The Effect of Race and Shear Stress on CRP-Induced Responses in Endothelial Cells

Background. C-reactive protein (CRP) is an independent biomarker of systemic inflammation and a predictor of future cardiovascular disease (CVD). More than just a pure bystander, CRP directly interacts with endothelial cells to decrease endothelial nitric oxide synthase (eNOS) expression and bioactivity, decrease nitric oxide (NO) production, and increase the release of vasoconstrictors and adhesion molecules. Race is significantly associated with CRP levels and CVD risks. With aerobic exercise, the vessel wall is exposed to chronic high laminar shear stress (HiLSS) that shifts the endothelium phenotype towards an anti-inflammatory, antioxidant, antiapoptotic, and antiproliferative environment. Thus, the purpose of this study was to assess the racial differences concerning the CRP-induced effects in endothelial cells and the potential role of HiLSS in mitigating these differences. Methods. Human umbilical vein endothelial cells (HUVECs) from four African American (AA) and four Caucasian (CA) donors were cultured and incubated under the following conditions: (1) static control, (2) CRP (10 μg/mL, 24 hours), (3) CRP receptor (FcγRIIB) inhibitor followed by CRP stimulation, (4) HiLSS (20 dyne/cm2, 24 hours), and (5) HiLSS followed by CRP stimulation. Results. AA HUVECs had significantly higher FcγRIIB receptor expression under both basal and CRP incubation conditions. Blocking FcγRIIB receptor significantly attenuated the CRP-induced decrements in eNOS expression only in AA HUVECs. Finally, HiLSS significantly counteracted CRP-induced effects. Conclusion. Understanding potential racial differences in endothelial function is important to improve CVD prevention. Our results shed light on FcγRIIB receptor as a potential contributor to racial differences in endothelial function in AA.

The Shear Stress/KLF2/Nrf2/ARE Pathway: A Hemodynamic Defense against Oxidative Stress

Many diseases have oxidative stress and inflammation as underlying pathological features, including metabolic and inflammatory/autoimmune disorders, diseases of the lung, liver, kidney, gastrointestinal tract, cardiovascular and nervous systems. A leading physiological mechanism for oxidative stress is the nuclear erythroid-related factor 2-like 2/antioxidant response element (Nrf2/ARE) signaling pathway. It maintains intracellular homeostasis and protects cells from oxidative damage by inducing phase II detoxifying and oxidative-stress responsive genes. Nrf2 transcription factor functions as the key controller of the redox homeostatic gene regulatory network, and is tightly controlled by the repressor protein, Kelch-like ECH-associated protein 1 (Keap1). Pharmacological agents to inhibit Keap1 and boost effectiveness of the Nrf2/ARE pathway have been developed and more are in development. This chapter elucidates the importance of hemodynamic laminar shear stress in oxidative homeostasis and examines hemodynamic induction of the shear stress (SS)/Krupple-like factor2 (KLF2) /Nrf2/ARE pathway as a means to combat oxidative stress through hemodynamics.

DNA motif analysis of shear stress responsive endothelial enhancers reveals differential association of KLF and ETV/ETS binding sites with gained and lost enhancers

Endothelial cells (EC) lining blood vessels are exposed to mechanical forces, such as shear stress exerted by the flowing blood. These forces control many aspects of EC biology, including vascular tone, cell migration and proliferation in addition to cell size and shape. Despite a good understanding of the genes and signaling pathways responding to shear stress, our insights into the transcriptional regulation of these responses is much more limited. In particular, we do not know the different sets of regulatory elements (enhancers) that might control increases or decreases in gene expression. Here, we set out to study changes in the chromatin landscape of human umbilical vein endothelial cells (HUVEC) exposed to laminar shear stress. To do so, we performed ChIP-Seq for H3K27 acetylation, indicative of active enhancer elements and ATAC-Seq to mark regions of open chromatin in addition to RNA-Seq on HUVEC exposed to 6 hours of laminar shear stress. Our results show a correlation of gained and lost enhancers with up- and downregulated genes, respectively. DNA motif analysis revealed an over-representation of KLF transcription factor (TF) binding sites in gained enhancers, while lost enhancers contained more ETV/ETS motifs. We validated a subset of flow responsive enhancers using luciferase-based reporter constructs and CRISPR-Cas9 mediated genome editing. Lastly, we characterized shear stress responsive genes in ECs of zebrafish embryos using RNA-Seq. Together, our results reveal the presence of shear stress responsive DNA regulatory elements and lay the groundwork for the future exploration of these elements and the TFs binding to them in controlling EC biology.

Passage dependent changes in nuclear and cytoskeleton structures of endothelial cells under laminar shear stress or cyclic stretch

<p class="abstract"><strong>Background:</strong> The ability of vascular endothelium to sense and respond to the mechanical stimuli generated by blood flow is pivotal in maintaining arterial homeostasis. A steady laminar flow tends to provide athero-protective effect via regulating endothelial functions, vascular tone, and further remodeling process. As arterial aging appeared to be an independent risk factor of cardiovascular diseases, it is critical to understand the effects of cell senescence on endothelial dysfunction under dynamic mechanical stimuli.</p><p class="abstract"><strong>Methods:</strong> In this study, we investigated the morphological responses of aortic endothelial cells toward laminar flow or cyclic stretch. Automated image recognition methods were applied to analyze image data to avoid bias. Differential patterns of morphological adaptations toward distinct mechanical stimuli were observed, and the shear-induced changes were found to be more associated with cell passages than that of cyclic strain. </p><p class="abstract"><strong>Results:</strong> Our results demonstrated that the cytoskeleton and nuclear structural adaptations in endothelial cells toward laminar flow were altered over prolonged culture, suggesting that the failure of senescent endothelial cells to adapt to the applied shear stress morphologically could be one of the contributors to endothelial dysfunctions during vascular aging.</p><p class="abstract"><strong>Conclusions:</strong> Results indicated that cells were able to adjust their cytoskeleton and nuclear alignment and nuclear shapes in response to the applied mechanical stimuli, and that the shear-induced changes were more dependent on PD levels, where cells with higher PDL were more responsive to external forces.</p>

Laminar Shear Stress Protects Against Premature Endothelial Senescence by SIRT1-Dependent Mechanisms

PURPOSE:Accumulation of senescent endothelial cells (ECs) and development of vascular aging have been implicated in the etiology of vascular dysfunction and disease. Aerobic exercise has been recognized as the single most effective non-pharmacological anti-aging intervention via increased laminar shear stress (LSS). This study aimed to determine the protective effects of LSS against premature senescence and the underlying mechanism.METHODS: Carotid artery partial ligation surgery was performed on the left carotid arteries (LCAs) of C57BL/6J male mice to determine the effect of disturbed flow on the development of endothelial senescence. Senescence-associated β-galactosidase (SA-β-gal) staining was performed to measure cellular senescence. Expression levels of protein markers for cell senescence including p21, p16, and p53 were measured by western blotting.RESULTS: <i>En face</i> SA-β-gal staining was identified only in the partially ligated LCAs of voluntary wheel-running mice, suggesting a direct relevance of LSS on the prevention of vascular senescence. In the <i>in vitro</i> H₂O₂-induced premature senescence model, preconditioning of high-flow LSS (20 dyne/cm², 36 hours) induced significant reduction in the percentage of SA-β-gal positive ECs. Expression of the molecular markers of cellular senescence such as p21, p16, and p53 was significantly decreased by LSS pretreatment. However, the protective effects of LSS against premature senescence were completely abolished by SIRT1 inhibition.CONCLUSIONS: The results suggest that high-flow LSS has protective effects against oxidative stress-induced premature endothelial senescence through a SIRT1-dependent mechanism.

Maintenance of HDACs and H3K9me3 Prevents Arterial Flow-Induced Venous Endothelial Damage

The transition of flow microenvironments from veins to arteries in vein graft surgery induces “peel-off” of venous endothelial cells (vECs) and results in restenosis. Recently, arterial laminar shear stress (ALS) and oscillatory shear stress (OS) have been shown to affect the cell cycle and inflammation through epigenetic controls such as histone deacetylation by histone deacetylases (HDACs) and trimethylation on lysine 9 of histone 3 (H3K9me3) in arterial ECs. However, the roles of H3K9me3 and HDAC in vEC damage under ALS are not known. We hypothesized that the different responses of HDACs and H3K9me3 might cause vEC damage under the transition of venous flow to arterial flow. We found that arterial ECs showed high expression of H3K9me3 protein and were retained in the G0 phase of the cell cycle after being subjected to ALS. vECs became round under ALS with a decrease in the expression of H3K9me3, HDAC3, and HDAC5, and an increase in the expression of vascular cell adhesion molecule 1 (VCAM-1). Inhibition of HDACs activity by a specific inhibitor, phenylbutyrate, in arterial ECs caused similar ALS-induced inflammation and cell loss as observed in vECs. Activation of HDACs and H3K9me3 by ITSA-1, an HDAC activator, could prevent ALS-induced peel-off and reduced VCAM-1 expression in vECs. Moreover, shear stress modulates EC morphology by the regulation of focal adhesion kinase (FAK) expression. ITSA-1 or EGF could increase phosphorylated (p)-FAK expression in vECs under ALS. We found that perturbation of the activity of p-FAK and increase in p-FAK expression restored ALS-induced H3K9me3 expression in vECs. Hence, the abnormal mechanoresponses of H3K9me3 and HDAC in vECs after being subjected to ALS could be reversed by ITSA-1 or EGF treatment: this offers a strategy to prevent vein graft failure.