Partial Carotid Ligation Research Articles

A Peptide Analogue of Selectin Ligands Attenuated Atherosclerosis by Inhibiting Monocyte Activation.

Background Circulating monocytes play a critical role in the pathogenesis of atherosclerosis. Monocyte homing to sites of atherosclerosis is primarily initiated by selectin. Thus, blockade of the interaction of selectins and their ligands holds a significant role in monocyte homing which might be a potential approach to treat atherosclerosis. Here, we investigated the efficacy of a novel peptide analogue of selectin ligands IELLQAR in atherosclerosis. Methods and Results In this study, we firstly measured the effect of the IELLQAR selectin-binding peptide on the inhibition of binding of selectins to monocytes by flow cytometry, which exhibited a dose-dependent inhibitory effect on the binding of the P-, E-, and L-selectins to monocytes, especially the inhibition of P-selectin binding to human peripheral blood monocytes (PBMCs) (half maximal inhibitory concentration (IC50~5 μM)) and THP-1 cells (IC50~10 μM). Furthermore, IELLQAR inhibited P-selectin-induced activation of CD11b on the surface of monocytes and decreased adhesion of monocytes to the endothelium. ApoE−/− mice with or without IELLQAR (1 or 3 mg/kg) fed a Western-type diet (WTD) or which had disturbed blood flow-induced shear stress underwent partial left carotid artery ligation (PLCA) to induce atherosclerosis. In the WTD- and PLCA-induced atherosclerosis models, atherosclerotic plaque formation and monocyte/macrophage infiltration of the arterial wall both decreased in ApoE−/− mice treated with the IELLQAR peptide. Our results also revealed that IELLQAR inhibited the differentiation of monocytes into macrophages through P-selectin-dependent activation of the nuclear factor- (NF-) κB and mammalian target of rapamycin (mTOR) pathways. Conclusion Collectively, our results demonstrated that IELLQAR, a peptide analogue of selectin ligands, inhibited selectin binding to monocytes, which led to subsequent attenuation of atherosclerosis via inhibition of monocyte activation. Hence, use of the IELLQAR peptide provides a new approach and represents a promising candidate for the treatment of atherosclerosis in the early stage of disease.

The polarity protein Scrib limits atherosclerosis development in mice.

The protein Scrib (Scribble 1) is known to control apico-basal polarity in epithelial cells. The role of polarity proteins in the vascular system remains poorly characterized; however, we previously reported that Scrib maintains the endothelial phenotype and directed migration. On this basis, we hypothesized that Scrib has anti-atherosclerotic functions. Tamoxifen-induced Scrib-knockout mice were crossed with ApoE-/- knockout mice and spontaneous atherosclerosis under high-fat diet (HFD), as well as accelerated atherosclerosis in response to partial carotid artery ligation and HFD, was induced. Deletion of Scrib resulted in increased atherosclerosis development in both models. Mechanistically, flow- as well as acetylcholine-induced endothelium-dependent relaxation and AKT phosphorylation was reduced by deletion of Scrib, whereas vascular permeability and leucocyte extravasation were increased after Scrib knockout. Scrib immune pull down in primary carotid endothelial cells and mass spectrometry identified Arhgef7 (Rho Guanine Nucleotide Exchange Factor 7, βPix) as interaction partner. Scrib or Arhgef7 down-regulation by siRNA reduced the endothelial barrier function in human umbilical vein endothelial cells. Gene expression analysis from murine samples and from human biobank material of carotid endarterectomies indicated that loss of Scrib resulted in endothelial dedifferentiation with a decreased expression of endothelial signature genes. By maintaining a quiescent endothelial phenotype, the polarity protein Scrib elicits anti-atherosclerotic functions.

KCa3.1 contributes to atherosclerotic lesion development induced by low, oscillatory flow

Increased expression of KCa3.1 has been found in vascular smooth muscle (VSM), macrophages and T cells in atherosclerotic lesions from humans and mice. Proliferating VSM cells increase expression of KCa3.1, such that it becomes a dominant K+ channel and contributes to VSM cell migration. Previously, we showed that the KCa3.1 inhibitor, TRAM‐34, could inhibit coronary neointimal development following coronary balloon injury in swine. In the current study, we tested the role of KCa3.1 in atherosclerotic lesion development using two mouse models of atherosclerosis. First, partial carotid ligation (PCL), which produces a low, oscillatory (i.e. atheroprone) flow pattern in the left carotid artery was performed on 6–8 week old male Apoe−/− mice. Mice were subsequently placed on a Western diet (WD; TD.88137, Teklad) for 4 weeks and received daily s.c. injections of TRAM‐34 (120 mg/kg) or equal volumes of vehicle (peanut oil). To directly test the role of KCa3.1 we used CRISPR/Cas9 to generate KCa3.1−/−Apoe−/− (DKO) mice. Subsequently, we examined lesion development in the brachiocephalic artery (BCA) of DKO vs. Apoe−/− male mice on a WD for 3 months. In PCL mice, TRAM‐34 treatment reduced lesion size ~50% (p<0.05). In addition, lesions from TRAM‐34 treated mice contained significantly less collagen (6 ± 1% v. 15 ± 2%; p<0.05), fibronectin (14±3% v. 32±3%; p<0.05) and smooth muscle content (19±2% v. 29±3%; p<0.05). Similarly, in BCAs of DKO mice, both lesion size (0.036 vs. 0.118 mm2, p<0.05) and relative stenosis (13.9% vs. 43.0%, p<0.05) were reduced 70% compared to Apoe−/− mice with no effect on medial or lumen area. Neither TRAM‐34 nor KCa3.1 silencing had any effect on total cholesterol or body weight. These data provide the first genetic validation defining a major role of KCa3.1 in lesion development and determining smooth muscle and matrix content of atherosclerotic lesions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Atherosclerosis characterization using lipid-specific photoacoustic imaging and 4D ultrasound strain mapping in mice

Dual-modality photoacoustic tomography (PAT) and four-dimensional ultrasound (4DUS) imaging have recently been used study atherosclerosis progression in small animals. PAT uses pulsed laser light to induce acoustic waves and reconstruct lipid-specific compositional images of tissue. 4DUS captures dynamic volumetric information and can be used to estimate three-dimensional (3D) Green-Lagrange strain using a direct deformation estimation method. Here, we hypothesized that PAT/4DUS can be used to correlate changes in arterial strain and hemodynamics with lipid localization and density in animals that have undergone partial carotid ligation (PCL) induced-atherosclerosis. A 40 MHz transducer (Vevo2100, VisualSonics) and a ND:YAG pulsed laser (Surelite EX, Continuum) were used to image five apolipoprotein-E deficient mice that underwent PCL of the left carotid artery while being fed a Western diet. Animals were imaged using 4DUS at days 0, 1, 4, 7, 10, and 14 to obtain pulsed-wave Doppler for hemodynamic characterization and 4DUS images for strain mapping. At day 14 all animals were euthanized and 3D in situ PAT images of the left carotid artery were acquired using 1210 nm light. The results show that atherosclerotic lesions can be characterized via PAT to localize both lipid accumulation and density and 4DUS to identify regions of low strain.

The pro-atherogenic response to disturbed blood flow is increased by a western diet, but not by old age

Atherogenic remodeling often occurs at arterial locations with disturbed blood flow (i.e., low or oscillatory) and both aging and western diet (WD) increase the likelihood for pro-atherogenic remodeling. However, it is unknown if old age and/or a WD modify the pro-atherogenic response to disturbed blood flow. We induced disturbed blood flow by partial carotid ligation (PCL) of the left carotid artery in young and old, normal chow (NC) or WD fed male B6D2F1 mice. Three weeks post-PCL, ligated carotid arteries had greater intima media thickness, neointima formation, and macrophage content compared with un-ligated arteries. WD led to greater remodeling and macrophage content in the ligated artery compared with NC mice, but these outcomes were similar between young and old mice. In contrast, nitrotyrosine content, a marker of oxidative stress, did not differ between WD and NC fed mice, but was greater in old compared with young mice in both ligated and un-ligated carotid arteries. In primary vascular smooth muscle cells, aging reduced proliferation, whereas conditioned media from fatty acid treated endothelial cells increased proliferation. Taken together, these findings suggest that the remodeling and pro-inflammatory response to disturbed blood flow is increased by WD, but is not increased by aging.

Focal TLR4 activation mediates disturbed flow-induced endothelial inflammation

Disturbed blood flow at arterial branches and curvatures modulates endothelial function and predisposes the region to endothelial inflammation and subsequent development of atherosclerotic lesions. Activation of the endothelial Toll-like receptors (TLRs), in particular TLR4, contributes to vascular inflammation. Therefore, we investigate whether TLR4 can sense disturbed flow (DF) to mediate the subsequent endothelial inflammation. En face staining of endothelium revealed that TLR4 expression, activation, and its downstream inflammatory markers were elevated in mouse aortic arch compared with thoracic aorta, which were absent in Tlr4mut mice. Similar results were observed in the partial carotid ligation model where TLR4 signalling was activated in response to ligation-induced flow disturbance in mouse carotid arteries, and such effect was attenuated in Tlr4mut mice. DF in vitro increased TLR4 expression and activation in human endothelial cells (ECs) and promoted monocyte-EC adhesion, which were inhibited in TLR4-knockdown ECs. Among endogenous TLR4 ligands examined as candidate mediators of DF-induced TLR4 activation, fibronectin containing the extra domain A (FN-EDA) expressed by ECs was increased by DF and was revealed to directly interact with and activate TLR4. Our findings demonstrate the indispensable role of TLR4 in DF-induced endothelial inflammation and pinpoint FN-EDA as the endogenous TLR4 activator in this scenario. This novel mechanism of vascular inflammation under DF condition may serve as a critical initiating step in atherogenesis.

Perivascular adipose tissue modulates carotid plaque formation induced by disturbed flow in mice

ObjectiveEmerging evidence shows that perivascular adipose tissue (PVAT) is crucially involved in inflammation and cardiovascular diseases. However, controversial results have been reported regarding the effect of PVAT in atherosclerosis. This study aimed to determine the role of PVAT in disturbed blood flow (d-flow)-induced carotid plaque formation. MethodsApoE−/− male mice underwent partial carotid ligation (PCL) to induce d-flow in the left carotid artery (LCA) and were fed a high-fat diet for 2 weeks. Oil Red O and hematoxylin and eosin stains were used to determine adipose tissue. Thoracic PVAT from ApoE−/− or wild-type female mice were transplanted to the LCA of PCL-treated ApoE−/− mice. Carotid arteries were stained with Sudan IV to detect atherosclerotic lesions. Quantitative real-time reverse transcription polymerase chain reaction and immunofluorescence staining were performed to assess macrophage infiltration. ResultsBy 2 weeks of the high-fat diet after PCL surgery, de novo adipose tissue was formed around the ligated LCA, where atherosclerotic plaques were also observed. Quantitative real-time reverse transcription polymerase chain reaction analysis of the newly formed PVAT revealed a similar transcription profile to native PVAT. Treatment with bisphenol A diglycidyl ether, a peroxisome proliferator-activated receptor γ inhibitor, diminished PVAT formation but increased plaque size and macrophage infiltration. Transplantation of thoracic PVAT from wild-type mice (PVAT-TWT) rather than from ApoE−/− mice (PVAT-TApoE−/−) nearly abrogated LCA plaque macrophage content without affecting plaque size. Mechanistically, PVAT-TApoE−/− showed higher messenger RNA levels of inflammatory cytokines compared with PVAT-TWT. ConclusionsOur findings suggest that regulated PVAT formation may confer protection against atherosclerosis-prone shear stress, probably through attenuation of focal inflammation.

Cystathionine γ Lyase Sulfhydrates the RNA Binding Protein Human Antigen R to Preserve Endothelial Cell Function and Delay Atherogenesis.

Hydrogen sulfide (H2S), generated by cystathionine γ lyase (CSE), is an important endogenous regulator of vascular function. The aim of the present study was to investigate the control and consequences of CSE activity in endothelial cells under physiological and proatherogenic conditions. Endothelial cell CSE knockout mice were generated, and lung endothelial cells were studied in vitro (gene expression, protein sulfhydration, and monocyte adhesion). Mice were crossed onto the apolipoprotein E-deficient background, and atherogenesis (partial carotid artery ligation) was monitored over 21 days. CSE expression, H2S bioavailability, and amino acid profiling were also performed with human material. The endothelial cell-specific deletion of CSE selectively increased the expression of CD62E and elevated monocyte adherence in the absence of an inflammatory stimulus. Mechanistically, CD62E mRNA was more stable in endothelial cells from CSE-deficient mice, an effect attributed to the attenuated sulfhydration and dimerization of the RNA-binding protein human antigen R. CSE expression was upregulated in mice after partial carotid artery ligation and in atheromas from human subjects. Despite the increase in CSE protein, circulating and intraplaque H2S levels were reduced, a phenomenon that could be attributed to the serine phosphorylation (on Ser377) and inhibition of the enzyme, most likely resulting from increased interleukin-1β. Consistent with the loss of H2S, human antigen R sulfhydration was attenuated in atherosclerosis and resulted in the stabilization of human antigen R-target mRNAs, for example, CD62E and cathepsin S, both of which are linked to endothelial cell activation and atherosclerosis. The deletion of CSE from endothelial cells was associated with the accelerated development of endothelial dysfunction and atherosclerosis, effects that were reversed on treatment with a polysulfide donor. Finally, in mice and humans, plasma levels of the CSE substrate l-cystathionine negatively correlated with vascular reactivity and H2S levels, indicating its potential use as a biomarker for vascular disease. The constitutive S-sulfhydration of human antigen R (on Cys13) by CSE-derived H2S prevents its homodimerization and activity, which attenuates the expression of target proteins such as CD62E and cathepsin S. However, as a consequence of vascular inflammation, the beneficial actions of CSE-derived H2S are lost owing to the phosphorylation and inhibition of the enzyme.

Reduced Plasma Kallistatin Is Associated With the Severity of Coronary Artery Disease, and Kallistatin Treatment Attenuates Atherosclerotic Plaque Formation in Mice.

BackgroundKallistatin exerts beneficial effects on organ injury by inhibiting oxidative stress and inflammation. However, the role of kallistatin in atherosclerosis is largely unknown. Here, we investigated the role and mechanisms of kallistatin in patients with coronary artery disease (CAD), atherosclerotic plaques of apoE−/− mice, and endothelial activation.Methods and ResultsPlasma kallistatin levels were analyzed in 453 patients at different stages of CAD. Kallistatin levels were significantly lower in patients with CAD and negatively associated with CAD severity and oxidative stress. Human kallistatin cDNA in an adenoviral vector was injected intravenously into apoE−/− mice after partial carotid ligation, with or without nitric oxide synthase inhibitor (Nω‐nitro‐L‐arginine methyl ester) or sirtuin 1 inhibitor (nicotinamide). Kallistatin gene delivery significantly reduced macrophage deposition, oxidative stress, and plaque volume in the carotid artery, compared with control adenoviral injection. Kallistatin administration increased endothelial nitrous oxide synthase, sirtuin 1, interleukin‐10, superoxide dismutase 2, and catalase expression in carotid plaques. The beneficial effects of kallistatin in mice were mitigated by Nω‐nitro‐L‐arginine methyl ester or nicotinamide. Furthermore, human kallistatin protein suppressed tumor necrosis factor‐α–induced NADPH oxidase activity and increased endothelial nitrous oxide synthase and sirtuin 1 expression in cultured human endothelial cells. These effects were also abolished by Nω‐nitro‐L‐arginine methyl ester or nicotinamide.ConclusionsThis was the first study to demonstrate that reduced plasma kallistatin levels in patients are associated with CAD severity and oxidative stress. Kallistatin treatment prevents carotid atherosclerotic plaque formation in mice by stimulating the sirtuin 1/endothelial nitrous oxide synthase pathway. These findings indicate the potential protective effects of kallistatin on atherosclerosis in human subjects and mouse models.

Cystathionine γ-Lyase Modulates Flow-Dependent Vascular Remodeling.

Objective- Flow patterns differentially regulate endothelial cell phenotype, with laminar flow promoting vasodilation and disturbed flow promoting endothelial proinflammatory activation. CSE (cystathionine γ-lyase), a major source of hydrogen sulfide (H2S) in endothelial cells, critically regulates cardiovascular function, by both promoting vasodilation and reducing endothelial activation. Therefore, we sought to investigate the role of CSE in the endothelial response to flow. Approach and Results- Wild-type C57Bl/6J and CSE knockout ( CSE-/-) mice underwent partial carotid ligation to induce disturbed flow in the left carotid. In addition, endothelial cells isolated from wild-type and CSE -/- mice were exposed to either laminar or oscillatory flow, an in vitro model of disturbed flow. Interestingly, laminar flow significantly reduced CSE expression in vitro, and only disturbed flow regions show discernable CSE protein expression in vivo, correlating with enhanced H2S production in wild-type C57BL/6J but not CSE-/- mice. Lack of CSE limited disturbed flow-induced proinflammatory gene expression (ICAM-1[intercellular adhesion molecule 1], VCAM-1 [vascular cell adhesion molecular 1]) and monocyte infiltration and CSE-/- endothelial cells showed reduced NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and proinflammatory gene expression in response to oscillatory flow in vitro. In addition, CSE-/- mice showed reduced inward remodeling after partial carotid ligation. CSE-/- mice showed elevated vascular nitrite levels (measure of nitric oxide [NO]) in the unligated carotids, suggesting an elevation in baseline NO production, and the NO scavenger 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide normalized the reduced inward remodeling, but not inflammation, of ligated carotids in CSE-/- mice. Conclusions- CSE expression in disturbed flow regions critically regulates both endothelial activation and flow-dependent vascular remodeling, in part through altered NO availability.

Vasoactive Intestinal Peptide Ameliorates Acute Myocarditis and Atherosclerosis by Regulating Inflammatory and Autoimmune Responses.

Vasoactive intestinal peptide (VIP) is a neuropeptide that exerts various vascular and cardioprotective functions and regulates immune function and inflammatory response at multiple levels. However, its role in inflammatory cardiovascular disorders is largely unknown. Myocarditis and atherosclerosis are two inflammatory and autoimmune cardiovascular diseases that cause important adverse circulatory events. In this study, we investigate the therapeutic effects of VIP in various well-established preclinical models of experimental autoimmune myocarditis and atherosclerosis. Intraperitoneal injection of VIP during the effector phase of experimental autoimmune myocarditis in susceptible BALB/c mice significantly reduced its prevalence, ameliorated signs of heart hypertrophy and injury, attenuated myocardial inflammatory infiltration, and avoided subsequent profibrotic cardiac remodeling. This effect was accompanied by a reduction of Th17-driven cardiomyogenic responses in peripheral lymphoid organs and in the levels of myocardial autoantibodies. In contrast, acute and chronic atherosclerosis was induced in apolipoprotein E-deficient mice fed a hyperlipidemic diet and subjected to partial carotid ligation. Systemic VIP treatment reduced the number and size of atherosclerotic plaques in carotid, aorta, and sinus in hypercholesterolemic mice. VIP reduced Th1-driven inflammatory responses and increased regulatory T cells in atherosclerotic arteries and their draining lymph nodes. VIP also regulated cholesterol efflux in macrophages and reduced the formation of foam cells and their presence in atherosclerotic plaques. Finally, VIP inhibited proliferation and migration of smooth muscle cells and neointima formation in a mouse model of complete carotid ligation. These findings encourage further studies aimed to assess whether VIP can be used as a pharmaceutical agent to treat heart inflammation and atherosclerosis.

Abstract 234: Nuclear Export of Telomeric Repeat Binding Factor 2 (terf2)-interacting Protein (terf2ip) Expression, One of the Sherterin Complex Molecule, is Crucial for Disturbed Flow-induced Endothelial Inflammation and Senescence via Upregulating Terf2ip S205 Phosphorylation

Atherosclerosis is a multifactorial disease mainly caused by endothelial cell (EC) dysfunction resulting from EC senescence. Both EC inflammation and senescence are increased by disturbed flow (d-flow) but the exact mechanism is still unknown. The potential role of TERF2IP in regulating inflammation has been reported, but it has been controversial, especially in ECs. We found that d-flow-induced NF-kB activation was significantly inhibited by the depletion of TERF2IP, but TNF-induced NF-kB activation under laminar flow (l-flow) was not inhibited by TERF2IP depletion. TERF2IP S205 phosphorylation was increased by d-flow, but not by s-flow. This TERF2IP S205 phosphorylation was crucial for d-flow-induced TERF2IP nuclear export, which also caused nuclear export of its binding partner TERF2 in a piggy-back manner. Both the depletion of TERF2IP (TERF2IP siRNA) and overexpression of TERF2IP S205A mutant inhibited d-flow-induced TERF2 nuclear export as well as subsequent EC senescence. In addition, we found the key role of p90RSK activation in regulating EC inflammation and senescence via TERF2IP S205 phosphorylation. To determine the role of TERF2IP and TERF2 cellular localization in vivo, we performed in vivo en face immunostaining of mouse aortic arch in EC specific TERF2IP knock out (EC-TERF2IP-KO) and control mice using anti-TERF2IP or TEFR2 and anti-VE-cadherin antibodies. In control mice TERF2IP expression was very low in the l-flow area. In contrast we found a significant increase of TERF2IP expression, especially in the extra-nuclear region of ECs in the d-flow area. More nuclear localization of TERF2 was observed in EC-TERF2IP-KO mice than those in wild type, which supports our finding in vitro. Significant decreases of VCAM-1 expression and apoptosis were found in the d-flow area in EC-TERF2IP-KO mice compared to control mice. Lastly, we found a significant decrease in d-flow-induced atherosclerotic plaque formation after partial carotid ligation in EC-TERF2IP-KO mice crossed with Ldlr-/- mice compared to control Ldlr-/- mice. These data suggest the key role of nuclear export of TERF2IP in up-regulating EC inflammation and senescence via p90RSK-mediated TERF2IP S205 phosphorylation.

Abstract 451: Radiation Induced Atherosclerosis on Mice

Rationale: The high prevalence of cardiovascular disease (CVD) in cancer survivors after ionizing radiation (IR) therapy has long been recognized, but the mechanism for this is unknown. Previous studies reported that the plaques in IR therapy survivors exhibit characteristics of increased instability without changes in the plaque size. Objective: To study the mechanism of IR induced CVD in humans, we decided to develop an IR mouse model that mimics human CV events. Methods and Results: We fed LDLR-/- mice or C57 mice with a single injection of adeno -associated virus vector(AAV) encoding a gain-of function of PCSK9 with a high fat diet, then irradiated the mice with different doses of IR (2 Gy, 5 Gy, 10 Gy). Whole body radiation was given 2 Gy, while localized IR to the neck and thoracic area was given at 5 Gy (1 time) or 10Gy (weekly 5 Gy, twice).About 2~3 weeks after the IR treatment, we performed left partial carotid artery ligation (PCL), and 3 weeks later examined the extent of plaque formation and cardiac abnormality by comparing those detected in non-irradiated control mice. The lesion area ratio between the partially ligated left carotid artery (LCA) and the right carotid artery (RCA) was significantly increased in the IR-treated group compared to the non-IR-treated group (3.1 ± 0.3 vs.1.8 ± 0.4, n = 7-8, p = 0.03). In addition, the lesion in the IR-treated of mice had significantly larger necrotic cores with a thinner fibrous cap when compared to those in non-irradiate mice. Furthermore, IR-treated mice showed cardiac hypertrophy, in which we found a significant increase of the wall thickness of mid-to small sized arteries as well as perivascular fibrosis, which were not noticed in the non-IR-treated mice. Conclusions: Our results demonstrate that IR not only increases atherosclerotic and vulnerable plaque formation but also cardiac hypertrophy with mid- and micro-vascular wall swelling. The increase of mid- and micro-vascular wall thickness may explain the heart failure preserved heart failure, which is observed in the majority of heart failure patients after IR treatment.

Abstract 606: The Differential Roles of the Adaptor Proteins Nck1 and Nck2 in Shear Stress-Induced Endothelial Activation in vitro and in vivo

Hemodynamic shear stress critically regulates endothelial activation and atherogenesis by affecting cytoskeletal dynamics and endothelial gene expression. The Nck adaptor proteins (Nck1 and Nck2) couple tyrosine kinase signaling to induction of cytoskeletal remodeling pathways, and we previously demonstrated that a peptide corresponding to a Nck-binding sequence in p21 activate kinase (PAK) blunts shear stress-induced proinflammatory signaling (PAK, NF-κB) and permeability in vitro and in vivo . However, the specific roles of Nck1 and Nck2 in flow-induced endothelial activation remain to be elucidated. Here, we demonstrate that Nck1, but not Nck2, critically regulates shear stress-induced endothelial activation. We show that Nck1 deficiency (siRNA knockdown, genetic knockout) decreases basal and shear stress-induced proinflammatory signaling (PAK2, NF-κB) and proinflammatory gene expression (ICAM-1, VCAM-1). In addition, only Nck1, but not Nck2, knockdown/knockout limited shear-induced endothelial permeability. However, other shear stress-dependent pathways, such as Akt activation, proved Nck1-independent. By contrast, selective Nck2 depletion paradoxically increased basal PAK2 activation without significant effects on NF-κB signaling or permeability. Using the partial carotid ligation model of disturbed flow, we found that Nck1 knockout mice showed significantly reduced proinflammatory gene expression that was not further diminished upon Nck2 deletion. However, Nck2 was not inconsequential to the flow response, as Nck2 knockdown or knockout reduced actin cytoskeletal alignment with laminar flow. Taken together, our data suggests that Nck1 plays a dominant role in flow-induced endothelial activation in response to shear stress, whereas Nck2 may critically regulate flow-induced cytoskeletal remodeling and endothelial alignment.

Disturbed Blood Flow induces Arterial Stiffening Through Thrombospondin‐1

BACKGROUNDArterial stiffness and wall shear stress are powerful determinants of cardiovascular health, and arterial stiffness is associated with increased cardiovascular mortality. Low and oscillatory wall shear stress, termed disturbed flow (d‐flow), promotes atherosclerotic arterial remodeling, but the relationship between d‐flow and arterial stiffness is not well understood. The objective of this study was to define the role of d‐flow on arterial stiffening and discover the relevant signaling pathways by which d‐flow stiffens arteries.METHODSD‐flow was induced in the carotid arteries of young and old mice of both sexes. Arterial stiffness was quantified ex vivo with cylindrical biaxial mechanical testing and in vivo from duplex ultrasound and compared with unmanipulated carotid arteries from 80‐week‐old mice. Gene expression and pathway analysis was performed on endothelial cell‐enriched RNA and validated by immunohistochemistry. In vitro testing of signaling pathways was performed under oscillatory and laminar wall shear stress conditions. Human arteries from regions of d‐flow and stable flow were tested ex vivo to validate critical results from the animal model.RESULTSD‐flow induced arterial stiffening through collagen deposition after partial carotid ligation, and the degree of stiffening was similar to that of unmanipulated carotid arteries from 80‐week‐old mice. Intimal gene pathway analyses identified transforming growth factor‐β pathways as having a prominent role in this stiffened arterial response, but this was attributable to thrombospondin‐1 (TSP‐1) stimulation of profibrotic genes and not changes to transforming growth factor‐β. In vitro and in vivo testing under d‐flow conditions identified a possible role for TSP‐1 activation of transforming growth factor‐β in the upregulation of these genes. TSP‐1 knockout animals had significantly less arterial stiffening in response to d‐flow than wild‐type carotid arteries. Human arteries exposed to d‐flow had similar increases TSP‐1 and collagen gene expression as seen in our model.CONCLUSIONSTSP‐1 has a critical role in shear‐mediated arterial stiffening that is mediated in part through TSP‐1's activation of the profibrotic signaling pathways of transforming growth factor‐β. Molecular targets in this pathway may lead to novel therapies to limit arterial stiffening and the progression of disease in arteries exposed to d‐flow.Support or Funding InformationFunding for this work was provided by the National Institutes of Health, KO8HL119592 (to Dr Brewster), HL119798 and HL124879 (to Dr Jo); the Society for Vascular Surgery/American College of Surgeons Mentored Clinical Scientist Research Career Development Award (to Dr Brewster); the American Heart Association, IRG1470001 (to Dr Brewster); departmental support from the Emory University Department of Surgery (Dr John F. Sweeney) (to Dr Brewster); and the John and Jan Portman Professorship (to Dr Jo) from Emory University School of Medicine and Georgia Institute of Technology.This study was supported in part by the Animal Physiology Core, which is subsidized by Emory University and Children's Healthcare of Atlanta. Additional support was provided by the Office of the Director of the National Institutes of Health under Award Number S10OD021748. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the National Institutes of Health.

Programmed cell death 5 suppresses AKT-mediated cytoprotection of endothelium

Programmed cell death 5 (PDCD5) has been associated with human cancers as a regulator of cell death; however, the role of PDCD5 in the endothelium has not been revealed. Thus, we investigated whether PDCD5 regulates protein kinase B (PKB/AKT)-endothelial nitric oxide synthase (eNOS)-dependent signal transduction in the endothelium and affects atherosclerosis. Endothelial-specific PDCD5 knockout mice showed significantly reduced vascular remodeling compared with wild-type (WT) mice after partial carotid ligation. WT PDCD5 competitively inhibited interaction between histone deacetylase 3 (HDAC3) and AKT, but PDCD5L6R, an HDAC3-binding-deficient mutant, did not. Knockdown of PDCD5 accelerated HDAC3-AKT interaction, AKT and eNOS phosphorylation, and nitric oxide (NO) production in human umbilical vein endothelial cells. Moreover, we found that serum PDCD5 levels reflect endothelial NO production and are correlated with diabetes mellitus, high-density lipoprotein cholesterol, and coronary calcium in human samples obtained from the cardiovascular high-risk cohort. Therefore, we conclude that PDCD5 is associated with endothelial dysfunction and may be a novel therapeutic target in atherosclerosis.

Genome-Wide Association and Functional Studies Identify SCML4 and THSD7A as Novel Susceptibility Genes for Coronary Artery Disease.

The genetic contribution to coronary artery disease (CAD) remains largely unclear. We combined genetic screening with functional characterizations to identify novel loci and candidate genes for CAD. We performed genome-wide screening followed by multicenter validation in 8 cohorts consisting of 21 828 participants of Han ethnicity and identified 3 novel intragenic SNPs (single nucleotide polymorphisms), rs9486729 (SCML4 [Scm polycomb group protein-like 4]; odds ratio, 1.25; 95% CI, 1.17-1.34; P=3.51×10-11), rs17165136 (THSD7A [thrombospondin type 1 domain-containing 7A]; odds ratio 1.28; 95% CI, 1.21-1.35; P<1.00×10-25), and rs852787 (DAB1 [disabled-1]; odds ratio, 1.29; 95% CI, 1.21-1.38; P=2.02×10-14), associated with CAD with genome-wide significance. The risk allele of rs9486729 and protective allele of rs17165136 were associated with the decreased expression of their host genes, SCML4 and THSD7A, respectively, whereas rs852787 did not have transcriptional effects on any gene. Knockdown of SCML4 activated endothelial cells by increasing the expression of IL-6, E-selectin, and ICAM and weakened their antiapoptotic activity, whereas the knockdown of THSD7A had little effect on these endothelial cell functions but attenuated monocyte adhesion via decreasing the expression of ICAM, L-selectin, and ITGB2. We further showed that inhibiting the expression of SCML4 exacerbated endothelial dysfunction and vascular remodeling in a rat model with partial carotid ligation. We identify 3 novel loci associated with CAD and show that 2 genes, SCML4 and THSD7A, make functional contributions to atherosclerosis. How rs852787 and its host gene DAB1 are linked to CAD needs further studies.

Fluid shear stress regulates the expression of Lectin-like oxidized low density lipoprotein receptor-1 via KLF2-AP-1 pathway depending on its intensity and pattern in endothelial cells

Background and aimsVascular endothelial cells (ECs) are exposed to fluid shear stress (FSS), which modulates vascular pathophysiology. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is crucial in endothelial dysfunction and atherosclerosis. We elucidated the mechanism regulating LOX-1 expression in ECs by FSS. MethodsHuman umbilical vein endothelial cells were exposed to laminar shear stress (LSS) of indicated intensities using a unidirectional steady flow, or to oscillatory shear stress (OSS) using a bidirectional disturbed flow. In vivo studies were performed in a mouse model of partial carotid ligation and human pulmonary artery sections. ResultsWithin ECs, OSS upregulated LOX-1 expression, while LSS (20 dyne/cm2) downregulated it. We confirmed that OSS-induced LOX-1 expression was suppressed when the mechanotransduction was inhibited by knockdown of the mechanosensory complex. In addition, we demonstrated that Kruppel-like factor 2 (KLF2) has an inhibitory role on OSS-induced LOX-1 expression. Next, we determined that activator protein-1 (AP-1) was the key transcription factor inducing LOX-1 expression by OSS, which was inhibited by KLF2 overexpression. To explore whether the intensity of LSS affects LOX-1 expression, we tested three different intensities (20, 60, and 120 dyne/cm2) of LSS. We observed higher LOX-1 expression with high shear stresses of 120 dyne/cm2 compared to 20 and 60 dyne/cm2, with OSS-like KLF2-AP-1 signaling patterns. Furthermore, ECs within disturbed flow regions showed upregulated LOX-1 expression in vivo. ConclusionsWe concluded that LOX-1 expression on ECs is regulated via FSS depending on its intensity as well as pattern. Furthermore, this is mediated through the KLF2-AP1 pathway of mechanotransduction.

Vascular Semaphorin 7A Upregulation by Disturbed Flow Promotes Atherosclerosis Through Endothelial β1 Integrin.

Accumulating evidence suggests a role of semaphorins in vascular homeostasis. Here, we investigate the role of Sema7A (semaphorin 7A) in atherosclerosis and its underlying mechanism. Using genetically engineered Sema7A-/-ApoE-/- mice, we showed that deletion of Sema7A attenuates atherosclerotic plaque formation primarily in the aorta of ApoE-/- mice on a high-fat diet. A higher level of Sema7A in the atheroprone lesser curvature suggests a correlation of Sema7A with disturbed flow. This notion is supported by elevated Sema7A expression in human umbilical venous endothelial cells either subjected to oscillatory shear stress or treated with the PKA (protein kinase A)/CREB (cAMP response element-binding protein) inhibitor H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCl hydrate). Further studies using the partial carotid artery ligation model showed that disturbed flow in the left carotid artery of Sema7A+/+ApoE-/- mice promoted the expression of endothelial Sema7A and cell adhesion molecules, leukocyte adhesion, and plaque formation, whereas such changes were attenuated in Sema7A-/-ApoE-/- mice. Further studies showed that blockage of β1 integrin, a known Sema7A receptor, or inhibition of FAK (focal adhesion kinase), MEK1/2 (mitogen-activated protein kinase kinase 1/2), or NF-κB (nuclear factor-κB) significantly reduced the expression of cell adhesion molecules and THP-1 (human acute monocytic leukemia cell line) monocyte adhesion in Sema7A-overexpressing human umbilical venous endothelial cells. Studies using chimeric mice suggest that vascular, most likely endothelial, Sema7A plays a major role in atherogenesis. Our findings indicate a significant role of Sema7A in atherosclerosis by mediating endothelial dysfunction in a β1 integrin-dependent manner.

Disturbed Flow Promotes Arterial Stiffening Through Thrombospondin-1.

Arterial stiffness and wall shear stress are powerful determinants of cardiovascular health, and arterial stiffness is associated with increased cardiovascular mortality. Low and oscillatory wall shear stress, termed disturbed flow (d-flow), promotes atherosclerotic arterial remodeling, but the relationship between d-flow and arterial stiffness is not well understood. The objective of this study was to define the role of d-flow on arterial stiffening and discover the relevant signaling pathways by which d-flow stiffens arteries. D-flow was induced in the carotid arteries of young and old mice of both sexes. Arterial stiffness was quantified ex vivo with cylindrical biaxial mechanical testing and in vivo from duplex ultrasound and compared with unmanipulated carotid arteries from 80-week-old mice. Gene expression and pathway analysis was performed on endothelial cell-enriched RNA and validated by immunohistochemistry. In vitro testing of signaling pathways was performed under oscillatory and laminar wall shear stress conditions. Human arteries from regions of d-flow and stable flow were tested ex vivo to validate critical results from the animal model. D-flow induced arterial stiffening through collagen deposition after partial carotid ligation, and the degree of stiffening was similar to that of unmanipulated carotid arteries from 80-week-old mice. Intimal gene pathway analyses identified transforming growth factor-β pathways as having a prominent role in this stiffened arterial response, but this was attributable to thrombospondin-1 (TSP-1) stimulation of profibrotic genes and not changes to transforming growth factor-β. In vitro and in vivo testing under d-flow conditions identified a possible role for TSP-1 activation of transforming growth factor-β in the upregulation of these genes. TSP-1 knockout animals had significantly less arterial stiffening in response to d-flow than wild-type carotid arteries. Human arteries exposed to d-flow had similar increases TSP-1 and collagen gene expression as seen in our model. TSP-1 has a critical role in shear-mediated arterial stiffening that is mediated in part through TSP-1's activation of the profibrotic signaling pathways of transforming growth factor-β. Molecular targets in this pathway may lead to novel therapies to limit arterial stiffening and the progression of disease in arteries exposed to d-flow.