Primary Human Aortic Endothelial Cells Research Articles

Toll-like receptor 4-induced endoplasmic reticulum stress contributes to impairment of vasodilator action of insulin.

Impairment of vasodilator action of insulin is associated with endothelial dysfunction and insulin resistance. Activation of Toll-like receptor 4 (TLR4) induces proinflammatory response and endoplasmic reticulum (ER) stress. Saturated fatty acids (SFA) activate TLR4, which induces ER stress and endothelial dysfunction. Therefore, we determined whether TLR4-mediated ER stress is an obligatory step mediating SFA-induced endothelial dysfunction. Palmitate stimulated proinflammatory responses and ER stress, and this was suppressed by knockdown of TLR4 in primary human aortic endothelial cells (HAEC). Next, we examined the role of TLR4 in vasodilatory responses in intact vessels isolated from wild-type (WT, C57BL/6) and TLR4-KO mice after feeding high-fat (HFD) or normal chow diet (NCD) for 12 wk. Arterioles isolated from HFD WT mice exhibited impaired insulin-stimulated vasodilation compared with arterioles isolated from NCD WT mice. Deficiency of TLR4 was protective from HFD-induced impairment of insulin-stimulated vasodilation. There were no differences in acetylcholine (Ach)- or sodium nitroprusside (SNP)-stimulated vasodilation between the two groups. Furthermore, we examined whether ER stress is involved in SFA-induced impairment of vasodilator actions of insulin. Infusion of palmitate showed the impairment of vasodilatory response to insulin, which was ameliorated by coinfusion with tauroursodeoxycholic acid (TUDCA), an ER stress suppressor. Taken together, the results suggest that TLR4-induced ER stress may be an obligatory step mediating the SFA-mediated endothelial dysfunction.

Differential role of integrin beta 4 in HLA class-I induced endothelial cell activation

Aim The development of anti-donor HLA class I (HLA-I) antibodies following solid organ transplant is associated with acute and chronic rejection and graft failure. Following ligation with antibody, HLA class I molecules form a molecular partnership with integrin β 4 (ITGB4) to transduce activation and proliferative signals in endothelial cells (EC) contributing to rejection. We hypothesize that determination of the domains on the integrin β 4 molecule that interact with HLA-I will provide critical information for the development of novel therapeutics for the treatment of chronic rejection. Methods To identify the ITGB4 intracellular domain(s) that associates with HLA-I, we used the ITGB4 full length (Wild Type) construct or deletion mutants that lack the Calx- β , fibronectin type III repeat FNIII 1-4 or connecting segment (CS) domain . These constructs were sub-cloned into an adenovirus-based vector and adenovirus encoding WT or mutants were generated. Endogenous ITGB4 expression was suppressed using siRNA transfection. Primary human aortic endothelial cells (HAEC) lacking endogenous ITGB4 were infected with adenovirus and then EC activation was determined by analyzing protein phosphorylation, stress fiber formation, cell migration and proliferation. Results Our results demonstrate that HLA-I binds to ITGB4 via the CS domain. The CS domain is required for the phosphorylation of Src, Paxillin, Akt, ERK1/2 and S6RP. The FNIII 3&4 domains are required for phosphorylating Paxillin, Akt, ERK1/2 and S6RP, but not Src. HLA-I phosphorylates ITGB4 at Tyr1494 and Tyr1526 in a Src dependent manner. Furthermore, deletion of CS, FNIII3&4, Calx- β inhibited HLA-I induced EC stress fiber formation, migration and proliferation. Moreover, HLA-I induces association between ITGB4 and the adaptor protein Shc which depends on CS and FNIII3&4 domains. Suppression of endogenous Shc protein by siRNA inhibited HLA-I induced EC migration, proliferation and phosphorylation of Akt, ERK1/2 and S6RP, but not Src. Conclusion Understanding the molecular mechanisms underlying HLA antibody-induced EC proliferation and migration should enable the development of novel therapeutic strategies for the treatment of antibody-mediated chronic transplant rejection.

OR37 Antibody ligation of Hla B*44:03 fails to stimulate endothelial cell proliferation due to a failure to recruit integrin beta4

Aim Antibody-mediated rejection (AMR) is an important clinical problem after solid organ transplantation. Recipients producing posttransplant HLA antibodies (Ab) are at a higher risk for acute and chronic AMR. Notably, not all patients producing post-transplant Ab develop AMR. We postulated that distinct HLA class I alleles may differ in their capacity to transduce signals. Our previous data showed that HLA-B*44:03 was less efficient than other B locus molecules, including B*44:02, to stimulate signal transduction networks in endothelial cells (EC) despite having a similar binding capacity. We tested the hypothesis that failure of B*44:03 to stimulate signal transduction and EC proliferation was due to its inability to recruit integrin β 4. Method Primary human aortic EC (HAEC) were stimulated with human HLA-B12 allele specific mAb, and phosphorylation of signaling proteins was detected by Western Blot. For protein:protein complex formation, HAEC were stimulated with F(ab’)2 fragment of anti-HLA class I (HLA I) mAb W6/32 or mouse IgG as a control. Cell lysates were immunoprecipitated with a mouse anti-HLA-B12 mAb recognizing both B*44:02 and B*44:03 alleles and complex formation between class I and integrin β 4 was detected by Western Blot. Gene silencing was determined with siRNA transfection. EC proliferation was measured by BrdU incorporation and analyzed by flow cytometry. Results Treatment of HAEC with anti-B12 mAb stimulated a increase in phosphorylation of Src, mTOR, ERK and Akt in B*44:02, but not in B*44:03 HAEC. Co-immunoprecipitation experiments showed complex formation between HLA I and integrin β 4 in B*44:02 HAEC, but not in HAEC carrying B*44:03. Furthermore, transfection of EC with integrin β 4 siRNA inhibited HLA-B12 mAb-induced protein phosphorylation and cell proliferation. Conclusions Our results demonstrate that HAEC carrying B*44:03 fail to transduce proliferative signals because they are unable to recruit integrin β 4 into a signaling complex. Disrupting complex formation between integrin β 4 and HLA I may provide a new therapeutic target to prevent AMR.

New Insight Into Metformin Action: Regulation of ChREBP and FOXO1 Activities in Endothelial Cells.

Metformin has been considered a potential adjunctive therapy in treating poorly controlled type 1 diabetes with obesity and insulin resistance, owing to its potent effects on improving insulin sensitivity. However, the underlying mechanism of metformin's vascular protective effects remains obscure. Thioredoxin-interacting protein (TXNIP), a key regulator of cellular redox state induced by high-glucose concentration, decreases thioredoxin reductase activity and mediates apoptosis induced by oxidative stress. Here we report that high glucose-induced endothelial dysfunction is associated with induction of TXNIP expression in primary human aortic endothelial cells exposed to high-glucose conditions, whereas the metformin treatment suppresses high-glucose-induced TXNIP expression at mRNA and protein levels. We further show that metformin decreases the high-glucose-stimulated nuclear entry rate of two transcription factors, carbohydrate response element-binding protein (ChREBP) and forkhead box O1 (FOXO1), as well as their recruitment on the TXNIP promoter. An AMP-activated protein kinase inhibitor partially compromised these metformin effects. Our data suggest that endothelial dysfunction resulting from high-glucose concentrations is associated with TXNIP expression. Metformin down-regulates high-glucose-induced TXNIP transcription by inactivating ChREBP and FOXO1 in endothelial cells, partially through AMP-activated protein kinase activation.

Assessment of the effect of IL-4 on IgE antibody high-affinity (FceRI) and low-affinity (FceRII) receptor mRNA expression on primary human aortic endothelial cells

Assessment of the effect of IL-4 on IgE antibody high-affinity (FceRI) and low-affinity (FceRII) receptor mRNA expression on primary human aortic endothelial cells

Deletion of Protein Tyrosine Phosphatase 1B (PTP1B) Enhances Endothelial Cyclooxygenase 2 Expression and Protects Mice from Type 1 Diabetes-Induced Endothelial Dysfunction.

Protein tyrosine phosphatase 1B (PTP1B) dephosphorylates receptors tyrosine kinase and acts as a molecular brake on insulin signaling pathway. Conditions of metabolic dysfunction increase PTP1B, when deletion of PTP1B protects against metabolic disorders by increasing insulin signaling. Although vascular insulin signaling contributes to the control of glucose disposal, little is known regarding the direct role of PTP1B in the control of endothelial function. We hypothesized that metabolic dysfunctions increase PTP1B expression in endothelial cells and that PTP1B deletion prevents endothelial dysfunction in situation of diminished insulin secretion. Type I diabetes (T1DM) was induced in wild-type (WT) and PTP1B-deficient mice (KO) with streptozotocin (STZ) injection. After 28 days of T1DM, KO mice exhibited a similar reduction in body weight and plasma insulin levels and a comparable increase in glycemia (WT: 384±20 vs. Ko: 432±29 mg/dL), cholesterol and triglycerides, as WT mice. T1DM increased PTP1B expression and impaired endothelial NO-dependent relaxation, in mouse aorta. PTP1B deletion did not affect baseline endothelial function, but preserved endothelium-dependent relaxation, in T1DM mice. NO synthase inhibition with L-NAME abolished endothelial relaxation in control and T1DM WT mice, whereas L-NAME and the cyclooxygenases inhibitor indomethacin were required to abolish endothelium relaxation in T1DM KO mice. PTP1B deletion increased COX-2 expression and PGI2 levels, in mouse aorta and plasma respectively, in T1DM mice. In parallel, simulation of diabetic conditions increased PTP1B expression and knockdown of PTP1B increased COX-2 but not COX-1 expression, in primary human aortic endothelial cells. Taken together these data indicate that deletion of PTP1B protected endothelial function by compensating the reduction in NO bioavailability by increasing COX-2-mediated release of the vasodilator prostanoid PGI2, in T1DM mice.

NOTCH1 regulates matrix gla protein and calcification gene networks in human valve endothelium

Valvular and vascular calcification are common causes of cardiovascular morbidity and mortality. Developing effective treatments requires understanding the molecular underpinnings of these processes. Shear stress is thought to play a role in inhibiting calcification. Furthermore, NOTCH1 regulates vascular and valvular endothelium, and human mutations in NOTCH1 can cause calcific aortic valve disease. Here, we determined the genome-wide impact of altering shear stress and NOTCH signaling on human aortic valve endothelium. mRNA-sequencing of primary human aortic valve endothelial cells (HAVECs) with or without knockdown of NOTCH1, in the presence or absence of shear stress, revealed NOTCH1-dependency of the atherosclerosis-related gene connexin 40 (GJA5), and numerous repressors of endochondral ossification. Among these, matrix gla protein (MGP) is highly expressed in aortic valve and vasculature, and inhibits soft tissue calcification by sequestering bone morphogenetic proteins (BMPs). Altering NOTCH1 levels affected MGP mRNA and protein in HAVECs. Furthermore, shear stress activated NOTCH signaling and MGP in a NOTCH1-dependent manner. NOTCH1 positively regulated endothelial MGP in vivo through specific binding motifs upstream of MGP. Our studies suggest that shear stress activates NOTCH1 in primary human aortic valve endothelial cells leading to downregulation of osteoblast-like gene networks that play a role in tissue calcification.

PTP1B Deletion Enhances Endothelial Cyclooxygenase 2 Expression and Protects Mice from Type 1 Diabetes‐Induced Endothelial Dysfunction

Protein tyrosine phosphatase 1B (PTP1B) dephosphorylates receptors tyrosine kinase and acts as a molecular brake on insulin signaling. Although vascular insulin signaling contributes to the control of glucose disposal, little is known regarding the role of PTP1B in the control of endothelial function. We hypothesized that metabolic dysfunctions increase PTP1B expression in endothelial cells and that PTP1B deletion prevents endothelial dysfunction in situation of diminished insulin secretion. Type I diabetes (T1DM) was induced in wild-type (WT) and PTP1B-deficient mice (KO) with streptozotocin (STZ). After 28 days of T1DM, body weight reduction, decrease in plasma insulin levels and increased in glycemia, cholesterol and triglycerides, were similar in WT and KO mice. T1DM increased PTP1B expression and impaired aortic endothelial NO-dependent relaxation. PTP1B deletion did not affect baseline endothelial NO-dependent relaxation, but preserved endothelium-dependent relaxation, in T1DM mice. NO synthase inhibition with L-NAME abolished endothelial relaxation in control and T1DM WT mice. L-NAME and the cyclooxygenases inhibitor indomethacin were required to abolish endothelium relaxation in T1DM KO mice. PTP1B deletion increased COX-2 expression and PGI2 levels, in mouse aorta and plasma respectively, in T1DM mice. In parallel, simulation of diabetic conditions increased PTP1B expression and knockdown of PTP1B increased COX-2 but not COX-1 expression, in primary human aortic endothelial cells. These data indicate that deletion of PTP1B protected endothelial function by compensating the reduction in NO bioavailability by increasing COX-2-mediated release of the vasodilator prostanoid PGI2, in T1DM mice.

Abstract 11678: Mitochondrial Reactive Oxygen Species Mediate Lysophosphatidylcholine-Induced Endothelial Cell Activation

Background: Lysophosphatidylcholines (LPCs) are a class of pro-inflammatory lipids that play important roles in atherogenesis. LPC activates endothelial cells (ECs) to upregulate adhesion molecules and chemokines, which is the initiation step of atherogenesis. However, the mechanisms underlying LPC-triggered EC activation are not fully understood. Previously considered as the toxic by-products of cellular metabolism, mitochondrial ROS (mtROS) are recently found to directly contribute to both the innate and adaptive immune responses. Here we tested a novel hypothesis that mtROS serve as signaling mediators for LPC-induced EC activation. Methods and Results: Using electron spin resonance and flow cytometry with fluorescence probe MitoSOX, we found that several LPC species including LPC 16:0, 18:0, and 18:1 induced mtROS in human primary aortic ECs (HAECs). Mechanistically, our analysis using mitochondrial calcium inhibitor and Seahorse XF96 mitochondrial function analyzer showed that LPC induced mtROS via increasing mitochondrial calcium entry which resulted in mitochondrial proton leakage. In addition, we found that mtROS scavenger MitoTEMPO abolished LPC-induced EC activation by downregulating Intercellular adhesion molecule 1 (ICAM-1) and Vascular cell adhesion protein 1 (VCAM-1) in HAECs. Moreover, our analysis with transcription factor profiling screening showed that MitoTEMPO acts by blocking LPC-induced nuclear translocation of pro-inflammatory transcription factor activator protein-1 (AP-1). Conclusions: Our results indicate that mtROS are responsible for LPC-induced EC activation and that mtROS may serve as a novel therapeutic target for vascular inflammation.

Glucose and palmitate uncouple AMPK from autophagy in human aortic endothelial cells.

Dysregulated autophagy and decreased AMP-activated protein kinase (AMPK) activity are each associated with atherogenesis. Atherogenesis is preceded by high circulating concentrations of glucose and fatty acids, yet the mechanism by which these nutrients regulate autophagy in human aortic endothelial cells (HAECs) is not known. Furthermore, whereas AMPK is recognized as an activator of autophagy in cells with few nutrients, its effects on autophagy in nutrient-rich HAECs has not been investigated. We maintained and passaged primary HAECs in media containing 25 mM glucose and incubated them subsequently with 0.4 mM palmitate. These conditions impaired basal autophagy and rendered HAECs more susceptible to apoptosis and adhesion of monocytes, outcomes attenuated by the autophagy activator rapamycin. Glucose and palmitate diminished AMPK activity and phosphorylation of the uncoordinated-51-like kinase 1 (ULK1) at Ser555, an autophagy-activating site targeted by AMPK. 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR)-mediated activation of AMPK phosphorylated acetyl-CoA carboxylase, but treatment with AICAR or other AMPK activators (A769662, phenformin) did not restore ULK1 phosphorylation or autophagosome formation. To determine whether palmitate-induced ceramide accumulation contributed to this finding, we overexpressed a ceramide-metabolizing enzyme, acid ceramidase. The increase in acid ceramidase expression ameliorated the effects of excess nutrients on ULK1 phosphorylation, without altering the effects of the AMPK activators. Thus, unlike low nutrient conditions, AMPK becomes uncoupled from autophagy in HAECs in a nutrient-rich environment, such as that found in patients with increased cardiovascular risk. These findings suggest that combinations of AMPK-independent and AMPK-dependent therapies may be more effective alternatives than either therapy alone for treating nutrient-induced cellular dysfunction.

Control of VEGF-A transcriptional programs by pausing and genomic compartmentalization.

Vascular endothelial growth factor A (VEGF-A) is a master regulator of angiogenesis, vascular development and function. In this study we investigated the transcriptional regulation of VEGF-A-responsive genes in primary human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) using genome-wide global run-on sequencing (GRO-Seq). We demonstrate that half of VEGF-A-regulated gene promoters are characterized by a transcriptionally competent paused RNA polymerase II (Pol II). We show that transition into productive elongation is a major mechanism of gene activation of virtually all VEGF-regulated genes, whereas only ∼40% of the genes are induced at the level of initiation. In addition, we report a comprehensive chromatin interaction map generated in HUVECs using tethered conformation capture (TCC) and characterize chromatin interactions in relation to transcriptional activity. We demonstrate that sites of active transcription are more likely to engage in chromatin looping and cell type-specific transcriptional activity reflects the boundaries of chromatin interactions. Furthermore, we identify large chromatin compartments with a tendency to be coordinately transcribed upon VEGF-A stimulation. We provide evidence that these compartments are enriched for clusters of regulatory regions such as super-enhancers and for disease-associated single nucleotide polymorphisms (SNPs). Collectively, these findings provide new insights into mechanisms behind VEGF-A-regulated transcriptional programs in endothelial cells.

OR56: PROTEIN TYROSINE KINASES SRC AND PI3K REGULATE HLA-II ANTIBODY-INDUCED SURVIVAL PROTEIN EXPRESSION IN ENDOTHELIAL CELLS

Antibody-mediated rejection (AMR) is caused by the development of donor-specific antibodies against polymorphic HLA molecules expressed by the transplanted organ. AMR is a sequential process leading to arterial and parenchymal damage, and ultimately transplant vasculopathy (TV) and graft dysfunction. Endothelial cell (EC) apoptosis followed by vascular repair and remodeling contribute to chronic rejection and development of TV. The pro-survival/anti-apoptotic molecules expressed by EC are members of the Bcl-2 protein family including Bcl-2 and Bcl-xL. The aim of this study was to determine whether HLA-II Ab triggers anti-apoptotic protein expression in EC. HLA-II expression was induced in primary human aortic EC by recombinant adenovirus pAd/PL-DEST encoding CIITA (Ad-CIITA), HLA-II transactivator, or treatment with TNFα and IFNγ. HLA-II expression was determined by flow cytometry, phosphorylation of protein kinase and expression of Bcl-2 and Bcl-xL was measured by Western Blot. Infection of EC with Ad-CIITA or pretreatment of EC with cytokines induced a marked increase in HLA-II expression, including DP, DQ, and DR. Ligation of HLA-II antigens on Ad-CIITA infected or cytokine pretreated EC stimulated a significant increase in phosphorylation of Src, PI3K and activation of mTORC1 pathway including increased phosphorylation of S6K and S6RP. Class II ligation also triggered phosphorylation of the mTORC2-targets Akt and ERK. To determine if class II-mediated mTORC2 activation regulates anti-apoptotic protein function we studied the effect of HLA-II ligation on Bcl-2 and Bcl-xL expression. Stimulation of EC with HLA-II Ab increased Bcl-2 and Bcl-xL expression at 24 h which was completely inhibited by pretreatment with the pharmacological inhibitors rapamycin and KU targeting mTORC2. Pretreatment of EC with the MEK inhibitor UO126 blocked HLA-II Ab-induced BclxL expression, but not Bcl-2 expression indicating ERK is upstream of Bcl-xL but not Bcl-2. Ligation of HLA-II with Ab on EC triggered intracellular signal cascades promoting cell survival via two distinct pathways. These studies support a role for HLA class II-mediated pro-survival signaling in the setting of chronic rejection in which antibodies promote endothelial cell survival contributing to TV.

OR23: THE INTEGRIN β4 CONNECTING SEGMENT DOMAIN IS REQUIRED FOR HLA CLASS I-MEDIATED ENDOTHELIAL CELL ACTIVATION

Endothelial cell (EC) activation is a major consequence of HLA class I (HLA-I) antibody-mediated rejection. Previous studies have shown that the molecular association between HLA-I molecule and integrin β 4 (ITGB4) is required for EC migration and proliferation by regulating ERK1/2 signaling pathway. However, the proximal signaling pathway that regulates HLA-I and ITGB4 association remains unclear. We determined the domains of the ITGB4 cytoplasmic tail required for HLA-I mediated phosphorylation pathways and correlated these findings with functional studies. Primary human aortic endothelial cells (HAEC) were used for this study. To identify the ITGB4 intracellular domain that associates with HLA-I, we generated constructs encoding ITGB4 full length (WT) or deletion mutants that lack the Calx- β , fibronectin type III repeat (FNIII 1–4) or connecting segment (CS). These constructs were sub-cloned into adenovirus-based vector and adenovirus encoding WT or mutants were generated. Endogenous ITGB4 expression was suppressed using siRNA transfection. HAEC lacking endogenous ITGB4 were infected with adenovirus and protein phosphorylation and cell migration were further analyzed. Flow cytometry and Western blot analysis showed that both the WT and deletion mutant-infected HAEC had similar levels of cell surface or total expression of ITGB4. Coimmunoprecipitation studies showed that stimulation with the F(ab′)2 fragment of HLA-I antibody increased HLA-I association with ITGB4 in a time dependent manner in WT adenovirus infected HAEC. In contrast, HAEC infected with the CS deletion mutant significantly inhibited HLA-I association with ITGB4 compared to WT, Calx- β or FNIII mutant infected cells. In addition, the CS deletion mutant decreased HLA-I induced ERK1/2 phosphorylation as measured by Western blot. Using in vitro wound healing assay, we found that over expression of ITGB4 increased EC migration compared to LacZ infected cells, whereas CS deletion decreased HLA-I induced EC migration. Furthermore, HLA-I antibody increased the association of ITGB4 with the adaptor protein Shc. This association was decreased by CS deletion. Our results demonstrate that HLA-I binds to ITGB4 via the connecting segment domain that is required for ERK1/2 signaling and EC activation.

OR24: MONOCYTE RECRUITMENT TO HUMAN LEUKOCYTE ANTIGEN CLASS I ANTIBODY-ACTIVATED ENDOTHELIAL CELLS IS DEPENDENT UPON MTOR

Aim A hallmark of antibody (Ab)-mediated rejection is the presence of intravascular monocytes in the allograft. The purpose of this study was to determine the effects of rapamycin on monocyte recruitment and M1/M2 polarization in a clinically relevant in vivo model of antibody mediated-rejection (AMR). Methods Rag1 knock-out mice heterotopically transplanted with mismatched donor hearts were treated with rapamycin and received passive transfer of anti-donor antibodies (MHC I Ab). After 10 days, grafts were examined for histological features of AMR including microvascular inflammation and swelling and mononuclear cell infiltration by immunohistochemistry. Cross-sections of allograft tissue were stained for Mac-2 to assess myeloid infiltration, and for Arginase 1, a marker of polarized M2 macrophage subset. In vitro experiments were used to confirm the role of mTOR in monocyte recruitment to HLA-activated primary human aortic endothelial cells. Results The positive control group, which received anti-donor MHC antibodies, manifested with capillary injury, epicardial and pericardial damage, as well as infiltrating mononuclear cells into the graft vasculature and epicardial regions. Mice in the experimental group, which received daily intraperitoneal rapamycin treatment followed by biweekly anti-donor MHC antibodies, demonstrated reduced intra-graft cellularity and endothelial damage. HE a marker of inflammatory macrophages. Arginase 1 staining revealed M2 + macrophages were present in MHC Ab-treated allografts, and furthermore, the distribution of this subset partially overlapped with that of Mac-2. Though Mac-2 staining was found diffusely throughout the allograft, Arg1 positive macrophages localized primarily to the pericardium and fibrotic regions. Conclusion Antibody-mediated rejection drives the polarization of M2 macrophages, which can promote cell proliferation and tissue repair. mTOR blockade prevents monocyte recruitment to activated endothelium of cardiac allografts. S. Salehi: Grant/Research Support; Company/Organization; NIH.

C. pneumoniae disrupts eNOS trafficking and impairs NO production in human aortic endothelial cells.

Endothelial nitric oxide synthase (eNOS) generated NO plays a crucial physiological role in the regulation of vascular tone. eNOS is a constitutively expressed synthase whose enzymatic function is regulated by dual acylation, phosphorylation, protein-protein interaction and subcellular localization. In endothelial cells, the enzyme is primarily localized to the Golgi apparatus (GA) and the plasma membrane where it binds to caveolin-1. Upon stimulation, the enzyme is translocated from the plasma membrane to the cytoplasm where it generates NO. When activation of eNOS ceases, the majority of the enzyme is recycled back to the membrane fraction. An inability of eNOS to cycle between the cytosol and the membrane leads to impaired NO production and vascular dysfunction. Chlamydia pneumoniae is a Gram-negative obligate intracellular bacterium that primarily infects epithelial cells of the human respiratory tract, but unlike any other chlamydial species, C. pneumoniae displays tropism toward atherosclerotic tissues. In this study, we demonstrate that C. pneumoniae inclusions colocalize with eNOS, and the microorganism interferes with trafficking of the enzyme from the GA to the plasma membrane in primary human aortic endothelial cells. This mislocation of eNOS results in significant inhibition of NO release by C. pneumoniae-infected cells. Furthermore, we show that the distribution of eNOS in C. pneumoniae-infected cells is altered due to an intimate association of the Golgi complex with chlamydial inclusions rather than by direct interaction of the enzyme with the chlamydial inclusion membrane.

Abstract 429: PTP1B, a Link Between Endoplasmic Reticulum Stress and Endothelial Dysfunction

Endothelial dysfunction is a common feature of diabetes and a major contributor to diabetic vascular complications. Despite aggressive diabetes management strategies, preventing diabetes-induced endothelial dysfunction remains a difficult challenge. Endothelium insulin resistance and endoplasmic reticulum (ER) stress have been reported to contribute to endothelial dysfunction. Protein tyrosine phosphatase 1B (PTP1B) is a phosphatase bound to the ER known to regulate insulin sensitivity and ER stress. We hypothesize that diabetes alters PTP1B regulation of endothelial cell function and that inhibiting PTP1B may allow a strategy to prevent diabetes-induced endothelial dysfunction. Using immuno-histochemistry we showed that PTP1B is expressed in the endothelial cells of human saphenous vein, and in human primary aortic endothelial cells. Western-blot and quantitative real-time PCR analysis revealed that diabetes increased PTP1B and ER stress markers expression (CHOP: 1.4±0.2, GRP78: 1.5±0.2, XBP1spliced: 2.1±0.3, XBP1: 1.4±0.2 fold changes), in human saphenous veins. Deletion of PTP1B in obese diabetic mice (db/db) significantly reduced the level of expression (mRNA) of ER stress markers in mouse aorta. Incubation of mouse aortic rings with tunicamycin (tunica) or thapsigargin (thapsi), two ER stress inducers, increased the level of expression of ER stress markers (mRNA) and significantly reduced endothelium-dependent relaxation. (Tunica: -17±5%, thapsi: -30±6%, vs. DMSO). NO synthase inhibition with LNAME completely abolished endothelium-dependent relaxation in vehicle and tunica-treated rings. Neither the reactive oxygen species (ROS) scavenger tempol, nor catalase restored endothelial function, in tunica-treated rings. This suggests that ER stress induces a reduction in NO bioavailability independent of ROS. Aortic rings from PTP1B KO mice were protected against ER stress-induced endothelial dysfunction and exhibited a modest increase in ER stress markers compared to control rings, in response to tunica. All together these data demonstrate that PTP1B deletion prevents ER stress-induced endothelial dysfunction and that targeting PTP1B might be a beneficial approach to restore diabetic endothelial dysfunction.

Nitinol-based nanotubular coatings for the modulation of human vascular cell function.

In this study, we describe the synthesis of an upright nanotubular coating with discrete, exposed nanotubes on top of superelastic Nitinol via anodization and characterization of the surface elemental composition and nickel release rates. We demonstrate, for the first time, that this coating could improve re-endothelialization by increasing the cell spreading and migration of primary human aortic endothelial cells on Nitinol. We also show the potential for reducing neointimal hyperplasia by decreasing the proliferation and expression of collagen I and MMP-2 in primary human aortic smooth muscle cells (HASMC). Furthermore, we did not observe the nanotubular surface to induce inflammation through ICAM-1 expression in HASMC as compared to the flat control. This coating could be used to improve Nitinol stents by reducing restenosis rates and, given the extensive use of Nitinol in other implantable devices, act as a generalized coating strategy for other medical devices.

TNT003, a Novel Anti-C1s Monoclonal Antibody, Inhibits Anti-HLA Antibody Induced Complement Deposition.

PURPOSE: To determine whether the anti-C1s antibody, TNT003, inhibits complement deposition induced by anti-HLA Class I and II antibodies (Ab). METHODS: HLA reference and transplant patient sera were used as sources of anti-HLA Ab. Complement deposition was evaluated using C1qScreen (OneLambda) and C3d solid phase assays (Immucor/Lifecodes). Briefly, single antigen beads were coated with anti HLA Ab, incubated with normal human serum complement, and C1q or C3d production/deposition were detected by monoclonal PE conjugates and quantified by Luminex analysis. For in vitro studies, either HLA-typed primary human aortic endothelial cells (HAECs) or EBV-immortalized B cells (B cells) were incubated with sera containing cognate anti-HLA Ab and normal human complement serum, and deposition of complement proteins was detected by flow cytometry using monoclonal antibodies. Complement split products produced during deposition reactions were measured by ELISA. TNT003 (True North Therapeutics Inc, CA) was titrated into these experiments in a concentration-dependent manner (0-50 μg/mL). RESULTS: The anti-C1s monoclonal Ab, TNT003, inhibited complement deposition in both solid phase and in vitro assays, and complement split product formation during in vitro assays. TNT003 reduced both anti-HLA Class I and II Ab induced C3d but not C1q deposition on single antigen beads in a concentration-dependent manner. In vitro, increasing concentrations of TNT003 were inversely proportional to levels of complement deposition on ECs that had been treated with anti-HLA Ab; complement split product production was also diminished with TNT003 treatment. Similar results were seen using B cells as a source of HLA, as TNT003 addition to the complement deposition reaction decreased levels of complement proteins on the cell surface, as well as split products in the supernatant of these reactions. CONCLUSIONS: We demonstrate here that TNT003, a novel anti-C1s monoclonal Ab, is capable of inhibiting complement activation, deposition, and split product formation induced by anti-HLA Class I and II Ab in both solid phase and cell based in vitro assays. These data suggest TNT003 may be useful in reducing DSA-induced complement-mediated damage in transplant patients experiencing AMR. DISCLOSURES:Thomas, K.: Grant/Research Support, True North Therapeutics Inc. Valenzuela, N.: Grant/Research Support, True North Therapeutics Inc. Parry, G.: Employee, True North Therapeutics Inc. Panicker, S.: Employee, True North Therapeutics Inc. Reed, E.: Grant/Research Support, Pfizer, True North Therapeutics Inc, Novartis, Immucor.

Monocyte Recruitment to HLA Class I Antibody-Activated Endothelial Cells Is Dependent Upon mTOR.

Aim: A hallmark of antibody (Ab)-mediated rejection is the presence of intravascular monocytes in the allograft. The purpose of this study was to identify the role of the mTOR signaling pathway in HLA class I Ab-mediated monocyte recruitment to endothelial cells (EC). Methods: Static and flow-based (ibidi) adhesion assays were used to assess monocyte (MM6) recruitment to HLA-activated primary human aortic endothelial cells. mTOR inhibition was characterized using pharmacological inhibitors (KU 0063794 and sirolimus) at different time points and doses in endothelial cells and monocytes. Results were verified using siRNA knock-down (ko) of rictor and raptor, components of the mTOR pathway. Cell-based ELISA was performed to quantitate Weibel-Palade Body exocytosis; protein phosphorylation was measured by Western blot and quantitated by densitometry. An established murine cardiac transplant model was used to determine the effect of sirolimus treatment on acute AMR. Briefly, Rag1 ko animals were transplanted with an H-2d mismatched heart transplant and received passive transfer of anti-donor Dd+Kd IgG antibodies. Results: Pretreatment of EC for 24 hours with sirolimus or Ku reduced monocyte adherence to EC by > 80%, whereas 2- hour mTOR inhibition resulted in partial inhibition of monocyte binding. siRNA ko of mTOR using siRNA yielded comparable results significantly decreasing monocyte adherence. siRNA against raptor and rictor revealed that both mTORC1 and mTORC2 are involved in HLA-mediated monocyte recruitment. Inhibition of mTOR had no effect on P-selectin induction. Instead, flow-based adhesion assays revealed that mTOR inhibition impairs the ability of EC to support firm adhesion of tethered monocytes by affecting late stage ICAM-1 clustering and its downstream signaling effects. mTOR inhibition in monocytes using sirolimus and Ku significantly impaired monocyte binding to activated endothelium. A reduction in endothelial damage and infiltrating mononuclear cells was observed in vivo in recipients of heart allografts treated with sirolimus. Conclusion: We found that mTOR blockade prevents monocyte recruitment to HLA-activated EC through mTORC1 and mTORC2 signaling pathways. In EC, mTOR inhibition prevents ICAM-1 clustering and blunts firm adhesion of monocytes. Our results provide novel insights into the mechanisms underlying monocyte recruitment in the setting of AMR. DISCLOSURE:Salehi, S.: Grant/Research Support, Pfizer. Reed, E.: Grant/Research Support, Pfizer.

Adaptor protein p66(Shc) mediates hypertension-associated, cyclic stretch-dependent, endothelial damage.

Increased cyclic stretch to the vessel wall, as observed in hypertension, leads to endothelial dysfunction through increased free radical production and reduced nitric oxide bioavailability. Genetic deletion of the adaptor protein p66(Shc) protects mice against age-related and hyperglycemia-induced endothelial dysfunction, as well as atherosclerosis and stroke. Furthermore, p66(Shc) mediates vascular dysfunction in hypertensive mice. However, the direct role of p66(Shc) in mediating mechanical force-induced free radical production is unknown; thus, we studied the effect of cyclic stretch on p66(Shc) activation in primary human aortic endothelial cells and aortic endothelial cells isolated from normotensive and hypertensive rats. Exposure of human aortic endothelial cells to cyclic stretch led to a stretch- and time-dependent p66(Shc) phosphorylation at Ser36 downstream of integrin α5β1 and c-Jun N-terminal kinase. In parallel, nicotinamide adenine dinucleotide phosphate oxidase activation, as well as production of reactive oxygen species, increased, whereas nitric oxide bioavailability decreased. Silencing of p66(Shc) blunted stretch-increased superoxide anion production and nicotinamide adenine dinucleotide phosphate oxidase activation and restored nitric oxide bioavailability. In line with the above, activation of p66(Shc) increased in isolated aortic endothelial cells of spontaneously hypertensive rats compared with normotensive ones. Pathological stretch by activating integrin α5β1 and c-Jun N-terminal kinase phosphorylates p66(Shc) at Ser36, augments reactive oxygen species production via nicotinamide adenine dinucleotide phosphate oxidase, and in turn reduces nitric oxide bioavailability. This novel molecular pathway may be relevant for endothelial dysfunction and vascular disease in hypertension.