Hayley Duckles

Disturbed Flow Promotes Endothelial Senescence via a p53-Dependent Pathway

Objective—Although atherosclerosis is associated with systemic risk factors such as age, high cholesterol, and obesity, plaque formation occurs predominately at branches and bends that are exposed to disturbed patterns of blood flow. The molecular mechanisms that link disturbed flow–generated mechanical forces with arterial injury are uncertain. To illuminate them, we investigated the effects of flow on endothelial cell (EC) senescence. Approach and Results—LDLR−/− (low-density lipoprotein receptor−/−) mice were exposed to a high-fat diet for 2 to 12 weeks (or to a normal chow diet as a control) before the assessment of cellular senescence in aortic ECs. En face staining revealed that senescence-associated &bgr;-galactosidase activity and p53 expression were elevated in ECs at sites of disturbed flow in response to a high-fat diet. By contrast, ECs exposed to undisturbed flow did not express senescence-associated &bgr;-galactosidase or p53. Studies of aortae from healthy pigs (aged 6 months) also revealed enhanced senescence-associated &bgr;-galactosidase staining at sites of disturbed flow. These data suggest that senescent ECs accumulate at disturbed flow sites during atherogenesis. We used in vitro flow systems to examine whether a causal relationship exists between flow and EC senescence. Exposure of cultured ECs to flow (using either an orbital shaker or a syringe-pump flow bioreactor) revealed that disturbed flow promoted EC senescence compared with static conditions, whereas undisturbed flow reduced senescence. Gene silencing studies demonstrated that disturbed flow induced EC senescence via a p53-p21 signaling pathway. Disturbed flow–induced senescent ECs exhibited reduced migration compared with nonsenescent ECs in a scratch wound closure assay, and thus may be defective for arterial repair. However, pharmacological activation of sirtuin 1 (using resveratrol or SRT1720) protected ECs from disturbed flow–induced senescence. Conclusions—Disturbed flow promotes endothelial senescence via a p53-p21–dependent pathway which can be inhibited by activation of sirtuin 1. These observations support the principle that pharmacological activation of sirtuin 1 may promote cardiovascular health by suppressing EC senescence at atheroprone sites.

Mechanoresponsive Networks Controlling Vascular Inflammation

Atherosclerosis is a chronic inflammatory disease of arteries that develops preferentially at branches and bends that are exposed to disturbed blood flow. Vascular function is modified by flow, in part, via the generation of mechanical forces that alter multiple physiological processes in endothelial cells. Shear stress has profound effects on vascular inflammation; high uniform shear stress prevents leukocyte recruitment to the vascular wall by reducing endothelial expression of adhesion molecules and other inflammatory proteins, whereas low oscillatory shear stress has the opposite effects. Here, we review the molecular mechanisms that underpin the effects of shear stress on endothelial inflammatory responses. They include shear stress regulation of inflammatory mitogen-activated protein kinase and nuclear factor-κB signaling. High shear suppresses these pathways through the induction of several negative regulators of inflammation, whereas low shear promotes inflammatory signaling. Furthermore, we summarize recent studies indicating that inflammatory signaling is highly sensitive to pulse wave frequencies, magnitude, and direction of flow. Finally, the importance of systems biology approaches (including omics studies and functional screening) to identify novel mechanosensitive pathways is discussed.

Heme oxygenase-1 regulates cell proliferation via carbon monoxide-mediated inhibition of T-type Ca2+ channels

Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) affords cellular protection and suppresses proliferation of vascular smooth muscle cells (VSMCs) associated with a variety of pathological cardiovascular conditions including myocardial infarction and vascular injury. However, the underlying mechanisms are not fully understood. Over-expression of Cav3.2 T-type Ca2+ channels in HEK293 cells raised basal [Ca2+]i and increased proliferation as compared with non-transfected cells. Proliferation and [Ca2+]i levels were reduced to levels seen in non-transfected cells either by induction of HO-1 or exposure of cells to the HO-1 product, carbon monoxide (CO) (applied as the CO releasing molecule, CORM-3). In the aortic VSMC line A7r5, proliferation was also inhibited by induction of HO-1 or by exposure of cells to CO, and patch-clamp recordings indicated that CO inhibited T-type (as well as L-type) Ca2+ currents in these cells. Finally, in human saphenous vein smooth muscle cells, proliferation was reduced by T-type channel inhibition or by HO-1 induction or CO exposure. The effects of T-type channel blockade and HO-1 induction were non-additive. Collectively, these data indicate that HO-1 regulates proliferation via CO-mediated inhibition of T-type Ca2+ channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically.

Heart rate reduction with ivabradine promotes shear stress-dependent anti-inflammatory mechanisms in arteries

Blood flow generates wall shear stress (WSS) which alters endothelial cell (EC) function. Low WSS promotes vascular inflammation and atherosclerosis whereas high uniform WSS is protective. Ivabradine decreases heart rate leading to altered haemodynamics. Besides its cardio-protective effects, ivabradine protects arteries from inflammation and atherosclerosis via unknown mechanisms. We hypothesised that ivabradine protects arteries by increasing WSS to reduce vascular inflammation. Hypercholesterolaemic mice were treated with ivabradine for seven weeks in drinking water or remained untreated as a control. En face immunostaining demonstrated that treatment with ivabradine reduced the expression of pro-inflammatory VCAM-1 (p<0.01) and enhanced the expression of anti-inflammatory eNOS (p<0.01) at the inner curvature of the aorta. We concluded that ivabradine alters EC physiology indirectly via modulation of flow because treatment with ivabradine had no effect in ligated carotid arteries in vivo, and did not influence the basal or TNFα-induced expression of inflammatory (VCAM-1, MCP-1) or protective (eNOS, HMOX1, KLF2, KLF4) genes in cultured EC. We therefore considered whether ivabradine can alter WSS which is a regulator of EC inflammatory activation. Computational fluid dynamics demonstrated that ivabradine treatment reduced heart rate by 20 % and enhanced WSS in the aorta. In conclusion, ivabradine treatment altered haemodynamics in the murine aorta by increasing the magnitude of shear stress. This was accompanied by induction of eNOS and suppression of VCAM-1, whereas ivabradine did not alter EC that could not respond to flow. Thus ivabradine protects arteries by altering local mechanical conditions to trigger an anti-inflammatory response.

Experimental Approaches to Study Endothelial Responses to Shear Stress

SIGNIFICANCE Shear stress controls multiple physiological processes in endothelial cells (ECs). RECENT ADVANCES The response of ECs to shear has been studied using a range of in vitro and in vivo models. CRITICAL ISSUES This article describes some of the experimental techniques that can be used to study endothelial responses to shear stress. It includes an appraisal of large animal, rodent, and zebrafish models of vascular mechanoresponsiveness. It also describes several bioreactors to apply flow to cells and physical methods to separate mechanoresponses from mass transport mechanisms. FUTURE DIRECTIONS We conclude that combining in vitro and in vivo approaches can provide a detailed mechanistic view of vascular responses to force and that high-throughput systems are required for unbiased assessment of the function of shear-induced molecules. Antioxid. Redox Signal. 25, 389-400.

T-Type Ca2+ Channel Regulation by CO: A Mechanism for Control of Cell Proliferation

T-type Ca(2+) channels regulate proliferation in a number of tissue types, including vascular smooth muscle and various cancers. In such tissues, up-regulation of the inducible enzyme heme oxygenase-1 (HO-1) is often observed, and hypoxia is a key factor in its induction. HO-1 degrades heme to generate carbon monoxide (CO) along with Fe(2+) and biliverdin. Since CO is increasingly recognized as a regulator of ion channels (Peers et al. 2015), we have explored the possibility that it may regulate proliferation via modulation of T-type Ca(2+) channels.Whole-cell patch-clamp recordings revealed that CO (applied as the dissolved gas or via CORM donors) inhibited all 3 isoforms of T-type Ca(2+) channels (Cav3.1-3.3) when expressed in HEK293 cells with similar IC(50) values, and induction of HO-1 expression also suppressed T-type currents (Boycott et al. 2013). CO/HO-1 induction also suppressed the elevated basal [Ca(2+) ](i) in cells expressing these channels and reduced their proliferative rate to levels seen in non-transfected control cells (Duckles et al. 2015).Proliferation of vascular smooth muscle cells (both A7r5 and human saphenous vein cells) was also suppressed either by T-type Ca(2+) channel inhibitors (mibefradil and NNC 55-0396), HO-1 induction or application of CO. Effects of these blockers and CO were non additive. Although L-type Ca(2+) channels were also sensitive to CO (Scragg et al. 2008), they did not influence proliferation. Our data suggest that HO-1 acts to control proliferation via CO modulation of T-type Ca(2+) channels.

192 The Induction of Homeobox Genes by Disturbed Flow Limits Inflammation at Atherosusceptible Sites

Introduction Atherosclerosis develops at branches and bends of arteries exposed to disturbed blood flow, whereas regions exposed to uniform flow are protected. Disturbed flow generates low, oscillatory wall shear stress (WSS) which promotes atherosclerosis by inducing endothelial cell (EC) expression of inflammatory molecules. Conversely, high unidirectional WSS is protective. We studied the transcriptome at low and high WSS regions in the porcine aorta using microarrays and observed differential expression of multiple Homeobox (Hox) genes, which regulate embryonic development and morphogenesis. We hypothesise that Hox genes influence EC responses to WSS. This project aimed to: (1) assess the effects of WSS on Hox gene expression, and (2) use gene silencing to determine the effects of Hox genes on inflammatory activation. Methods EC were isolated from the outer (high WSS) and inner (low WSS) curvatures of porcine aortae prior to measurement of Hox gene expression by quantitative real-time PCR (qPCR). The expression of HOXA9 and HOXB9 proteins in the murine aortic arch was assessed at the protein level by en face fluorescence staining. Human umbilical vein EC (HUVEC) were exposed to flow for 72 h using an in vitro orbital shaking system, which generates high WSS at the periphery of a cell culture well and low WSS at the centre. Alternatively, cells were exposed to oscillatory or unidirectional WSS using an IBIDI pump system. The expression of Hox genes in sheared HUVEC was assessed by qPCR. Hox genes were silenced in sheared EC prior to the assessment of inflammatory activation by qPCR and immunofluorescence staining. Results qPCR revealed that EC expression of multiple Hox genes (HOXA1, HOXA9, HOXA10, HOXB4, HOXB7, HOXB9, HOXD8, HOXD9) was increased at the low WSS region compared to the high WSS region of the porcine aorta (p < 0.05). Similarly, en face staining demonstrated that endothelial expression of HOXA9 and HOXB9 proteins was higher at a low WSS site compared to a high WSS region (p < 0.05). Consistent with these observations, low oscillatory WSS induced the expression of the above Hox genes in cultured HUVEC while high unidirectional WSS did not. Silencing of HOXB9 significantly enhanced the expression of E-selectin, MCP1 and VCAM-1 in EC exposed to low oscillatory WSS (p < 0.05), indicating that HOXB9 exerts anti-inflammatory effects. Conversely, silencing of other Hox genes had little or no effect on inflammatory molecule expression. Conclusions We conclude that disturbed flow induces multiple Hox genes and that HOXB9 limits inflammatory activation of EC. Thus, although disturbed flow promotes inflammation, it also activates anti-inflammatory HOXB9 which promotes physiological homeostasis. These findings highlight the complex effects of WSS on vascular inflammation and indicate that inflammation at atheroprone sites is governed by a balance between the activities of pro- and anti-inflammatory molecules.