AVE0991 treatment in both the Mas1 mutant and the AT1 mutant knock outs relative to the SS/Mcwi. The RAS is a complex physiological system that can affect a wide range of processes in the cardiovascular system. Even though the AT1 and Mas1 receptor axes largely have antagonistic functions, we have shown that capillary angiogenesis can both be restored by AngII‐AT1 or Ang‐(1–7)‐Mas1 activity, and the restoration depends on the presence of functioning Mas1 and AT1 receptors.Support or Funding InformationNIH Heart, Lung and Blood Institute 082798

Hyperglycemia‐induced Glycosylation: A Driving Force for Vascular Dysfunction in Diabetes?

Vascular endothelial dysfunction leading to increased risk of coronary artery disease, peripheral vascular disease, and impaired ability to respond to tissue ischemia remains an important clinical problem in type 2 diabetic mellitus (T2DM) patients. Hyperglycemia, a major causative factor in T2DM, is among causes leading to these vascular complications, although the underlying mechanisms of dysfunction are not well understood. It is known that early intensive glycemic control reduces the risk of vascular complications during T2DM; however, there is a large gap in studies of the etiology of hyperglycemia‐induced endothelial dysfunction. T2DM patients with a high glycation index of hemoglobin A1C (HbA1c) have a greater risk of microvascular and macrovascular complications, suggesting there is a correlation between hyperglycemia‐induced glycosylation and the progression of T2DM vascular complications. Clinically, HbA1c glycosylation levels have been used as a predictor of retinopathy, nephropathy, and cardiovascular complications and lowering HbA1c glycosylation levels correlates with improved vascular phenotypes. These studies suggest that high levels of blood glucose correlate with biochemically favorable Maillard reactions that cause spontaneous glycosylation events in the vasculature. The objective of this study was to expand our knowledgebase on the influence of hyperglycemia‐induced glycosylation events in the vascular endothelium and its contribution to the onset and progression of T2DM. Initial characterization of the effects of hyperglycemia on ex vivo vasodilation and in vitro tube formation assays exhibited impaired endothelial phenotypes. In addition, enzymatic removal of glycosylations on the endothelial cell surface was able to restore normal phenotypes. In order to identify specific mechanisms behind the observed endothelial impairment a combination of glycoproteomic, phosphoproteomic, and genomic methodology was used on normal and high glucose treated rat microvascular endothelial cells. These analyses revealed differential glycosylation of important endothelial cell surface receptors, ion channels, and adhesive protein targets; including the angiotensin II (AT1) and serotonin (5HT‐2A) receptors. Additionally, further analysis of gene expression and the synergistic balance between intracellular O‐GlcNAcylation and phosphorylation of treated rat endothelial cells indicated alterations of vasoreactive pathways, inflammatory factors (IL6, IL1R1a), and VEGF signaling important for endothelial function. Overall, this study identified multiple hyperglycemia‐induced endothelial signaling alterations that potentially contribute to peripheral vascular disease in T2DM.Support or Funding InformationThis work was supported by the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (K01DK105043 to BRH) and the Medical College of Wisconsin (MCW) Research Affairs Committee through a new faculty pilot grant (to BRH). Additional support was provided by the MCW Biotechnology and Bioengineering Center.

A protective role of ciglitazone in ox-LDL-induced ratmicrovascular endothelial cells via modulating PPARγ-dependent AMPK/eNOS pathway.

Thiazolidinediones, the antidiabetic agents such as ciglitazone, has been proved to be effective in limiting atherosclerotic events. However, the underlying mechanism remains elucidative. Ox-LDL receptor-1 (LOX-1) plays a central role in ox-LDL-mediated atherosclerosis via endothelial nitric oxide synthase (eNOS) uncoupling and nitric oxide reduction. Therefore, we tested the hypothesis that ciglitazone, the PPARγ agonist, protected endothelial cells against ox-LDL through regulating eNOS activity and LOX-1 signalling. In the present study, rat microvascular endothelial cells (RMVECs) were stimulated by ox-LDL. The impact of ciglitazone on cell apoptosis and angiogenesis, eNOS expression and phosphorylation, nitric oxide synthesis and related AMPK, Akt and VEGF signalling pathway were observed. Our data showed that both eNOS and Akt phosphorylation, VEGF expression and nitric oxide production were significantly decreased, RMVECs ageing and apoptosis increased after ox-LDL induction for 24 hrs, all of which were effectively reversed by ciglitazone pre-treatment. Meanwhile, phosphorylation of AMP-activated protein kinase (AMPK) was suppressed by ox-LDL, which was also prevented by ciglitazone. Of interest, AMPK inhibition abolished ciglitazone-mediated eNOS function, nitric oxide synthesis and angiogenesis, and increased RMVECs ageing and apoptosis. Further experiments showed that inhibition of PPARγ significantly suppressed AMPK phosphorylation, eNOS expression and nitric oxide production. Ciglitazone-mediated angiogenesis and reduced cell ageing and apoptosis were reversed. Furthermore, LOX-1 protein expression in RMVECs was suppressed by ciglitazone, but re-enhanced by blocking PPARγ or AMPK. Ox-LDL-induced suppression of eNOS and nitric oxide synthesis were largely prevented by silencing LOX-1. Collectively, these data demonstrate that ciglitazone-mediated PPARγ activation suppresses LOX-1 and moderates AMPK/eNOS pathway, which contributes to endothelial cell survival and function preservation.

Calreticulin Protects Rat Microvascular Endothelial Cells against Microwave Radiation‐induced Injury by Attenuating Endoplasmic Reticulum Stress

This study was designed to evaluate whether exogenous CRT was beneficial for alleviating MR-induced injury by suppressing ER stress in rat MMECs. MMECs were pretreated with CRT (25 pg/mL) for 12 hours, followed by the exposure to 2.856 GHz radiation at a mean power density of 30 mW/cm(2) for six minutes. MR-induced injury in MMECs was evaluated by LDH leakage, apoptosis, and cell viability analysis. The expression of GRP78, CRT, CHOP, Bcl-2, and Bax were examined by Western blot analysis to reflect ER stress response and ER stress-related apoptosis. MR induced marked MMECs injury, as shown by increased LDH leakage and apoptosis rate and decreased cell viability. MR also induced excessive ER stress, characterized by increased expression of GRP78 and CRT, and ER stress-related apoptotic signaling as well, as shown by the upregulation of CHOP and Bax and the downregulation of Bcl-2. Exogenous CRT pretreatment remarkably attenuated MR-induced cell apoptosis and LDH leakage, ER stress, and activation of the ER stress-related apoptotic signaling. Exogenous CRT attenuates MR-induced ER stress-related apoptosis by suppressing CHOP-mediated apoptotic signaling pathways in MMECs.

Hyperglycemia‐driven glycosylation of the AT1 receptor in the vascular endothelium (1086.8)

Vascular dysfunction, leading to coronary artery and peripheral vascular disease, remains an important problem in type 2 diabetes mellitus (T2DM) patients. In T2DM, hyperglycemia is a major causative factor for vascular endothelial dysfunction, yet there is a large gap in studies of the etiology behind the underlying mechanisms. This study evaluated the hypothesis that hyperglycemia‐induced endothelial glycoproteome changes lead to alterations in homeostatic endothelial signaling pathways. To test this hypothesis, innovative advances in glycoproteome and phosphoproteome enrichment, highly sensitive mass spectrometry, and relevant functional assays were utilized to assess alterations of rat microvascular endothelial cells (RMVECs) cultured in normal (5 mM) or high glucose (25 mM). Differential glycosylation of the type‐1A angiotensin II receptor (AT1; p=0.029) on the RMVEC surface was observed during hyperglycemia, along with glycosylation on other important vascular targets. Additionally, hyperglycemia‐induced changes in the synergistic balance of intracellular O‐GlcNAcylation and phosphorylation, along with inhibition of RMVEC tube formation, revealed alterations in AT1‐receptor signaling processes. Overall, this study identified glycoproteome alterations converging to cause endothelial dysfunction and potentially peripheral vascular disease during hyperglycemia and T2DM.Grant Funding Source: Supported by HL082798 and T32 HL094273

Abstract 230: Evaluation of Hyperglycemia-Driven Alterations on Vascular Endothelial Function

Type 2 diabetes mellitus (T2DM) afflicts ~25 million people in the United States and hyperglycemia is a major causative factor contributing to vascular dysfunction among this population. This study evaluated the hypothesis that hyperglycemia-induced changes in endothelial glycoproteome leads to alterations of important homeostatic signaling pathways that result in vascular dysfunction. Utilizing targeted glycoprotein enrichment coupled with tandem mass spectrometry for protein identification and quantification, the cell surface glycoproteins were assessed from rat microvascular endothelial cells (RMVECs) cultured in ‘normal’ glucose (5 mM) or ‘high’ glucose (25 mM). Altogether, 273 N-glycosylation modified proteins were identified; 65 proteins uniquely N-glycosylated and 22 proteins significantly increased in N-glycosylation (p<0.05) from ‘high’ glucose RMVECs. Additionally, 499 O-glycoproteins were identified; 78 proteins uniquely O-glycosylated and 65 proteins significantly increased in O-glycoslyation (p<0.05) from ‘high’ glucose RMVECs. The type-1A angiotensin II receptor (AT1; p=0.029), along with numerous cell adhesion molecules, had increased N-glycosylation in ‘high’ glucose RMVECs. Numerous ion channels important for vascular function and the 5-HT2A serotonin receptor (p=5.3e-28), recently shown to contribute to vascular dysfunction in T2DM, had elevated O-glycosylation in ‘high’ glucose RMVECs. In order to evaluate these targets, functional assays were developed to measure endothelial and vascular function. Immobilization assays in a parallel plate flow chamber (PPFC) indicated that ‘high’ glucose significantly increased RMVEC adhesion to gelatin substrate under laminar flow (p<0.05). In addition, incubation of RMVECs under ‘high’ glucose conditions increased the adherence of healthy platelets to RMVECs, suggesting the importance of adhesion protein glycosylation in endothelial dysfunction. Finally, studies in human subcutaneous arterioles demonstrated that acute exposure to ‘high’ glucose impaired acetylcholine-induced vasodilation. Utilizing these assays, differentially hyperglycemia-regulated targets will be functionally evaluated for potential therapeutic intervention.

Abstract 232: High Glucose Attenuates In Vitro Tube Growth of Microvascular Endothelial Cells

Aim: To investigate how high glucose affects the ability of microvascular endothelial cells to form tube-like structures in vitro and to develop open source software that automates the analysis of tube formation assays. Background: Over 300 million people worldwide suffer from diabetes. Patients with diabetes experience impaired angiogenesis. Tube formation assays (TFAs) mimic in vivo angiogenesis. During a TFA, endothelial cells that are placed onto a matrigel coated surface arrange into tube-like structures. Quantification of TFAs is difficult; automation typically requires a fluorescent stain and a specific software package. Manual quantification is tedious and subject to inter-user variability. In this study, we describe how high glucose affects the ability of rat microvascular endothelial cells (RMVECs) to form tube-like structures and the development of an open source tool called “Pipeline” that analyzes brightfield TFA images quantifying the total tube length and number of branchpoints. Methods: RMVECs were cultured in media with normal (5.6 mM) or high glucose (25 mM) for one and two week intervals. RMVECs were added to four-chamber slides coated with growth factor reduced Matrigel. After 24 and 48 hours, brightfield images were taken of the TFA. Images were analyzed by manually tracing the total tube length in MetaMorph image processing software or automatically using Pipeline. Results from automatic and manual analysis were compared. The algorithm was validated by measuring the total length of images generated in silico . The code was compiled in C and will run on any computer with the freely available MATLAB R2012a runtime environment. Results: A significant decrease in the total tube length was found when comparing groups treated with high glucose for 2 weeks versus 1 weeks (20.3 +/- 1.4mm vs 25.2 +/- 1.3mm, p<0.05). Additionally, cells in high glucose for 2 weeks versus 1 week were found to have significantly less branch points (68 +/- 6.1 vs 90.9 +/- 5.8, p<0.05). When validating the Pipeline algorithm against images generated in silico (at multiple orientations), Pipeline was able to detect total image length within 1% (9216μm vs 9348 +/- 98μm). Analysis using pipeline was found to be 25X faster than manual analysis (7 minutes versus 180 minutes).

Ang‐(1–7) Induced MAS1 Receptor‐Mediated Angiogenesis in the Rat Microvasculature

The renin‐angiotensin system influences hypertension and angiogenesis. Specifically, angiotensin II (AngII) has been shown to regulate angiogenesis and cause vasoconstriction, fibrosis, cellular growth, and migration through the AT1 receptor. Angiotensin 1–7 (Ang(1–7)), whose actions are mediated by the Mas1 receptor, can act antagonistically to AngII by inducing vasodilation, anti‐fibrotic, anti‐hypertrophic, and anti‐proliferative effects. However, Ang‐(1–7) induced MAS1 receptor signaling is much less defined than AngII signaling, especially during angiogenesis. Here we show that Ang‐(1–7) promotes angiogenesis in the microvasculature of the rat hind limb through the Mas1 receptor independently of AngII and the AT1 receptor. Additionally, we show in an ex vivo tube formation assay that Ang‐(1–7) induces an increased angiogenic response among rat microvascular endothelial cells. Utilizing advanced proteomic methodology, downstream components of the Mas1 receptor signaling pathway were also identified to determine possible signaling pathways in the angiogenic response. Signaling pathway components identified included proteins involved in nascent protein synthesis, exocytosis, and cellular reorganization, along with regulators of nitric oxide, G‐protein, and NF‐kappaB signaling.

Vitamin E attenuates P. aeruginosa‐mediated injury in pulmonary endothelial cells

Infections caused by the opportunistic pathogen P. aeruginosa are a serious complication in critically ill patients with often lethal consequences. The high mortality of patients who develop P. aeruginosa pneumonia is associated with acute lung injury, characterized by airspace flooding. We have previously shown that in endothelial cells P. aeruginosa induces the small G‐protein RhoA, increasing actin stress fiber formation, and permeability. Herein, we hypothesize that the antioxidant vitamin, Vitamin E (Vit E) will be beneficial in preventing P. aeruginosa‐mediated damage. In rat microvascular endothelial cells (RMVEC), exposure to the PAK‐strain of P. aeruginosa (MOI 80 for 10 minutes), increased ROS production. This was completely prevented with pretreatment with 2 doses of Vit E, (18 and 1 hr prior to administration of PAK). Electric cell‐substrate impedance sensing (ECIS) was used as a functional assay to measure changes in resistance in confluent monolayers following exposure to PAK (MOI 80). PAK exposure decreased resistance indicating increased permeability. This decrease was attenuated by pretreatment with Vit E. RhoA activation and stress fiber formation by PAK was also completely blocked by pre‐treatment with Vit E. These data suggest a ROS‐component to PAK‐mediated damage and indicate a possible therapeutic role for Vit E.