Role Of Endothelial Cells Research Articles

A potential role for EphB:Ephrin B expression in the regulation of beta cell and islet endothelial cell communication

Eph receptor tyrosine kinases and their associated ligands Ephrins are localised on cell membranes and require direct cell-cell contact for their activation. EphA-EphrinA mediates bi-directional communication between beta cells within islets to regulate insulin secretion [1], while EphB4 and EphrinB2 play vital signalling roles in endothelial cells during the regulation of angiogenesis. Islet endocrine and endothelial cells maintain direct contact in situ, but the role(s) of Ephrin signalling in islet endocrine-endothelial communication currently remains uninvestigated. Our aim was therefore to investigate whether the EphB signalling pathways are expressed in islets to mediate beta cell- endothelial communication. RT-PCR analysis revealed the expression of EphB4 and EphrinB2/B1 mRNA expression in islets. We have previously shown a rapid loss of endothelial cell (EC) populations during mouse and human islet culture in vitro (72hrs) independently of EC-specific CD31 which was reduced to 16±8% (n=3, p<0.001) after 48 hrs and 5±1.8% (p<0.001) after 72 hrs of culture. These observations are consistent with differenzial expression of EphB4 receptor and the EphrinB1/2 ligands by islet endocrine cells and ECs, respectively. In accordance with this, insulin producing MIN6 cells express EphB4 mRNA. Heterotypic contact co-culture of MIN6 cells with the mouse EC line CCEC-2 resulted in up regulation of EphB4 mRNA expression (6.8-fold), which was associated with increased insulin secretion (MIN6 monoculture 161±53ng/50,000cells/24h; co-culture, 385±86, p<0.05). Our results suggest that modulation of endocrine cell-endothelial cell communication via EphB:EphrinB signalling could provide a novel target to manipulate beta cell function. [1] „EphA-Ephrin-A-Mediated βCell Communication Regulates Insulin Secretion from Pancreatic Islets“ Konstantinova et al., Cell 129, 359–370, April 20, 2007 Funded by Diabetes UK, Diabetes Foundation (UK) and the EU programme „Leonardo da Vinci“

Targeted siRNA delivery to diseased microvascular endothelial cells—Cellular and molecular concepts

Increased insight in the role of endothelial cells in the pathophysiology of cancer, inflammatory and cardiovascular diseases, has drawn great interest in pharmacological interventions aiming at the endothelium in diseased sites. Their location in the body makes them suitable targets for therapeutic approaches based on targeted drug delivery. Functional heterogeneity of the microvascular bed in normal organ homeostasis has been appreciated for a long time, and more recent studies have revealed heterogeneity in endothelial reactivity to inflammatory stimuli as well. Upon stimulation, each organ displays a vascular bed specific pattern of cell adhesion molecules providing challenging opportunities to deliver drugs or small RNAs to organ specific (micro)vascular endothelial subsets. In this review we introduce general concepts of endothelial heterogeneity in relation to disease state and its consequences for targeted therapeutic interventions. Furthermore, we will describe novel approaches to interfere with endothelial cell engagement in disease with a main focus on siRNA therapeutics and currently used nonviral lipid and polymer-based siRNA delivery systems. The last part of this review addresses some technical issues that are essential in proving the concept of target mRNA knock down in a vascular bed specific manner, and the further development of effective endothelial cell specific drug delivery devices.

Dopamine Receptor Type 5 in the Primary Cilia Has Dual Chemo- and Mechano-Sensory Roles

Polycystic kidney disease is characterized by cardiovascular irregularities, including hypertension. Dopamine, a circulating hormone, is implicated in essential hypertension in humans and animal models. Vascular endothelial primary cilia are known to function as mechano-sensory organelles. Although both primary cilia and dopamine receptors play important roles in vascular hypertension, their relationship has never been explored. To determine the roles of the dopaminergic system and mechano-sensory cilia, we studied the effects of dopamine on ciliary length and function in wild-type and mechano-insensitive polycystic mutant cells (Pkd1(-/)(-) and Tg737(orpk/orpk)). We show for the first time that mouse vascular endothelia exhibit dopamine receptor-type 5 (DR5), which colocalizes to primary cilia in cultured cells and mouse arteries in vivo. DR5 activation increases cilia length in arteries and endothelial cells through cofilin and actin polymerization. DR5 activation also restores cilia function in the mutant cells. In addition, silencing DR5 completely abolishes mechano-ciliary function in WT cells. We found that DR5 plays very important roles in ciliary length and function. Furthermore, the chemo-sensory function of cilia can alter the mechano-sensory function through changes in sensitivity to fluid-shear stress. We propose that ciliary DR5 has functional chemo- and mechano-sensory roles in endothelial cells.

Discovery of shear- and side-specific mRNAs and miRNAs in human aortic valvular endothelial cells

The role of endothelial cells (ECs) in aortic valve (AV) disease remains relatively unknown; however, disease preferentially occurs in the fibrosa. We hypothesized oscillatory shear (OS) present on the fibrosa stimulates ECs to modify mRNAs and microRNAs (miRNAs) inducing disease. Our goal was to identify mRNAs and miRNAs differentially regulated by OS and laminar shear (LS) in human AVECs (HAVECs) from the fibrosa (fHAVECs) and ventricularis (vHAVECs). HAVECs expressed EC markers as well as some smooth muscle cell markers and functionally aligned with the flow. HAVECs were exposed to OS and LS for 24 h, and total RNA was analyzed by mRNA and miRNA microarrays. We found over 700 and 300 mRNAs down- and upregulated, respectively, by OS; however, there was no side dependency. mRNA microarray results were validated for 26 of 28 tested genes. Ingenuity Pathway Analysis revealed thrombospondin 1 (Thbs1) and NF-κB inhibitor-α (Nfkbia) as highly connected, shear-sensitive genes. miRNA array analysis yielded 30 shear-sensitive miRNAs and 3 side-specific miRNAs. miRNA validation confirmed 4 of 17 shear-sensitive miRNAs and 1 of 3 side-dependent miRNAs. Using miRWalk and several filtering steps, we identified shear-sensitive mRNAs potentially targeted by shear-sensitive miRNAs. These genes and signaling pathways could act as therapeutic targets of AV disease.

Thrombospondin-1: An Islet Endothelial Cell Signal of Importance for β-Cell Function

OBJECTIVELoss of thrombospondin (TSP)-1 in pancreatic islets has been shown to cause islet hyperplasia. This study tested the hypothesis that endothelial-derived TSP-1 is important for β-cell function.RESEARCH DESIGN AND METHODSIslet function was evaluated both in vivo and in vitro. Messenger RNA and protein expression were measured by real-time PCR and Western blot, respectively. The role of endothelial-derived TSP-1 for β-cell function was determined using a transplantation design in which recipient blood vessels either were allowed to grow or not into the transplanted islets.RESULTSTSP-1–deficient mice were glucose intolerant, despite having an increased β-cell mass. Moreover, their islets had decreased glucose-stimulated insulin release, (pro)insulin biosynthesis, and glucose oxidation rate, as well as increased expression of uncoupling protein-2 and lactate dehydrogenase-A when compared with control islets. Almost all TSP-1 in normal islets were found to be derived from the endothelium. Transplantation of free and encapsulated neonatal wild-type and TSP-1–deficient islets was performed in order to selectively reconstitute with TSP-1–positive or –negative blood vessels in the islets and supported that the β-cell defects occurring in TSP-1–deficient islets reflected postnatal loss of the glycoprotein in the islet endothelial cells. Treatment of neonatal TSP-1–deficient mice with the transforming growth factor (TGF)β-1–activating sequence of TSP-1 showed that reconstitution of TGFβ-1 activation prevented the development of decreased glucose tolerance in these mice. Thus, endothelial-derived TSP-1 activates islet TGFβ-1 of importance for β-cells.CONCLUSIONSOur study indicates a novel role for endothelial cells as functional paracrine support for pancreatic β-cells.

Anti-inflammatory and anti-angiogenic effect of long chain n-3 polyunsaturated fatty acids in intestinal microvascular endothelium

The role of endothelial cells in inflammatory bowel disease has been recently emphasized. Endothelial activation and expression of adhesion molecules are critical for leukocytes recruitment into the inflammatory wall. Compelling evidence demonstrated anti-inflammatory effects of long chain n-3 PUFA in inflammatory models. We previously showed that long chain n-3 PUFA (EPA and DHA) inhibited inflammatory response in epithelial and dendritic cells. As long chain n-3 PUFA treatment led to a decreased expression of adhesion molecules in endothelial cells from other organs, we have now investigated their effect on intestinal endothelial cells invitro and in colitic rats. In vitro study: Primary culture of human intestinal microvascular endothelial cells (HIMEC) were pre-treated with DHA and then incubated with IL-1β. In vivo study: Colitis was induced in 2 groups at day0 by intrarectal injection of 2-4-6-trinitrobenzen sulfonic acid (TNBS). Rats received by gavage either fish oil, rich in EPA and DHA (TNBS+n-3) or an isocaloric isolipidic oil formula for 14 days. DHA led to a decreased VCAM-1, TLR4, cyclooxygenase-2 and VEGFR2 expression and a decreased production of IL-6, IL-8 and GM-CSF and a reduced production of PGE(2) and LTB(4) (p<0.001) in IL-1β-induced HIMEC. Similarly, dietary intervention with fish oil rich in EPA and DHA significantly decreased colon production of PGE(2) and LTB(4,) endothelial VCAM-1 and VEGFR2 in rats with colitis. Data obtained from invitro and invivo studies reveal a potential anti-angiogenic role of long chain n-3 PUFA in intestinal endothelial cells. This protective effect of long chain n-3 PUFA may partly explain the observed benefit of dietary intake of long chain n-3 PUFA in IBD development.

Inflammation and Acute Venous Thrombosis

Inflammation is known as a controlled response to an injury that protects against further injury and clears damaged tissue. However, uncontrolled inflammation can lead to a marked breakdown of the extracellular matrix as well as organ destruction. Stewart et al. demonstrated in a vein thrombosis model that leukocytes migrate into vein walls with an intact layer of endothelial cells, subsequently linking inflammation and thrombosis. Since then, multiple studies have reported on the interaction between inflammation and vein thrombosis. This article summarizes published material that assesses the relationship between inflammation and acute vein thrombosis. The article will focus on the role of endothelial cells, leukocytes, platelets, and microparticles in acute vein thrombosis and discuss the role of thrombin, P-selectin, platelet-activating factor, endothelin-1, and nitric oxide from the perspective of inflammation.

Nuclear Receptor COUP-TFII Controls Pancreatic Islet Tumor Angiogenesis by Regulating Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor-2 Signaling

The significance of angiogenesis in cancer biology and therapy is well established. In this study, we used the prototypical RIP-Tag model of multistage pancreatic islet tumorigenesis to show that the nuclear receptor COUP-TFII is essential to regulate the balance between pro- and anti-angiogenic molecules that influence the angiogenic switch in cancer. Conditional ablation of COUP-TFII in the tumor microenvironment severely compromised neoangiogenesis and lymphangiogenesis during pancreatic tumor progression and metastasis. We found that COUP-TFII plays a cell-autonomous role in endothelial cells to control blood vessel sprouting by regulating cell proliferation and migration. Mechanistic investigations revealed that COUP-TFII suppressed vascular endothelial growth factor (VEGF)/VEGF receptor-2 (VEGFR-2) signaling by transcriptionally repressing the expression of VEGFR-1, thereby curtailing a central angiogenic driver of vascular growth. Taken together, our results implicate COUP-TFII as a critical factor in tumor angiogenesis through regulation of VEGF/VEGFR-2 signaling, suggesting COUP-TFII as a candidate target for antiangiogenic therapy.

Role of Endothelial Cells in Myocardial Ischemia-Reperfusion Injury.

Minimizing myocardial ischemia-reperfusion injury has broad clinical implications and is a critical mediator of cardiac surgical outcomes. "Ischemic injury" results from a restriction in blood supply leading to a mismatch between oxygen supply and demand of a sufficient intensity and/or duration that leads to cell necrosis, whereas ischemia-reperfusion injury occurs when blood supply is restored after a period of ischemia and is usually associated with apoptosis (i.e. programmed cell death). Compared to vascular endothelial cells, cardiac myocytes are more sensitive to ischemic injury and have received the most attention in preventing myocardial ischemia-reperfusion injury. Many comprehensive reviews exist on various aspects of myocardial ischemia-reperfusion injury. The purpose of this review is to examine the role of vascular endothelial cells in myocardial ischemia-reperfusion injury, and to stimulate further research in this exciting and clinically relevant area. Two specific areas that are addressed include: 1) data suggesting that coronary endothelial cells are critical mediators of myocardial dysfunction after ischemia-reperfusion injury; and 2) the involvement of the mitochondrial permeability transition pore in endothelial cell death as a result of an ischemia-reperfusion insult. Elucidating the cellular signaling pathway(s) that leads to endothelial cell injury and/or death in response to ischemia-reperfusion is a key component to developing clinically applicable strategies that might minimize myocardial ischemia-reperfusion injury.

Leptin Signalling Coordinates Lipid Oxidation with Thermogenesis and Defence Against Oxidative Stress

[See original article on page 955]

혈관내피세포 탈착에 미치는 melatonin의 병리학적 영향

항산화 기능과 면역 억제 기능을 갖는 것으로 알려진 melatonin이 혈관 내피층에서는 어떤 기능을 갖는지 알기 위한 일련의 실험을 수행하였다. 본 연구의 실험 결과, 혈관기능과 관련된 혈관내피세포의 성장, 사멸, 이동에 melatonin은 특이적인 효과를 나타내지 않았고, 백혈구의 혈관내피세포 부착과 백혈구 동종간의 응집에도 melatonin의 역할이 관찰되지 않았다. 이와는 대조적으로 melatonin은 PP2A를 통해 eNOS의 활성을 억제하여 산화질소의 양을 감소시키고, 이로 인해 혈관내피세포의 탈착이 유발되는 효과가 있음을 확인할 수 있었다. 종합해보면, 혈액 내 고농도의 melatonin은 PP2A 및 eNOS의 활성을 변화시켜 혈관내피세포의 탈착을 상승시킴으로써 혈관내에서 발생할 수 있는 혈전 형성에 의한 병리적 현상을 유발할 수 있다. In this study, we carried out a series of experiments to know whether melatonin, an anti-oxidative and immunosuppressive agent, played an important role in endothelial cells. It was revealed that melatonin had little or no effect on endothelial proliferation, cell death or migration. Additionally, melatonin had no effect on adhesion of THP-1 leukocytes to bovine aortic endothelial cells (BAECs) and THP-1 homotypic cell aggregation. In contrast, it was shown that melatonin diminished the basal level of nitric oxide by PP2A-mediated dephosphorylation of endothelial nitric oxide synthase (eNOS), leading to enhanced detachment of BAEC from the extracellular matrix. Collectively, melatonin in high doses decreases the NO production via regulations of PP2A and eNOS activities, inducing detachment of endothelial cells, a possible initial step for thrombosis.

Bioinformatic identification and characterization of human endothelial cell-restricted genes

BackgroundIn this study, we used a systematic bioinformatics analysis approach to elucidate genes that exhibit an endothelial cell (EC) restricted expression pattern, and began to define their regulation, tissue distribution, and potential biological role.ResultsUsing a high throughput microarray platform, a primary set of 1,191 transcripts that are enriched in different primary ECs compared to non-ECs was identified (LCB >3, FDR <2%). Further refinement of this initial subset of transcripts, using published data, yielded 152 transcripts (representing 109 genes) with different degrees of EC-specificity. Several interesting patterns emerged among these genes: some were expressed in all ECs and several were restricted to microvascular ECs. Pathway analysis and gene ontology demonstrated that several of the identified genes are known to be involved in vasculature development, angiogenesis, and endothelial function (P < 0.01). These genes are enriched in cardiovascular diseases, hemorrhage and ischemia gene sets (P < 0.001). Most of the identified genes are ubiquitously expressed in many different tissues. Analysis of the proximal promoter revealed the enrichment of conserved binding sites for 26 different transcription factors and analysis of the untranslated regions suggests that a subset of the EC-restricted genes are targets of 15 microRNAs. While many of the identified genes are known for their regulatory role in ECs, we have also identified several novel EC-restricted genes, the function of which have yet to be fully defined.ConclusionThe study provides an initial catalogue of EC-restricted genes most of which are ubiquitously expressed in different endothelial cells.

Abstract 1281: Endothelial cells promote the cancer stem cell phenotype of human colorectal cancer cells

Abstract Background: Cancer stem cells (CSC), which possess the properties of self-renewal, multi-potential differentiation and tumor initiation, can arise either from normal stem cells or due to the influence of the tumor micro-environment. This study was done to determine the potential role of endothelial cell (EC) derived paracrine factors on promoting the CSC phenotype in human colorectal cancer (CRC) cells. Methods: RFP-labeled human CRC cells HCT116 were co-cultured with RF24 ECs (immortalized HUVECs) under low serum conditions. After FAC-sorting for the CRC cells, the CSC population was analyzed by 1) the Aldefluor assay, 2) sphere forming ability, and 3) Western blot analysis for the CSC markers CD133 and CD44. In addition, low serum condition media obtained from either ECs or parental CRC cells (control), was added to CRC cells to determine its effect on the CSC phenotype. In order to characterize the apoptotic effect of EC derived factors on CRC cells, we performed Annexin V staining by FACS, PARP cleavage, Caspase 3 cleavage, and Bcl2 levels by Western blotting. Using MTT assay, we determined the effect of EC derived factors on chemo-sensitivity of CRC cells exposed to 5-FU. Results: Co-culturing of CRC cells with ECs markedly increased the ALDH-positive population from 5% to 18%, and the sphere forming ability by more than 4-fold in CRC cells. In addition, CRC cells also displayed increased CD133 (6-fold) and CD44 (4-fold) protein levels. Furthermore, this effect could be mimicked simply by co-culturing CRC cells with conditioned media obtained from ECs. Similarly, treatment of CRC cells with conditioned medium from ECs significantly increased the ALDH-positive population from 4% to 8%, sphere forming ability &amp;gt;3-fold, and the expression of CD133 (7-fold) and CD44 (6-fold). Conditioned medium from ECs, concomitantly decreased spontaneous apoptosis in CRC cells as demonstrated by a 3-fold decrease in Annexin V-positive population (21% to 7%), down-regulation of cleaved PARP and Caspase 3, and up-regulation of Bcl2. CRC cells exposed to EC conditioned medium also displayed decreased sensitivity to 5-FU [&amp;gt;10-fold increase of the IC50 from 2.0 μg/ml to 22.3 μg/ml (p&amp;lt;0.05)]. Conclusions: Exposure of CRCs to ECs increased CSC properties, and decreased spontaneous apoptosis. Since, this effect can also be mimicked by the conditioned media, additional experiments to identify and validate the soluble factors that mediate this effect are in progress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1281.

The role of endothelial cells and their progenitors in intimal hyperplasia

Intimal hyperplasia leading to restenosis is the major process that limits the success of cardiovascular intervention. The emergence of vascular progenitor cells and, in particular, endothelial progenitor cells has led to great interest in their potential therapeutic value in preventing intimal hyperplasia. We review the mechanism of intimal hyperplasia and highlight the important attenuating role played by a functional endothelium. The role of endothelial progenitor cells in maintaining endothelial function is reviewed and we describe how reduced progenitor cell number and function and reduced endothelial function lead to an increased risk of intimal hyperplasia. We review other potential sources of endothelial cells, including monocytes, mesenchymal stem cells and tissue resident stem cells. Endothelial progenitor cells have been used in clinical trials to reduce the risk of restenosis with varied success. Progenitor cells have huge therapeutic potential to prevent intimal hyperplasia but a more detailed understanding of vascular progenitor cell biology is necessary before further clinical trials are commenced.

Notch regulates the angiogenic response via induction of VEGFR-1

Notch is a critical regulator of angiogenesis and arterial specification. We show that ectopic expression of activated Notch1 induces endothelial morphogenesis in human umbilical vein endothelial cells (HUVEC) in a VEGFR-1-dependent manner. Notch1-mediated upregulation of VEGFR-1 in HUVEC increased their responsiveness to the VEGFR-1 specific ligand, Placental Growth Factor (PlGF). In mice and human endothelial cells, inhibition of Notch signaling resulted in decreased VEGFR-1 expression during VEGF-A-induced neovascularization. In summary, we show that Notch1 plays a role in endothelial cells by regulating VEGFR-1, a function that may be important for physiological and pathological angiogenesis.

Endothelial IQGAP1 regulates efficient lymphocyte transendothelial migration

Leukocyte movement from the blood to the tissues is a fundamental process in acute and chronic inflammatory diseases. While the role of endothelial cells (EC) to recruit leukocytes to sites of inflammation is well known, the mechanisms that control remodeling of EC shape and adhesive contacts during leukocyte transendothelial migration (TEM) are not completely understood. We studied the role of IQGAP1, an adaptor protein that binds to filamentous-actin and microtubules (MT) at interendothelial junctions, during lymphocyte TEM. EC IQGAP1 knockdown decreases MT tethered to the adherens junction, and decreases lymphocyte TEM to approximately 70% (p<0.05) versus control. Similarly, loss of adherens junction-associated MT induced by brief nocodazole (ND) treatment decreases lymphocyte TEM to approximately 65% of control (p<0.01). Confocal microscopy imaging indicates that EC IQGAP1 knockdown and MT depolymerization both result in lymphocyte accumulation above the vascular endothelial cadherin (VE-cadherin) junctions and reduces the fraction of adherent lymphocytes that complete diapedesis across interendothelial cell junctions. However, we observe no change in VE-cadherin gap formation underlying adherent lymphocytes among control, IQGAP1 knockdown, or MT depolymerised EC monolayers. These data indicate that IQGAP1 contributes to MT stability at endothelial junctions. Further, IQGAP1 is involved in junction remodeling required for efficient lymphocyte diapedesis, independent of VE-cadherin gap formation.

Heat Shock Cognate Protein 70 Is Essential for Akt Signaling in Endothelial Function

Heat shock protein 70s (Hsp70s) are molecular chaperones that protect cells from damage in response to various stress stimuli. However, the functions and mechanisms in endothelial cells (ECs) have not been examined. Herein, we investigate the role of Hsp70s, including heat shock cognate protein 70 (Hsc70), which is constitutively expressed in nonstressed cells (ie, ECs). The Hsp70 inhibitor, KNK437, significantly decreased vascular endothelial growth factor (VEGF)-induced cell migration and tube formation in vitro. KNK437 inhibited the phosphorylation of VEGF-induced Akt and endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells. In a mouse hind limb model of vascular insufficiency, intramuscular inhibition of Hsp70s attenuated collateral and capillary vessel formation. Silencing the Hsc70 gene by short interfering RNA abolished VEGF-induced Akt phosphorylation and VEGF-stimulated human umbilical vein endothelial cell migration and tube formation. As the molecular mechanisms, Hsc70 knockdown reduced the expression of phosphatidylinositol 3-kinase. Collectively, Hsc70 plays a significant role in ECs via the phosphatidylinositol 3-kinase/Akt pathway. Hsc70 may provide the basis for the development of new therapeutic strategies for angiogenesis.

Abstract 5139: Critical Role of CalDAG-GEFI Signaling in Myocardial Ischemia-Reperfusion Injury

Background: Myocardial infarction (MI) is a national health concern, with an occurrence of over 1 million cases per year. An event central to MI is reperfusion injury, i.e. the damage to tissue caused when blood supply returns to the tissue after the period of ischemia. An important role for both leukocytes and platelets in the development of reperfusion injury has been described. Our recent studies demonstrated that the signaling molecule CalDAG-GEFI (Calcium and diacyglycerol-regulated guanine nucleotide exchange factor I) is predominantly expressed in neutrophils and platelets, where it plays a critical role for integrin activation and cell adhesion. Objective: We evaluated if CalDAG-GEFI is a potential target for intervention with reperfusion injury. Methods/Results: Myocardial ischemia was induced for 30 minutes in wild-type (WT) and CalDAG-GEFI−/− mice by a minimally invasive operative procedure. After 24 hours of reperfusion, the mice were sacrificed and the hearts were extracted for further analysis. CalDAG-GEFI-deficient mice showed an approximately 40% reduction in infarct size compared to WT mice (48% vs. 30% in WT and CalDAG-GEFI−/− respectively). In order to rule out that CalDAG-GEFI affects mI/R injury due to its role in endothelial cell and/or cardiomyocyte function, we first studied its expression in these cell types. Compared to the strong expression in both platelets and neutrophils, only trace amounts of CalDAG-GEFI expression were observed in cardiomyocytes or endothelial cells. In a next step, mI/R injury was studied in lethally irradiated CalDAG-GEFI−/− mice reconstituted with bone marrow isolated from WT (KO/WT) or CalDAG-GEFI−/− (KO/KO) mice. As expected, the infarct size in KO/WT chimeras was similar to that observed in WT mice and significantly larger than that of KO/KO chimeras, strongly suggesting that CalDAG-GEFI is critical for the development of mI/R injury due to its role in platelet and/or neutrophil function. Interestingly, CalDAG-GEFI−/− mice showed a stronger protection from mI/R injury than neutrophil-depleted WT mice. Conclusion: Our studies identify CalDAG-GEFI as a novel and promising intracellular therapeutic target for the reduction of myocardial reperfusion injury.

Translation-Linked mRNA Destabilization Accompanying Serum-Induced Nox4 Expression in Human Endothelial Cells

NADPH oxidase is involved in cell signaling, regulating proliferation of vascular cells, especially in endothelium. The Nox4 catalytic subunit has a major role in endothelial cells, but growth arrest of cultured endothelial cells following serum deprivation paradoxically increases mRNA for Nox4. We investigated the relationships between Nox4 mRNA stability and protein expression in human microvascular endothelial cells. Serum starvation increased the steady-state level of Nox4 mRNA but paradoxically diminished Nox4 protein expression. mRNA transcription in the absence of serum is maintained by the p38MAP kinase pathway, for inhibition of p38MAP kinase reduced both Nox4 mRNA and Nox4 promoter activity. In serum-starved cells, reintroduction of serum increased Nox4 protein levels within 30 min and up to 24 h. In contrast, the mRNA decreased equally rapidly after serum stimulation. Inhibition of Nox4 translation by cycloheximide blocked serum-induced mRNA degradation and Nox4 protein synthesis, and actinomycin-D also delayed Nox4 mRNA decay. Therefore, Nox4 mRNA level falls after serum stimulation because of a translation-initiated mRNA destabilization program. Clearly Nox4 mRNA is regulated at both transcriptional and post-transcriptional levels, and the steady state level of Nox4 mRNA does not accurately reflect Nox4 protein abundance and functions, with implications for regulation of cell proliferation and survival.

Hyperglycemia attenuates angiogenic capability of survivin in endothelial cells

Survivin, an anti-apoptotic protein, can be induced by hypoxia and contributes to angiogenic activity in endothelial cells. To determine the potential mechanism of survivin in endothelial dysfunction caused by hyperglycemia in diabetes, we evaluated the role of survivin in hyperglycemia and its effect on endothelial homeostasis. We demonstrated that an increase of d-glucose was sufficient to down-regulate survivin expression, impacting survivin's angiogenic role in endothelial cells. We additionally showed that survivin expression was increased in response to hypoxia yet this reaction was mitigated when the endothelial cells were in hyperglycemic conditions prior to hypoxia. Hyperglycemia also affected survivin-related proliferation and migration of endothelial cells and increased the number of apoptotic cells. In the ischemic porcine myocardium, the expression of survivin was induced. Moreover, survivin expression in the aorta, myocardium, and isolated endothelial cells was attenuated in a porcine model of diabetes in comparison to non-diabetes, which correlated negatively with the levels of fasting blood sugars and positively with territory perfusion. These results demonstrate that hyperglycemia critically alters survivin expression in vitro and in vivo, which leads to attenuation of angiogenic activity and impacts endothelial metabolism.