Areas Of Disturbed Flow Research Articles

Exercise may be a Novel Strategy to Reduce Vascular Toxicity of Immune Checkpoint Inhibitors in a Mouse Melanoma Model

Background: Immune checkpoint inhibitors (ICIs) are a cornerstone in advanced melanoma treatment. Unfortunately, ICI-associated cardiovascular toxicity, particularly atherosclerotic events, poses a significant challenge. Identifying interventions to mitigate the cardiovascular toxicity of ICI is critical for enhancing the quality and duration of life in cancer survivors. Given the established benefits of exercise in atherosclerosis prevention, this study explores its potential to reduce ICI-induced vascular toxicity in the context of melanoma. We hypothesized that aerobic exercise would decrease ICI's vascular toxicity in a mouse melanoma model. The primary experimental outcome was endothelial mesenchymal transition (EndMT), an early atherosclerosis indicator. Methods: C57BL/6 mice were subcutaneously injected with 0.8 x 106 BP melanoma cells ( BrafV600E/WT and Pten−/−). Upon tumor palpability, mice were assigned into four groups: IgG, Exercise, anti-PD-1, and anti-PD-1+Exercise. Mice were treated with bi-weekly administration of anti-PD-1 or IgG antibody (150 μg) for three weeks, combined with or without treadmill exercise (12 meters/minute for 45 min per day, five days/week). Body weight was monitored weekly. Following the final treatment, aortic tissues were collected and en face immunofluorescence staining for endothelial marker VE-cadherin (VE-Cad) and vimentin was performed to assess EndMT, then analyzed via confocal microscopy. Results: No significant weight differences were observed between groups. Notably, the EndMT marker vimentin expression was significantly higher in anti-PD-1 treated tumor-bearing mice compared to IgG controls (3.8% vs 0.6%, p=.03) in the atheroprotective laminar flow area. This was reduced by exercise; the anti-PD-1+Exercise group showed significantly reduced endothelial vimentin levels relative to the anti-PD-1 alone (0.8% vs 3.8%, p=.04). Disturbed flow areas did not exhibit notable differences between any group. Conclusions: Aerobic exercise may be protective against vascular toxicity in mice treated with ICIs by promoting atheroprotective flow and potentially inhibiting EndMT. Although these initial results are promising, comprehensive studies are required to ascertain the impact of exercise on preventing ICI-induced EndMT and subsequent atherosclerosis in this mouse melanoma model. Future research will aim to more deeply characterize the process of EndMT following ICI treatment and evaluate the preventive capacity of exercise against atherosclerosis in this context. MD Anderson Cancer Center Melanoma SPORE grant (NCI P50CA221703-05). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Highlighting Hemodynamic Risks for Bioresorbable Stents in Coronary Arteries

A three-dimensional, transient computational fluid dynamics analysis was conducted on an idealised geometry of a coronary artery fitted with representative geometries of an Absorb bioresorbable vascular scaffold (BVS) or a Xience drug-eluting stent (DES) in order to identify and compare areas of disturbed flow and potential risk sites. A non-Newtonian viscosity model was used with a transient velocity boundary condition programmed with user-defined functions. At-risk areas were quantified in terms of several parameters linked to restenosis: wall shear stress, time-averaged wall shear stress, oscillatory shear index, particle residence time, and shear rate. Results indicated that 71% of the BVS stented surface area had time-averaged wall shear stress values under 0.4 Pa compared to 45% of the DES area. Additionally, high particle residence times were present in 23% and 8% of the BVS and DES areas, respectively, with risk areas identified as being more prominent in close proximity to crowns and link struts. These results suggest an increased risk for thrombosis and neointimal hyperplasia for the BVS compared to the DES, which is in agreement with the outcomes of clinical trials. It is intended that the results of this study may be used as a pre-clinical tool to aid in the design of bioresorbable coronary stents.

The interleukin-1 receptor type-1 in disturbed flow-induced endothelial mesenchymal activation.

Atherosclerosis is a progressive disease that develops in areas of disturbed flow (d-flow). Progressive atherosclerosis is characterized by bulky plaques rich in mesenchymal cells and high-grade inflammation that can rupture leading to sudden cardiac death or acute myocardial infarction. In response to d-flow, endothelial cells acquire a mesenchymal phenotype through endothelial-to-mesenchymal transition (EndMT). However, the signaling intermediaries that link d-flow to EndMT are incompletely understood. In this study we found that in human atherosclerosis, cells expressing SNAI1 (Snail 1, EndMT transcription factor) were highly expressed within the endothelial cell (EC) layer and in the pre-necrotic areas in unstable lesions, whereas stable lesions did not show any SNAI1 positive cells, suggesting a role for EndMT in lesion instability. The interleukin-1 (IL-1), which signals through the type-I IL-1 receptor (IL-1R1), has been implicated in plaque instability and linked to EndMT formation in vitro. Interestingly, we observed an association between SNAI1 and IL-1R1 within ECs in the unstable lesions. To establish the causal relationship between EndMT and IL-1R1 expression, we next examined IL-1R1 levels in our Cre-lox endothelial-specific lineage tracing mice. IL-1R1 and Snail1 were highly expressed in ECs under atheroprone compared to athero-protective areas, and oscillatory shear stress (OSS) increased IL-1R1 protein and mRNA levels in vitro. Exposure of ECs to OSS resulted in loss of their EC markers and higher induction of EndMT markers. By contrast, genetic silencing of IL-1R1 significantly reduced the expression of EndMT markers and Snail1 nuclear translocation, suggesting a direct role for IL-1R1 in d-flow-induced EndMT. In vivo, re-analysis of scRNA-seq datasets in carotid artery exposed to d-flow confirmed the IL-1R1 upregulation among EndMT population, and in our partial carotid ligation model of d-flow, endothelial cell specific IL-1R1 KO significantly reduced SNAI1 expression. Global inhibition of IL-1 signaling in atherosclerosis as a therapeutic target has recently been tested in the completed CANTOS trial, with promising results. However, the data on IL-1R1 signaling in different vascular cell-types are inconsistent. Herein, we show endothelial IL-1R1 as a novel mechanosensitive receptor that couples d-flow to IL-1 signaling in EndMT.

Endothelial PHACTR1 Promotes Endothelial Activation and Atherosclerosis by Repressing PPARγ Activity Under Disturbed Flow in Mice.

Numerous genome-wide association studies revealed that SNPs (single nucleotide polymorphisms) at the PHACTR1 (phosphatase and actin regulator 1) locus strongly correlate with coronary artery disease. However, the biological function of PHACTR1 remains poorly understood. Here, we identified the proatherosclerotic effect of endothelial PHACTR1, contrary to macrophage PHACTR1. We generated global (Phactr1-/-) and endothelial cell (EC)-specific (Phactr1ECKO) Phactr1 KO (knockout) mice and crossed these mice with apolipoprotein E-deficient (Apoe-/-) mice. Atherosclerosis was induced by feeding the high-fat/high-cholesterol diet for 12 weeks or partially ligating carotid arteries combined with a 2-week high-fat/high-cholesterol diet. PHACTR1 localization was identified by immunostaining of overexpressed PHACTR1 in human umbilical vein ECs exposed to different types of flow. The molecular function of endothelial PHACTR1 was explored by RNA sequencing using EC-enriched mRNA from global or EC-specific Phactr1 KO mice. Endothelial activation was evaluated in human umbilical vein ECs transfected with siRNA targeting PHACTR1 and in Phactr1ECKO mice after partial carotid ligation. Global or EC-specific Phactr1 deficiency significantly inhibited atherosclerosis in regions of disturbed flow. PHACTR1 was enriched in ECs and located in the nucleus of disturbed flow areas but shuttled to cytoplasm under laminar flow in vitro. RNA sequencing showed that endothelial Phactr1 depletion affected vascular function, and PPARγ (peroxisome proliferator-activated receptor gamma) was the top transcription factor regulating differentially expressed genes. PHACTR1 functioned as a PPARγ transcriptional corepressor by binding to PPARγ through the corepressor motifs. PPARγ activation protects against atherosclerosis by inhibiting endothelial activation. Consistently, PHACTR1 deficiency remarkably reduced endothelial activation induced by disturbed flow in vivo and in vitro. PPARγ antagonist GW9662 abolished the protective effects of Phactr1 KO on EC activation and atherosclerosis in vivo. Our results identified endothelial PHACTR1 as a novel PPARγ corepressor to promote atherosclerosis in disturbed flow regions. Endothelial PHACTR1 is a potential therapeutic target for atherosclerosis treatment.

Disturbed Flow-Facilitated Margination and Targeting of Nanodisks Protect against Atherosclerosis.

Disturbed blood flow induces endothelial pro-inflammatory responses that promote atherogenesis. Nanoparticle-based therapeutics aimed at treating endothelial inflammation in vasculature where disturbed flow occurs may provide a promising avenue to prevent atherosclerosis. By using a vertical-step flow apparatus and a microfluidic chip of vascular stenosis, herein, it is found that the disk-shaped versus the spherical nanoparticles exhibit preferential margination (localization and adhesion) to the regions with the pro-atherogenic disturbed flow. By employing a mouse model of carotid partial ligation, superior targeting and higher accumulation of the disk-shaped particles are also demonstrated within disturbed flow areas than that of the spherical particles. In hyperlipidemia mice, administration of disk-shaped particles loaded with hypomethylating agent decitabine (DAC) displays greater anti-inflammatory and anti-atherosclerotic effects compared with that of the spherical counterparts and exhibits reduced toxicity than "naked" DAC. The findings suggest that shaping nanoparticles to disk is an effective strategy for promoting their delivery to atheroprone endothelia.

Abstract MP23: Knockout Of Pgc1α In Endothelial Cells Augments Abdominal Aortic Aneurysm Formation

Introduction: It is well described that changes in the aortic media are crucial in the development of Abdominal Aortic Aneurysms (AAA), however the involvement of the endothelium is not as clear. The endothelium is the first vascular layer exposed to blood flow and aneurysms preferentially form in areas of disturbed flow. The regulatory mechanisms as to why aneurysms form at these sites remains unclear. Here we propose that the transcription coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) plays a pivotal role in the endothelium’s response to AAA formation in a shear responsive manner. Methods: 10-12 week old male mice deficient in endothelial specific PGC1α (KO) and their corresponding wild type background mice (WT) were treated with 0.75 mg/kg/day of Ang II. Our primary endpoints were measurement of the maximal width of the suprarenal aortas, frequency, and complexity based on the Daugherty classification. To determine the effects of shear, HAECs exposed to steady flow (15 dyn/cm 2 ) and disturbed flow (0±6 dyn/cm 2 at 1 Hz) for 12 hours were compared to determine if mRNA expression were different for PGC1α and SIRT1. Results: AAA incidence and severity were increased in ANG II-infused KO compared to the WT mice (100% vs. 20%, respectively). Measurements of excised aortas showed that KO mice exhibited larger AAA fold-increase compared to WT (2.00 ± 0.11 fold increase vs. 0.43 ± 0.19 fold increase, p-value 0.0002). The KO male mice also exhibited a more complex phenotype. HAECs that underwent disturbed flow had a significant decrease in expression of SIRT1 (p = 0.02) and trended towards a decrease in expression of PGC1α (p = 0.06). Conclusions: Our data strongly suggests that knockout of endothelial specific PGC1α leads to more frequent, complex and larger AAAs primarily in the suprarenal aorta, which is an area of disturbed flow. Interestingly, the addition of ANG II alone was able to induce AAA formation in our model. Additionally, we show that PGC1α and SIRT1 are regulated in a shear responsive manner. Taken together, endothelial PGC1α likely plays a regulatory role in AAA formation through shear-related mechanisms.

An integrated fluid-chemical model toward modeling the thrombus formation in an idealized model of aortic dissection.

Type B aortic dissection is a major aortic catastrophe that can be acutely complicated by rapid expansion, rupture, and malperfusion syndromes. The separation of the intima from aortic walls will form a second blood-filled lumen defined as "false lumen (FL)", where the thrombus is more likely to form due to the local stasis hemodynamic conditions. Complete thrombosis of FL is associated with a beneficial outcome while patency and partial thrombosis will lead to later complications. However, the thrombosis mechanism is still unclear and little is known about the impact of chemical species transported by blood flow on this process. The proteins involved in the coagulation cascade (CC) may play an important role in the process of thrombosis, especially in the activation and stabilization of platelets. Based on this hypothesis, a reduced-order fluid-chemical model was established to simulate CC in an aortic dissection phantom with two tears. A high level of fibrin is continuously observed at the top of the FL and some time-varying areas between two tears, indicating a high likelihood of thrombus formation there. This finding is consistent with the clinical observation. The time evolution of coagulation factors is greatly affected by local hemodynamics, especially in the high disturbance zone where the evolution has characteristics of periodic changes consistent with the flow field. The ability of the proposed model to reproduce the CC response provides a potential application to integrate with a model that can simulate platelet activities, forming a biochemical-based model which would help unveil the mechanisms of thrombosis in FL and the clinical decision of appropriate treatment.

Mechanical forces regulate endothelial-to-mesenchymal transition and atherosclerosis via an Alk5-Shc mechanotransduction pathway.

The response of endothelial cells to mechanical forces is a critical determinant of vascular health. Vascular pathologies, such as atherosclerosis, characterized by abnormal mechanical forces are frequently accompanied by endothelial-to-mesenchymal transition (EndMT). However, how forces affect the mechanotransduction pathways controlling cellular plasticity, inflammation, and, ultimately, vessel pathology is poorly understood. Here, we identify a mechanoreceptor that is sui generis for EndMT and unveil a molecular Alk5-Shc pathway that leads to EndMT and atherosclerosis. Depletion of Alk5 abrogates shear stress-induced EndMT responses, and genetic targeting of endothelial Shc reduces EndMT and atherosclerosis in areas of disturbed flow. Tensional force and reconstitution experiments reveal a mechanosensory function for Alk5 in EndMT signaling that is unique and independent of other mechanosensors. Our findings are of fundamental importance for understanding how mechanical forces regulate biochemical signaling, cell plasticity, and vascular disease.

Disturbed flow disrupts the blood-brain barrier in a 3D bifurcation model

The effect of disturbed flow profiles on the endothelium have been studied extensively in systemic vasculature, but less is known about the response of the blood-brain barrier (BBB) to these flow regimes. Here we investigate the effect of disturbed flow on the integrity of the BBB using a three-dimensional, perfusable bifurcation model consisting of a co-culture of endothelial cells with mural and glial cells. Experimental flow patterns predicted by computational fluid dynamics mimic in vivo flow regimes, specifically the presence of a recirculation zone immediately downstream of the bifurcation. Dextran permeability assays and immunostaining with markers for tight junctions show that barrier disruption is significantly greater in areas of disturbed flow compared to fully developed regions downstream of the bifurcation. Probing crosstalk between cell types suggests that disturbed flow causes barrier breakdown independent of endothelial-mural and endothelial-glial interaction. Overall, disturbed flow-induced disruption of the blood-brain barrier suggests that flow-mediated mechanisms may contribute to vascular pathologies in the central nervous system.

A fully coupled computational fluid dynamics – agent-based model of atherosclerotic plaque development: Multiscale modeling framework and parameter sensitivity analysis

BackgroundPeripheral Artery Disease (PAD) is an atherosclerotic disorder that leads to impaired lumen patency through intimal hyperplasia and the build-up of plaques, mainly localized in areas of disturbed flow. Computational models can provide valuable insights in the pathogenesis of atherosclerosis and act as a predictive tool to optimize current interventional techniques. Our hypothesis is that a reliable predictive model must include the atherosclerosis development history. Accordingly, we developed a multiscale modeling framework of atherosclerosis that replicates the hemodynamic-driven arterial wall remodeling and plaque formation. MethodsThe framework was based on the coupling of Computational Fluid Dynamics (CFD) simulations with an Agent-Based Model (ABM). The CFD simulation computed the hemodynamics in a 3D artery model, while 2D ABMs simulated cell, Extracellular Matrix (ECM) and lipid dynamics in multiple vessel cross-sections. A sensitivity analysis was also performed to evaluate the oscillation of the ABM output to variations in the inputs and to identify the most influencing ABM parameters. ResultsOur multiscale model qualitatively replicated both the physiologic and pathologic arterial configuration, capturing histological-like features. The ABM outputs were mostly driven by cell and ECM dynamics, largely affecting the lumen area. A subset of parameters was found to affect the final lipid core size, without influencing cell/ECM or lumen area trends. ConclusionThe fully coupled CFD-ABM framework described atherosclerotic morphological and compositional changes triggered by a disturbed hemodynamics.

ZBTB46 is a shear-sensitive transcription factor inhibiting endothelial cell proliferation via gene expression regulation of cell cycle proteins

ZBTB46 is a transcription factor identified in classical dendritic cells and keeps dendritic cells in a quiescent state. Chromatin immunoprecipitation sequencing in dendritic cells has identified over 1300 potential gene targets of ZBTB46, affecting many processes including cell cycle. Endothelial cells (ECs) also express ZBTB46 and are mostly in a quiescent non-proliferative state. While EC proliferation is a critical process in development, dysregulation of EC proliferation as seen in areas of disturbed flow play an important role in many disease processes such as atherosclerosis, pulmonary hypertension, transplant vasculopathy, neointimal hyperplasia, and in-stent restenosis. We studied the role of ZBTB46 in ECs, hypothesizing that it inhibits EC proliferation. Using a model of disturbed flow in mice, we found that ZBTB46 is expressed in murine arterial ECs in vivo, and is downregulated by disturbed flow. In vitro results using HAECs showed that cell confluence and laminar shear stress, both known physiological conditions promoting EC quiescence, led to upregulation of ZBTB46 expression. Adenoviral-mediated overexpression of ZBTB46 in vitro caused reduced EC proliferation, and increased number of cells in the G0/G1 phase of cell cycle, without affecting apoptosis or senescence, while siRNA knockdown of ZBTB46 negated the known inhibitory role of unidirectional laminar shear stress on EC proliferation. ZBTB46 overexpression also led to a broad suppression of genes involved in cell cycle progression including multiple cyclins and cyclin-dependent kinases, but an increase in the CDK inhibitor CDKN1A. Phosphorylation of the retinoblastoma protein was also decreased as assessed by Western blot. Tube formation on Matrigel was reduced, suggesting an inhibitory role for ZBTB46 in angiogenesis. Further research is required to investigate the potential role of ZBTB46 in specific pathologic conditions and whether it can be targeted in a therapeutic manner.

Abstract 229: Small GTPase Rap1 deficiency Accelerates Development of Atherosclerosis in ApoE Deficient Mice

Rap1, a ubiquitously expressed small GTPase, integrates signals from multiple receptors and promotes activation and signaling by cell adhesion receptors. In vivo , endothelial (EC) Rap1 is required for normal vessel formation, angiogenesis and dynamic regulation of EC barrier. Two Rap1 isoforms, Rap1A and Rap1B, share 95% identity, and EC deletion of both isoforms leads to embryonic lethality due to cardiovascular defects. We have recently demonstrated that Rap1 is a novel regulator of shear sensing in endothelium and that it is essential for nitric oxide release, and that Rap1 deficiency leads to EC dysfunction. These findings demonstrated that Rap1 plays an essential role in vasoprotective response of endothelium in response to shear stress. The objective of this study is to determine the specific role of Rap1 response to laminar, vasoprotective flow and pro-inflammatory, disturbed flow by examining the effect of EC deletion of Rap1B, the more prominent Rap1 isoform, on progression of atherosclerosis. To investigate the role of Rap1 in progression of atherosclerosis, we crossed apolipoprotein E-deficient (ApoE -/- ) mice with tamoxifen-inducible, EC specific Rap1B-knockout (Cadh5-CreERT +/0 ; Rap1B f/f ) to generate atherogenic EC-Rap1B KO (Athero-Rap1B KO) mice. We hypothesized that Rap1B deficiency will exacerbate progression of atherosclerosis. Athero-Rap1B KO and littermate controls (Cadh5-Cre-negative mice or mice injected with carrier oil only) were fed high fat, western diet (21.2% fat, 0.2% cholesterol) for 16 weeks. No difference in total cholesterol levels were observed between Athero- Rap1B KO and control mice. Atherosclerotic lesion formation was visualized with oil O red and plaque area was quantified in en-face preparations. (n=5-9). We observed increased lesion area in the descending aorta of Athero-Rap1B KO mice (0.7351 ± 0.1065 vs 0.4866 ± 0.09812; p =0.0584, one-tailed t-test, Welch corrected) and a strong trend towards larger lesion area in the total aorta (2.142 ± 0.5058 vs 1.267 ± 0.1718; p=0.0679), and the arch of the aorta (1.489 ± 0.4343 vs 0.7807 ± 0.1152; p=0.0747) compared with control mice. These findings demonstrate that Rap1B deficiency accelerates progression of atherosclerosis and show that Rap1 plays a vasoprotective role in both laminar flow and disturbed flow areas.

Disturbed flow disrupts eNOS and caveolin‐1 expression and colocalization via glycocalyx degradation

Atherosclerosis is the main cause of cardiovascular diseases such as coronary artery and carotid artery disease and is known to occur in specific, atheroprone areas of the vasculature characterized by disturbed flow patterns. The endothelial glycocalyx, a mechanotransductive carbohydrate layer that lines the endothelium, helps maintain vascular homeostasis through flow‐induced regulation of vasculoprotective molecules such as nitric oxide. Areas of atherosclerosis development have been shown to contain diminished glycocalyx expression; however, it is unclear whether glycocalyx degradation contributes to atherosclerosis or is a result of its progression. Here, we investigated the effects of disturbed flow on glycocalyx‐mediated nitric oxide regulation in rat fat pad endothelial cells, which robustly express glycocalyx components. Using a custom parallel‐plate flow chamber, we found that disturbed flow (DF) patterns characteristic of atheroprone regions of the vasculature decrease glycocalyx coverage and thickness in comparison to uniform flow (UF). This decrease was accompanied by a reduction in endothelial nitric oxide synthase (eNOS) expression. For the first time, we discovered that DF also decreased the expression of caveolin‐1 (cav‐1), a core protein of caveolae microvesicles that assist in eNOS activation. Additionally, fluorescence microscopy also demonstrated decreased cav‐1 membrane and upstream distribution as well as decreased eNOS‐cav‐1 colocalization in areas of disturbed flow. Finally, UF exposure upon enzymatic degradation of heparan sulfate, a major glycocalyx component, resulted in DF‐like expression colocalization of eNOS and cav‐1, implicating the glycocalyx in eNOS and cav‐1 regulation. These findings provide new evidence to the body of information on glycocalyx‐mediated endothelial regulation, which collectively suggests that glycocalyx degradation may contribute to atherosclerosis development.Support or Funding InformationThis study was funded by National Institute of Health Grant K01 HL125499, National Science Foundation Grant DGE 1451070, Northeastern University Provost Start‐up funds, and an Engineering Dean's Fellowship provided by Northeastern University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

172 β2-catenin mediates the anti-apoptotic effects of no in endothelial cells

β-Catenin Mediates the Anti-Apoptotic Effects of NO in Endothelial CellsApoptosis is implicated in a number of cardiovascular diseases. Increased endothelial cell apoptosis is associated with the development of atherosclerotic plaques....

Abstract 362: Ccn1 Induction and Its Role in the Neointima Formation-induced by Carotid Artery Ligation in Mice

Aim: Atherosclerosis, characterized by neointima formation, occurs preferentially at areas of disturbed flow. Extracellular matrix protein CCN1 is upregulated at the site of atherosclerotic lesions in humans and in animal models. Our aim was to examine the expression of CCN1-regulated by disturbed flow and its role in neointima formation. Methods and Results: Ccn1+/lacZ mice, in which a lacZ reporter gene was inserted to be driven by Ccn1-promoter, were subjected to complete ligation of the left common carotid artery. Progressive reduction in luminal diameters of the ligated artery were observed using B mode ultrasound compared with the right common carotid artery. Ccn1 expression, assessed by whole-mount X-gal staining, was detected in the intima and the media of carotid arteries 3 days after ligation, and the staining intensified between 1-4 weeks in the neointima after ligation. Specifically, Ccn1 expression was observed in endothelial cells, smooth muscle cells, and macrophages in the neointima. To dissect the role of CCN1 in the development of arterial lesions, knock-in mice carrying the integrin α6β1-binding-deficient mutant allele Ccn1-dm were tested in the carotid artery ligation model. Compared with wild-type mice, Ccn1dm/dm mice displayed significantly less neointima after ligation with improved endothelial function, less endothelial apoptosis, and lower oxidative stress, suggesting that the arterial expression of CCN1 induces endothelial dysfunction through its receptor integrin α6β1. Conclusions: CCN1 is a critical pathophysiological regulator mediating neointima formation induced by disturbed flow-generated after carotid artery ligation. CCN1 and its receptor integrin α6β1 represent potential therapeutic targets for atherosclerosis.

Phage-display-guided nanocarrier targeting to atheroprone vasculature.

In regions of the circulation where vessels are straight and unbranched, blood flow is laminar and unidirectional. In contrast, at sites of curvature, branch points, and regions distal to stenoses, blood flow becomes disturbed. Atherosclerosis preferentially develops in these regions of disturbed blood flow. Current therapies for atherosclerosis are systemic and may not sufficiently target these atheroprone regions. In this study, we sought to leverage the alterations on the luminal surface of endothelial cells caused by this atheroprone flow for nanocarrier targeting. In vivo phage display was used to discover unique peptides that selectively bind to atheroprone regions in the mouse partial carotid artery ligation model. The peptide GSPREYTSYMPH (PREY) was found to bind 4.5-fold more avidly to the region of disturbed flow and was used to form targeted liposomes. When administered intravenously, PREY-targeted liposomes preferentially accumulated in endothelial cells in the partially occluded carotid artery and other areas of disturbed flow. Proteomic analysis and immunoblotting indicated that fibronectin and Filamin-A were preferentially bound by PREY nanocarriers in vessels with disturbed flow. In additional experiments, PREY nanocarriers were used therapeutically to deliver the nitric oxide synthase cofactor tetrahydrobiopterin (BH4), which we have previously shown to be deficient in regions of disturbed flow. This intervention increased vascular BH4 and reduced vascular superoxide in the partially ligated artery in wild-type mice and reduced plaque burden in the partially ligated left carotid artery of fat fed atheroprone mice (ApoE(-/-)). Targeting atheroprone sites of the circulation with functionalized nanocarriers provides a promising approach for prevention of early atherosclerotic lesion formation.

Abstract 151: ZBTB46 is a Shear Stress Sensitive Transcription Factor in Endothelial Cells

Background: ZBTB46, also known as zDC or BTBD4, is a member of the BTB-ZF protein family which comprises a diverse group of transcription factors. Recently, ZBTB46 was identified as a transcription repressor specific to classical dendritic cells (cDCs) among immune cells and responsible for keeping cDCs in a quiescent state. ZBTB46 down-regulation by TLR signaling leads to cDC activation. Interestingly, endothelial cells also express ZBTB46, and may be viewed as part of the immune system; expressing functional TLRs and interacting with other immune cells. However, the role of ZBTB46 in endothelial cells has not been studied. Endothelial cell activation in areas of disturbed flow is a critical step in initiation of atherosclerosis, leading to heart attacks and stroke. Hypothesis: ZBTB46 is expressed in quiescent endothelial cells under laminar shear conditions and is down-regulated by low and oscillatory shear stress patterns as seen in areas of disturbed flow, leading to endothelial cell activation. Methods: We used the mouse partial carotid ligation model to assess ZBTB46 shear responsiveness in vivo and a cone and plate cell culture model to assess shear responsiveness in vitro . We measured endothelial cell mRNA using qPCR and protein via western blot. We used immunofluorescence to show localization of ZBTB46 in the arterial wall. Results: We showed that ZBTB46 is expressed in both human and mouse endothelial cells by mRNA and protein measurements. We also showed that ZBTB46 is down-regulated under low and oscillatory shear conditions after 48 hours both in vivo (Figure 1-A) and in vitro (Figure 1-B). Further functional studies including monocyte adhesion and migration assays as well as mechanistic studies assessing upstream and downstream pathways are currently underway.

Thioredoxin-Interacting Protein Is a Biomechanical Regulator of Src Activity

Rationale: Fluid shear stress differentially regulates endothelial cell stress fiber formation with decreased stress fibers in areas of disturbed flow compared with steady flow areas. Importantly, stress fibers are critical for several endothelial cell functions including cell shape, mechano-signal transduction, and endothelial cell–cell junction integrity. A key mediator of steady flow–induced stress fiber formation is Src that regulates downstream signaling mediators such as phosphorylation of cortactin, activity of focal adhesion kinase, and small GTPases. Objective: Previously, we showed that thioredoxin-interacting protein (TXNIP, also VDUP1 [vitamin D upregulated protein 1] and TBP-2 [thioredoxin binding protein 2]) was regulated by fluid shear stress; TXNIP expression was increased in disturbed flow compared with steady flow areas. Although TXNIP was originally characterized for its role in redox and metabolic cellular functions, recent reports show important scaffold functions related to its α-arrestin structure. Based on these findings, we hypothesized that TXNIP acts as a biomechanical sensor that regulates Src kinase activity and stress fiber formation. Methods and Results: Using en face immunohistochemistry of the aorta and cultured endothelial cells, we show inverse relationship between TXNIP expression and Src activity. Specifically, steady flow increased Src activity and stress fiber formation, whereas it decreased TXNIP expression. In contrast, disturbed flow had opposite effects. We studied the role of TXNIP in regulating Src homology phosphatase-2 plasma membrane localization and vascular endothelial cadherin binding because Src homology phosphatase-2 indirectly regulates dephosphorylation of Src tyrosine 527 that inhibits Src activity. Using immunohistochemistry and immunoprecipitation, we found that TXNIP prevented Src homology phosphatase-2–vascular endothelial cadherin interaction. Conclusions: In summary, these data characterize a fluid shear stress–mediated mechanism for stress fiber formation that involves a TXNIP-dependent vascular endothelial cadherin–Src homology phosphatase-2–Src pathway.

Novel paradigms for dialysis vascular access: upstream hemodynamics and vascular remodeling in dialysis access stenosis.

Failure of hemodialysis access is caused mostly by venous intimal hyperplasia, a fibro-muscular thickening of the vessel wall. The pathogenesis of venous neointimal hyperplasia in primary arteriovenous fistulae consists of processes that have been identified as upstream and downstream events. Upstream events are the initial events producing injury of the endothelial layer (surgical trauma, hemodynamic shear stress, vessel wall injury due to needle punctures, etc.). Downstream events are the responses of the vascular wall at the endothelial injury that consist of a cascade of processes including leukocyte adhesion, migration of smooth muscle cells from the media to the intimal layer, and proliferation. In arteriovenous fistulae, the stenoses occur in specific sites, consistently related to the local hemodynamics determined by the vessel geometry and blood flow pattern. Recent findings that the localization of these sites matches areas of disturbed flow may add new insights into the pathogenesis of neointimal hyperplasia in the venous side of vascular access after the creation of the anastomosis. The detailed study of fluid flow motion acting on the vascular wall in anastomosed vessels and in the arm vasculature at the patient-specific level may help to elucidate the role of hemodynamics in vascular remodeling and neointimal hyperplasia formation. These computational approaches may also help in surgical planning for the amelioration of clinical outcome. This review aims to discuss the role of the disturbed flow condition in acting as upstream event in the pathogenesis of venous intimal hyperplasia and in producing subsequent local vascular remodeling in autogenous arteriovenous fistulae used for hemodialysis access. The potential use of blood flow analysis in the management of vascular access is also discussed.

The Role of Platelet-Endothelial Cell Adhesion Molecule-1 in Atheroma Formation Varies Depending on the Site-Specific Hemodynamic Environment

Polymorphisms in the platelet-endothelial cell adhesion molecule (PECAM-1)-1 gene are linked to increased risk of coronary artery disease. Because PECAM-1 has been demonstrated to form a mechanosensory complex that can modulate inflammatory responses in murine arterial endothelial cells, we hypothesized that PECAM-1 contributes to atherogenesis in a shear-dependent and site-specific manner. ApoE(-/-) mice that were wild-type, heterozygous, or deficient in PECAM-1 were placed on a high-fat diet. Detailed analysis of the aorta at sites with differing hemodynamics revealed that PECAM-1-deficient mice had reduced disease in areas of disturbed flow, whereas plaque burden was increased in areas of steady, laminar flow. In concordance with these observations, bone marrow chimera experiments revealed that hematopoietic PECAM-1 resulted in accelerated atheroma formation in areas of laminar and disturbed flow, however endothelial PECAM-1 moderated disease progression in areas of high sheer stress. Moreover, using shear stress-modifying carotid cuffs, PECAM-1 was shown to promote macrophage recruitment into lesions developing in areas of low shear stress. PECAM-1 on bone marrow cells is proatherogenic irrespective of the hemodynamic environment, however endothelial cell PECAM-1 is antiatherogenic in high shear environments. Thus, targeting this pathway therapeutically would require a cell-type and context-specific strategy.