Human Cardiac Microvascular Endothelial Cells Research Articles

Abstract 18331: Endothelial Microrna-155 Promotes Myocardial Microvascular Permeability and Inflammatory Cell Adhesion in Experimental Septic Cardiomyopathy

Introduction: Septic cardiomyopathy remains a leading cause of death in critically ill patients. Myocardial edema and inflammatory cell recruitment to cardiac interstitial space are proposed mechanisms underlying sepsis-induced contractile dysfunction. Hypothesis: Evaluate the role of endothelial microRNA-155 (miR-155) in experimental septic cardiomyopathy. Methods: Experimental sepsis was induced using LPS injection in WT (n=20) and miR-155-/- (KO; n=20) male mice. Cardiac function was evaluated through echocardiography. Expression of NOS2, NOS3, VASP phosphorylation and myocardial nitrite/nitrate content was evaluated. Cardiac microvascular (MV) permeability was assessed with evans blue-albumin conjugate leaking to extravascular space and myocardial water content. Mouse cardiac MV endothelial cells (MCMVEC; CD31+Sca-1+CD45-) were isolated with FACS. Quantification of miR-155, cytokine mRNA profile and adhesion molecules (ICAM1, VCAM1 and E-selectin) was performed. Human cardiac MV endothelial cells (HCMVEC) were transfected with anti-miR-155 or scramble LNA probe and incubated with LPS (100ng/mL) or vehicle for 24h. Evaluation of cytokine mRNA profile, NOS isoforms, VASP phosphorylation and adhesion molecules was performed. Monocyte adhesion assay was performed using Dil-stained THP1 monocytic cells. Results: LPS exposure increased miR-155 expression in the myocardium, MCMVEC and HCMVEC. KO mice presented with attenuated LPS-induced cardiac dysfunction, MV permeability, NOS2 expression and VASP phosphorylation, compared with WT. KO MCMVEC presented with attenuated LPS-induced cytokine expression and adhesion molecules, compared with WT cells. Mir-155 inhibition in HCMVEC prevented LPS-induced overexpression of cytokines and adhesion molecules, nitric oxide production and monocyte adhesion, through SOCS1-mediated inhibition of STAT3 and NF-kB signaling pathways. Conclusions: Endothelial miR-155 is upregulated in experimental septic cardiomyopathy. MiR-155 knockout attenuates LPS-induced myocardial dysfunction, edema and MV permeability. In MCMVEC and HCMVEC, miR-155 inhibition blunted LPS-induced nitric oxide production, cytokine and cell adhesion molecules overexpression.

Macrophages Aggravate Hypoxia-Induced Cardiac Microvascular Endothelial Cell Injury via Peroxynitrite: Protection by Tongxinluo.

Activated macrophages contribute to endothelial dysfunction; however, it is unclear how peroxynitrite contributes to macrophage-mediated human cardiac microvascular endothelial cell (HCMEC) injury in hypoxia. In macrophage-HCMEC co-cultures subjected to hypoxia, there was an increase in hypoxia-inducible factor (HIF)-1α, HIF-2α, inducible nitric oxide synthase (iNOS), endothelin-converting enzyme (ECE)-1 and cyclooxygenase-2 (COX-2), and concomitant decrease in prostacyclin synthase (PGIS). This was mimicked by a peroxynitrite donor and attenuated by its decomposition catalyst. Tongxinluo (TXL) could decrease HIF-2α, iNOS, ECE-1 and COX-2 and increase PGIS in a dose-dependent manner, with increase of vascular endothelial growth factor. The protein alterations verified the remarkably affected mRNAs, indicating that the effects of TXL were similar to but better than that of peroxynitrite decomposition catalyst. Furthermore, TXL inhibited macrophage-mediated nitrotyrosine accumulation and attenuated HCMEC injury. The results suggest that peroxynitrite contributes to macrophage-mediated HCMEC injury in hypoxia, and TXL attenuates HCMEC injury mainly by inhibiting peroxynitrite.

Scutellarin's Cardiovascular Endothelium Protective Mechanism: Important Role of PKG-Iα.

Scutellarin (SCU), a flavonoid glycoside compound, has been successfully used in clinic for treatment of ischemic diseases in China. In this report, we checked the effects of SCU on endothelium dysfunction (ED) of coronary artery (CA) against myocardial ischemia reperfusion (MIR) injury in vivo. The involvement of PKG-Iα was further studied using cultured endothelial cells subjected to hypoxia reoxygenation (HR) injury in vitro. In rat MIR model, SCU (45 and 90 mg/kg, iv) significantly reduced ischemic size and restored the endothelium-dependent vasodilation of isolated CA rings. PKG inhibitor Rp-8-Br-cGMP (50 μg/kg, iv) could ameliorate the protective effects of SCU. Increase in phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a main substrate of PKG, at Ser 239 was observed in both heart tissue and serum of SCU-treated animals. In cultured human cardiac microvascular endothelial cells (HCMECs), SCU (1 and 10 μM) dose-dependently protected cell viability and increased the mRNA and protein level of PKG-Iα against HR injury. The activity of PKG was also increased by SCU treatment. The activation of PKG–1α was then studied using targeted proteomic analysis (MRM-MS) checking the phosphorylation state of the autophosphorylation domain (aa42-94). Significant decrease in phosphorylation of PKG-Iα at Ser50, Ser72, Ser89 was induced by HR injury while SCU treatment significantly increased the phosphorylation of PKG-Iα, not only at Ser50, Ser72 and Ser89, but also at Ser44 and Thr58 (two novel phosphorylation domains). Our results demonstrate PKG-Iα might play an important role in the protective effects of SCU on ED against MIR injury.

GW26-e1335 siRNA Inhibits AT2 Receptor In decreasing NO Generation by Recombinant Human Angiotensin converting Enzyme 2 in Cardiac Microvascular Endothelial Cells

SiRNA was used to silence AT2 receptor to explore the effect of Ang (1-9) -ACE2-AT2 pathway on NO formation after the impact of recombinant human Angiotensin Converting Enzyme 2 (rhACE2) on the cardiac microvascular endothelial cells (CMVEC). Human cardiac microvascular endothelial cells (CMVEC)

Abstract 272: Hyperhomocysteinemia Potentiated the Impairment of Hydrogen Sulfide-induced Endothelium-derived Hyperpolarization-mediated Vascular Relaxation in Diabetic Db/db Mice

Background: Cumulative evidence indicates that plasma homocysteine (Hcy) level is positively correlated with cardiovascular mortality in Type 2 diabetic patients. Aims: To explore the effects and mechanisms of elevated plasma Hcy level[[Unable to Display Character: ―]] hyperhomocysteinemia (HHcy) on endothelial function in db/db mice. Methods and Results: HHcy was induced in diabetic db/db and non-diabetic db/+ mice fed with a high methionine diet (HM, 2% methionine) for 8 weeks (plasma tHcy=54.31±5.4 and 34.21±4.15 μM). Endothelial function was examined in small mesenteric arteries (SMA) using myographs. In non-diabetic mice, HHcy did not change vascular relaxation to acetylcholine (Ach); whereas, nitric oxide (NO)- and prostacyclin (PGI2)-mediated relaxation to Ach were impaired. Interestingly, endothelium-derived hyperpolarization factor (EDHF)-mediated relaxation to Ach was improved. In diabetic mice, HHcy potentiated the impairment of NO-, PGI2- and EDHF-mediated relaxation to Ach. Moreover, sodium hydrogen sulfide (NaHS), a donor of hydrogen sulfide (H2S), induced EDHF-mediated relaxation which was impaired in diabetic mice and potentiated by HHcy. NS309, a non-specific calcium-activated potassium channel (Kca) activator, significantly improved H2S- and Ach-induced EDHF-mediated relaxation in diabetic mice with HHcy. Tram-34, an intermediate conductance Kca (IK) blocker, but not small conductance Kca blocker apamin, diminished HHcy-induced impairment of EDHF-mediated relaxation in diabetic mice, suggesting that IK inactivation plays a major role. Free sulfide was decreased in plasma and SMA of diabetic mice which was potentiated with HHcy. Superoxide generation was increased and potentiated by HHcy in lung ECs from diabetic mice. Moreover, PEG-SOD improved vascular relaxation in diabetic mice with HHcy. Finally, tyrosine nitration of IK was increased in human cardiac microvascular endothelial cells (ECs) treated with either D-glucose (25 mM) or DL-Hcy (500 μM) for 48h, which was potentiated by a combination of D-glucose and DL-Hcy. Conclusions: H2S is a major EDHF in resistant arteries in mouse. H2S-contributed EDHF-mediated vascular relaxation was impaired in diabetes and was potentiated by HHcy via oxidative stress and IK inactivation.

SRT1720 counteracts glucosamine-induced endoplasmic reticulum stress and endothelial dysfunction.

We hypothesized that a disproportionate activation of the glucosamine (GlcN) pathway, caused by a prolonged exposure to hyperglycaemia, could impair endothelial integrity promoting endoplasmic reticulum (ER) stress. We also tested the possibility that SRT1720 may be able to counteract GlcN-induced ER stress. Human umbilical vein endothelial cells (HUVECs), human cardiac microvascular endothelial cells, and human retinal endothelial cells were treated with GlcN in the presence or absence of the chemical chaperone phenyl butyric acid (PBA) or SRT1720. Expression of ER stress markers, activation of apoptosis, and pro-inflammatory/pro-thrombotic pathways were evaluated by western blot, real-time RT-PCR, and ELISA assays. GlcN treatment resulted in a significantly increased expression of the major ER stress mediators. ER stress activation was paralleled by increased levels of apoptotic markers and by pro-inflammatory/pro-coagulant pathway activation. In HUVECs, ER stress inhibition by PBA alleviated a GlcN-induced pro-apoptotic and pro-inflammatory/pro-thrombotic state, suggesting a crucial role of ER stress in endothelial dysfunction caused by GlcN. Furthermore, SRT1720 treatment abolished GlcN-induced ER stress and reversed its effects on apoptosis and pro-inflammatory/pro-coagulant pathways. This SRT1720 action was mediated by its ability to modulate Raptor acetylation, thus inhibiting mammalian target of Rapamycin complex 1 (mTORC1)-dependent protein synthesis and alleviating ER overload. Our data show that GlcN promotes a pro-apoptotic and pro-inflammatory/pro-thrombotic phenotype in endothelial cells by activating ER stress. The observation that SRT1720, inhibiting ER stress, was able to counteract GlcN effects lays the basis for future studies aimed to exploit this drug and cognate compounds in the treatment of endothelial dysfunction and atherosclerosis.

Tongxinluo (TXL), a Traditional Chinese Medicinal Compound, Improves Endothelial Function After Chronic Hypoxia Both In Vivo and In Vitro.

:Vascular injury after chronic hypoxia leads to endothelial injury and structural damage to tight junctions (TJs), thereby resulting in a variety of cardiovascular diseases. Thus, attenuating hypoxia-induced damage has great significance for the prevention and treatment of cardiovascular disease. The aim of this study was to investigate whether the endothelial protection conferred by tongxinluo (TXL), a traditional Chinese medicinal compound, is related to its regulation of TJ protein expression. In vivo, we found that TXL could promote hypoxia-induced angiogenesis in lung and liver tissue. In vitro, we found that CoCl2 treatment significantly reduced the expression of the TJ proteins occludin, claudin-1, VE-cadherin, and beta-catenin in cultured human cardiac microvascular endothelial cells. TXL pretreatment abrogated the CoCl2-induced downregulation of these TJ proteins. Conversely, overexpression of Krüppel-like factor 4 (KLF4) inhibited the expression of TJ proteins in human cardiac microvascular endothelial cells, an effect that was reversed by TXL pretreatment. Further experiments showed that TXL could promote endothelial cell proliferation by increasing KLF4 phosphorylation, thereby reversing the effect of KLF4 on the expression of TJ proteins. These findings provide a new molecular mechanism for the TXL-induced increase in TJ protein expression.

Chinese medicine Tongxinluo increases tight junction protein levels by inducing KLF5 expression in microvascular endothelial cells.

Tongxinluo (TXL) is a compound prescription formulated according to the meridian theory of traditional Chinese medicine. It may play an important role in cardiovascular protection by improving endothelial cell function. The aim of present study was to investigate whether endothelial protection with TXL is related to its regulation of tight junction protein expression. Human cardiac microvascular endothelial cells (HCMECs) were cultured and treated with 10(-7) mol l(-1) angiotensin II (Ang II) and the different doses of TXL; the expression of tight junction proteins occludin, claudin, VE-cadherin and beta-catenin was determined by Western blotting and real-time PCR. Gain-of-function and loss-of-function of Krüppel-like factor 5 (KLF5) were carried out in HCMEC transfected with either KLF5 adenovirus pAd-KLF5 or siRNA specific for KLF5. Angiotensinogen transgenic mice were treated with TXL by oral administration of TXL of 0.75 g kg(-1) day(-1) , and immunohistochemical staining was performed with antioccludin, anticlaudin, anti-VE-cadherin, antibeta-catenin and anti-KLF5 antibodies. Ang II treatment significantly reduced the expression of tight junction proteins occludin, claudin, VE-cadherin and beta-catenin in cultured HCMECs. TXL pretreatment could abrogate the down-regulation of these tight junction proteins induced by Ang II. Ang II treatment also decreased KLF5 expression at the mRNA and protein levels; TXL pretreatment markedly reversed the inhibitory effect of Ang II on KLF5 expression. Gain-of-function and loss-of-function of KLF5 showed that KLF5 mediated the expression of tight junction proteins in HCMECs. TXL-enhanced expression of the tight junction proteins was mediated by KLF5. In angiotensinogen transgenic mice, TXL also increased the tight junction protein levels by inducing KLF5 expression. Chinese medicine TXL increases tight junction protein levels by inducing KLF5 expression in microvascular endothelial cells.

Tongxinluo inhibits cyclooxygenase-2, inducible nitric oxide synthase, hypoxia-inducible factor-2α/vascular endothelial growth factor to antagonize injury in hypoxia-stimulated cardiac microvascular endothelial cells.

Background:Endothelial dysfunction is considered as the initiating process and pathological basis of cardiovascular disease. Cyclooxygenase-2 (COX-2) and prostacyclin synthase (PGIS), inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) are key enzymes with opposing actions in inflammation and oxidative stress, which are believed to be the major driver of endothelial dysfunction. And in hypoxia (Hx), Hx-inducible factor (HIF)-1α and HIF-2α are predominantly induced to activate vascular endothelial growth factor (VEGF), resulting in abnormal proliferation. Whether and how Tongxinluo (TXL) modulates COX-2, PGIS, iNOS, eNOS, HIF-1α, HIF-2α, and VEGF in Hx-stimulated human cardiac microvascular endothelial cells (HCMECs) have not been clarified.Methods:HCMEC were treated with CoCl2 to mimic Hx and the mRNA expressions of COX-2, PGIS, iNOS, eNOS, HIF-1α, HIF-2α, and VEGF were first confirmed, and then their mRNA expression and protein content as well as the cell pathological alterations were evaluated for TXL treatment with different concentrations. In addition, the effector molecular of inflammation prostaglandin E2 (PGE2) and the oxidative marker nitrotyrosine (NT) was adopted to reflect HCMEC injury.Results:Hx could induce time-dependent increase of COX-2, iNOS, HIF-2α, and VEGF in HCMEC. Based on the Hx-induced increase, TXL could mainly decrease COX-2, iNOS, HIF-2α, and VEGF in a concentration-dependent manner, with limited effect on the increase of PGIS and eNOS. Their protein contents verified the mRNA expression changes, which was consistent with the cell morphological alterations. Furthermore, high dose TXL could inhibit the Hx-induced increase of PGE2 and NT contents, attenuating the inflammatory and oxidative injury.Conclusions:TXL could inhibit inflammation-related COX-2, oxidative stress-related iNOS, and HIF-2α/VEGF to antagonize Hx-induced HCMEC injury.

Ionizing radiation induces immediate protein acetylation changes in human cardiac microvascular endothelial cells.

Reversible lysine acetylation is a highly regulated post-translational protein modification that is known to regulate several signaling pathways. However, little is known about the radiation-induced changes in the acetylome. In this study, we analyzed the acute post-translational acetylation changes in primary human cardiac microvascular endothelial cells 4 h after a gamma radiation dose of 2 Gy. The acetylated peptides were enriched using anti-acetyl conjugated agarose beads. A total of 54 proteins were found to be altered in their acetylation status, 23 of which were deacetylated and 31 acetylated. Pathway analyses showed three protein categories particularly affected by radiation-induced changes in the acetylation status: the proteins involved in the translation process, the proteins of stress response, and mitochondrial proteins. The activation of the canonical and non-canonical Wnt signaling pathways affecting actin cytoskeleton signaling and cell cycle progression was predicted. The protein expression levels of two nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases, sirtuin 1 and sirtuin 3, were significantly but transiently upregulated 4 but not 24 h after irradiation. The status of the p53 protein, a target of sirtuin 1, was found to be rapidly stabilized by acetylation after radiation exposure. These findings indicate that post-translational modification of proteins by acetylation and deacetylation is essentially affecting the radiation response of the endothelium.

Protective Effects of Scutellarin on Human Cardiac Microvascular Endothelial Cells against Hypoxia-Reoxygenation Injury and Its Possible Target-Related Proteins.

Scutellarin (SCU) is one of the main components of traditional Chinese medicine plant Erigeron breviscapus (Vant.) Hand.-Mazz. In this paper, we studied the protective effects of SCU on human cardiac microvascular endothelial cells (HCMECs) against hypoxia-reoxygenation (HR) injury and its possible target-related proteins. Results of MTT assay showed that pretreatment of SCU at doses of 1, 5, and 10 μM for 2 h could significantly inhibit the decrease in cell viability of HCMECs induced by HR injury. Subcellular fractions of cells treated with vehicle control, 1 μM SCU, HR injury, or 1 μM SCU + HR injury were separated by ultracentrifugation. The protein expression profiles of cytoplasm and membrane/nuclei fractions were checked using protein two-dimensional electrophoresis (2-DE). Proteins differentially expressed between control and SCU-treated group, control and HR group, or HR and SCU + HR group were identified using mass spectrometry (MS/MS). Possible interaction network of these target-related proteins was predicted using bioinformatic analysis. The influence of SCU on the expression levels of these proteins was confirmed using Western blotting assay. The results indicated that proteins such as p27BBP protein (EIF6), heat shock 60 kDa protein 1 (HSPD1), and chaperonin containing TCP1 subunit 6A isoform (CCT6A) might play important roles in the effects of SCU.

Inhibition of protein kinase C enhances angiogenesis induced by platelet-derived growth factor C in hyperglycemic endothelial cells.

BackgroundDiabetes is a risk factor for the development of cardiovascular diseases with impaired angiogenesis. We have previously shown that platelet-derived growth factor C (PDGF-C) and its receptor, PDGF receptor α (PDGFR-α) were downregulated in ischemic limbs of diabetic mice, although the underlying mechanisms remained elusive. Protein kinase C (PKC) is a family of serine/threonine kinases and is known to be involved in angiogenesis. The purpose of this study is to elucidate the mechanisms of how PDGF-C/PDGFR-α axis is impaired in diabetes.MethodsHuman umbilical vein endothelial cells (HUVECs) and human cardiac microvascular endothelial cells (HMVECs) cultured in normoglycemic or hyperglycemic conditions were examined. We also examined the effects of PKC inhibition on the PDGF-C/PDGFR-α axis in endothelial cells exposed to hyperglycemia.ResultsHyperglycemia inhibited proliferation and decreased viability of both HUVECs and HMVECs. Hyperglycemic endothelial cells exhibited decreased PDGFR-α expression both at messenger RNA (mRNA) and protein levels, while there was no significant change in expression of PDGF-C. We also found that expression of PKC-α, one of the PKC isoforms, was increased in hyperglycemic endothelial cells and that inhibition of PKC upregulated PDGFR-α expression in these cells. Phosphorylation of extracellular signal-regulated kinase (ERK) and Akt induced by PDGF-C was significantly attenuated in hyperglycemic endothelial cells, whereas inhibition of PKC effectively reversed these inhibitory effects. Moreover, inhibition of PKC also promoted angiogenesis induced by PDGF-C in hyperglycemic endothelial cells, which was not observed in vascular endothelial growth factor-A (VEGF-A)-induced angiogenesis.ConclusionsThese findings suggest that downregulation of the PDGF-C/PDGFR-α axis is involved in impaired angiogenesis of hyperglycemia through upregulation of PKC. Targeting PKC to restore PDGF-C signaling might be a novel therapeutic strategy for the treatment of vascular complications in diabetes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12933-015-0180-9) contains supplementary material, which is available to authorized users.

Mechanism Study of Traditional Medicine Using Proteomics Alone or Integrated with Other Systems Biology Technologies.

The efficacy of traditional medicine such as traditional Chinese medicine (TCM) had been confirmed by many years of clinical use. However, the mechanisms of traditional medicine remained obscure. Since the development of systems biology in 2000, systems biology technologies have been popularly used in the mechanism study of traditional medicine. In this special issue, we particularly picked reviews and research papers related to mechanism study of TCM, TCM diagnosis, or TCM therapy methods using proteomics as well as other systems biology technologies. There are 10 papers collected in this special issue, in which 3 papers are reviews and 7 papers are original research papers. The 3 reviews in the present issue introduce the advancements in applications of proteomics technologies in the study of TCM and the achievements in the mechanism study of TCM. Q. Ji et al. summarized the applications of proteomics technologies in TCM syndrome research as well as the mechanistic study of TCM treatments, including Chinese herbal medicine, Chinese herbal formula, and acupuncture. Furthermore, they introduced the combined analyses of proteomics with other “-omics” technologies in the study of TCM. Y. A. Sulistio and K. Heese discussed the utility of comparative proteomics for a better understanding of the mechanisms involved in TCM activities and its potential application as complementary therapy for the treatment of Alzheimer's disease (AD). Additionally, they reviewed the data from comparative proteomics studies of AD patients and established the relevance of the data with available AD hypotheses and potential TCM-based treatments, most notably regarding the ubiquitin proteasome system. Y. Yang et al. focused on discussing various results of proteomics studies of mechanisms of anticancer TCM, including terpenes, flavonoids, and glycosides. And they suggested future research strategies such as integrating translating mRNA analysis with proteomics in the study of the mechanisms of anticancer TCM. Four of the research papers reported results of mechanism studies of TCM. J.-Y. Shiau et al. studied the mechanism of 2-β-D-glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne, an active component of Bidens pilosa, in human T-cell acute lymphocytic leukemia cells by using combined differential proteomics and bioinformatics approaches. M.-N. Shi et al. reported the protective effects of scutellarin, a flavone isolated from Erigeron breviscapus (Vant.) Hand.-Mazz, on human cardiac microvascular endothelial cells against hypoxia-reoxygenation injury. Possible target-related proteins of scutellarin were searched using proteomics analysis and a possible interaction network was predicted using bioinformatics analysis. C.-C. Guo et al. studied the antiosteoporotic effects and possible target-related proteins of Huangqi Sanxian decoction, a traditional Chinese formula composed of Radix astragali, Epimedii folium, Cistanche herba, Radix notoginseng, Radix Salviae Miltiorrhiae, Corydalis rhizoma, Radix Angelicae sinensis, and Radix Clematidis, in cultured rat osteoblasts. Z. Bo et al. investigated the molecular mechanism of fibroblast regulation and the treatment of recurrent oral ulcer by Shuizhongcao granule-containing serum. Shuizhongcao granule is a TCM formula composed of buffalo horn, urine sediment, callicarpa, and so forth, and it exhibited great efficacy in the clinical treatment of recurrent oral ulcer. In the present issue, there are also 3 research papers reporting about the mechanisms of TCM used for the diagnosis of diseases or specialized TCM therapy methods such as moxibustion. In the TCM system, tongue diagnosis has been an important diagnostic method. X. Liu et al. used the GC/MS technology to determine the potential changes of metabolites and identify special metabolic biomarkers in the tongue coating of H. pylori infected chronic gastritis patients. W.-T. Dang et al. studied the expression of caspase-1 gene transcript variant mRNA in peripheral blood mononuclear cells of patients with primary gout in different TCM syndromes. X.-L. Shi et al. investigated the effects of moxibustion, a traditional Chinese practice that involves heated Artemisia vulgaris (mugwort) stimulation, on hormonal imbalance and ovarian granulosa cell apoptosis in a rat model of perimenopause. In summary, this special issue provides a snapshot of the current status of mechanism studies of traditional medicine, especially TCM, using proteomics and other systems biology technologies. Authors of the present issue highlight both the achievements and challenges faced in the field of the mechanism study of traditional medicine. Hopefully, this publication will help readers to follow mechanism studies of traditional medicine and will contribute to improved applications of proteomics or other systems biology technologies in research of traditional medicine.

Abstract 12083: Lymphatic Vasculature Mediates Cardiac Progenitor Cell Trafficking After Myocardial Infarction

Myocardial infarction (MI) and post-MI heart failure are major causes of morbidity and mortality. Cardiac progenitor cells (CPC) regenerate injured myocardium, due to their ability to migrate and engraft in the wounded area. Yet, the mechanisms governing CPC trafficking in the diseased myocardium are largely unknown. We examined a hitherto unaddressed role of cardiac lymphatic microvessels (LMV) in CPC mobilization to the site of infarction. We detected that in acute and chronic MI, mouse CPC accumulated in the infarct border zone in proximity of LMV and traversed their wall. We showed that human CPC actively intravasated into the lumen of three-dimensional LMV formed by human lymphatic endothelial cells (LEC) in vitro. The binding of CPC to LEC was hindered in the presence of N-cadherin inhibitory peptide or E-selectin neutralizing antibody. CPC displayed unique pattern of interactions with LEC. Specifically, the propensity of CPC to adhere to LEC at basal conditions and after pre-stimulation with an inflammatory mediator TNFα was higher comparing to human cardiac microvascular endothelial cells (CMEC). Trans-migration of CPC through LEC but not CMEC monolayer was enhanced following treatment with TNFα. Interference with the SDF-1/CXCR4 pathway diminished CPC intravasation via LEC but did not affect the CPC ability to traverse CMEC. Intriguingly, we found that the bioactive lipid S1P, secreted by endothelial cells, had a significant impact on CPC migration. S1P binds to 5 different S1P receptors (S1P1-5). We established that the expression of S1P3 was enriched in CPC. Although exposure to S1P mitigated CPC motility, an S1P3-selective agonist evoked strong chemotaxis of CPC, whereas down-regulation of S1P3 blocked CPC translocation. Accordingly, CPC activation with S1P3 agonist reduced their contacts with the extracellular matrix, while S1P ligand induced the organization of adhesion complexes containing β1-integrin subunit. The direct role of S1P3 in CPC trafficking in vivo is supported by our observations that S1P3 expression was increased in the cardiac cells associated with LMV after MI. In conclusion, we provide evidence that specific interactions with LMV may promote CPC trafficking in the infarcted heart facilitating myocardial repair.

GW25-e5361 Traditional Chinese medicine Tongxinluo inhibits ischemia/reperfusion induced apoptosis of human cardiac microvascular endothelial cells via activating the RISK pathways

GW25-e5361 Traditional Chinese medicine Tongxinluo inhibits ischemia/reperfusion induced apoptosis of human cardiac microvascular endothelial cells via activating the RISK pathways

Oxygen consumption of human heart cells in monolayer culture

Tissue engineering in cardiovascular regenerative therapy requires the development of an efficient oxygen supply system for cell cultures. However, there are few studies which have examined human cardiomyocytes in terms of oxygen consumption and metabolism in culture. We developed an oxygen measurement system equipped with an oxygen microelectrode sensor and estimated the oxygen consumption rates (OCRs) by using the oxygen concentration profiles in culture medium. The heart is largely made up of cardiomyocytes, cardiac fibroblasts, and cardiac endothelial cells. Therefore, we measured the oxygen consumption of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), cardiac fibroblasts, human cardiac microvascular endothelial cell and aortic smooth muscle cells. Then we made correlations with their metabolisms. In hiPSC-CMs, the value of the OCR was 0.71±0.38pmol/h/cell, whereas the glucose consumption rate and lactate production rate were 0.77±0.32pmol/h/cell and 1.61±0.70pmol/h/cell, respectively. These values differed significantly from those of the other cells in human heart. The metabolism of the cells that constitute human heart showed the molar ratio of lactate production to glucose consumption (L/G ratio) that ranged between 1.97 and 2.2. Although the energy metabolism in adult heart in vivo is reported to be aerobic, our data demonstrated a dominance of anaerobic glycolysis in an in vitro environment. With our measuring system, we clearly showed the differences in the metabolism of cells between in vivo and in vitro monolayer culture. Our results regarding cell OCRs and metabolism may be useful for future tissue engineering of human heart.

Induction of Autophagy by Tongxinluo Through the MEK/ERK Pathway Protects Human Cardiac Microvascular Endothelial Cells From Hypoxia/Reoxygenation Injury

: In contrast to cardiomyocytes, autophagy in cardiac microvascular endothelial cells (CMECs) during ischemia/reperfusion (I/R) injury has not been fully investigated. Tongxinluo (TXL), a traditional Chinese medicine, was shown to be vascular protective. We aimed to elucidate the role of autophagy and its regulatory mechanisms by TXL in CMECs subjected to I/R injury. CMECs were exposed to different treatments for 30 minutes and subjected to hypoxia/reoxygenation each for 2 hours. The results indicated that hypoxia/reoxygenation significantly induced autophagy, as identified by an increased number of monodansylcadaverine-positive CMECs, increased autophagosome formation, and a higher type II/type I of light chain 3 ratio, but not Beclin-1 expression. Autophagy inhibition using 3-methyladenine was proapoptotic, but rapamycin-induced autophagy was antiapoptotic. TXL enhanced autophagy and decreased apoptosis in a dose-dependent manner, reaching its largest effect at 800 μg/mL. 3-methyladenine attenuated the TXL-promoted autophagy and antiapoptotic effects, whereas rapamycin had no additional effects compared with TXL alone. TXL upregulated mitogen-activated protein kinase and extracellular signal-regulated kinase (ERK) phosphorylation; however, PD98059 abrogated ERK phosphorylation and decreased autophagy and increased apoptosis compared with TXL alone. These results suggest that autophagy is a protective mechanism in CMECs subjected to I/R injury and that TXL can promote autophagy through activation of the mitogen-activated protein kinase/ERK pathway.

TCTAP A-147 TongXinLuo Protects Human Cardiac Microvascular Endothelial Cells from Hypoxia/Reoxygenation Injury by Inducing Autophagy via the MEK/ERK Pathway

In contrast to cardiomyocytes, autophagy in cardiac microvascular endothelial cells (CMECs) during ischemia/reperfusion (I/R) injury has not been fully investigated. Tongxinluo (TXL), which is a traditional Chinese medicine formulation consists of extractions or powders from Radix ginseng, Buthus

Involvement of p38 MAP kinase in alcohol‐induced endothelial hyperpermeability (672.5)

Recently we have demonstrated using a rat model of alcohol binge drinking that alcohol intoxication increases microvascular leakage in the gut. To investigate the endothelial mechanisms involved, we utilized a cultured endothelial cell (EC) model, utilizing human umbilical vein EC, human dermal microvascular EC, or human cardiac microvascular EC. We hypothesized that alcohol‐induced endothelial hyperpermeability is mediated by p38 MAP kinase signaling. Transendothelial electrical resistance (TER) of EC monolayers served as an index of barrier function. The pharmacological inhibitor SB203580 was used to test the role of p38 MAPK. In addition, localization of the intercellular junction protein VE‐cadherin was determined by immunofluorescence labeling. The results show that alcohol at concentrations greater than 20 mM significantly decrease TER and disrupt VE‐Cadherin continuity. Addition of 6 µM of SB203580, 30 min before addition of alcohol significantly inhibited the maximum decrease in TER (‐14.9% ± 1.9 vs. ‐36.8% ± 1.8, p<0.001). These data suggest that alcohol‐induced increase in endothelial permeability occurs due to activation of p38 MAP kinase and disruption of junctional VE‐Cadherin. Supported by NIH R01HL098215, R21AA020049, and the ABMRF/Foundation for Alcohol Research.

Wnt5a inhibits angiogenic function via soluble VEGF receptor 1 expression in human microvascular endothelial cells

Alternative splicing of the gene encoding vascular endothelial growth factor receptor 1(VEGFR1/FLT1) generates an anti‐angiogenic soluble form (sVEGFR1/sFLT). sVEGFR1 has a high affinity for VEGF‐A and can act as a decoy receptor, preventing signaling through the pro‐angiogenic VEGFR2 receptor. Regulation of sVEGFR1 expression in endothelial cells is not fully understood. Herein we show that the non‐canonical Wnt ligand, Wnt5a, attenuates endothelial function through regulation of sVEGFR1. Recombinant Wnt5a induced rapid and sustained expression of the soluble form of VEGFR1 in human cardiac microvascular endothelial cells. In accordance, Wnt5a treatment inhibited VEGF‐induced endothelial migration and tube formation, as well as attenuating endothelial cell proliferation. sVEGFR1 expression in endothelial cells was downregulated by Box5, a Wnt5a competitive antagonist. Wnt5a regulation of VEGFR1 has been implicated in myeloid cells. Since both inflammatory and endothelial cells release Wnt5a, our data suggests Wnt5a may be important in the autocrine and paracrine signals contributing to endothelial dysregulation. These findings provide a new understanding in physiological and pathological regulation of VEGF‐induced angiogenesis. (R37 HL104017)