An in silico study of the influence of vessel wall deformation on neointimal hyperplasia progression in peripheral bypass grafts

Neointimal hyperplasia (NIH) is a major obstacle to graft patency in the peripheral arteries. A complex interaction of biomechanical factors contribute to NIH development and progression, and although haemodynamic markers such as wall shear stress have been linked to the disease, these have so far been insufficient to fully capture its behaviour. Using a computational model linking computational fluid dynamics (CFD) simulations of blood flow with a biochemical model representing NIH growth mechanisms, we analyse the effect of compliance mismatch, due to the presence of surgical stitches and/or to the change in distensibility between artery and vein graft, on the haemodynamics in the lumen and, subsequently, on NIH progression. The model enabled to simulate NIH at proximal and distal anastomoses of three patient-specific end-to-side saphenous vein grafts under two compliance-mismatch configurations, and a rigid wall case for comparison, obtaining values of stenosis similar to those observed in the computed tomography (CT) scans. The maximum difference in time-averaged wall shear stress between the rigid and compliant models was 3.4 Pa, and differences in estimation of NIH progression were only observed in one patient. The impact of compliance on the haemodynamic-driven development of NIH was small in the patient-specific cases considered

NF-κB inhibition prevents acute shear stress-induced inflammation in the saphenous vein graft endothelium

The long saphenous vein (LSV) is commonly used as a conduit in coronary artery bypass grafting. However, long term patency remains limited by the development of vascular inflammation, intimal hyperplasia and accelerated atherosclerosis. The impact of acute exposure of venous endothelial cells (ECs) to acute arterial wall shear stress (WSS) in the arterial circulation, and the subsequent activation of inflammatory pathways, remain poorly defined. Here, we tested the hypothesis that acute exposure of venous ECs to high shear stress is associated with inflammatory responses that are regulated by NF-κB both in-vitro and ex-vivo. Analysis of the LSV endothelium revealed that activation of NF-κB occurred within 30 min after exposure to arterial rates of shear stress. Activation of NF-κB was associated with increased levels of CCL2 production and enhanced binding of monocytes in LSVECs exposed to 6 h acute arterial WSS. Consistent with this, ex vivo exposure of LSVs to acute arterial WSS promoted monocyte interactions with the vessel lumen. Inhibition of the NF-κB pathway prevented acute arterial WSS-induced CCL2 production and reduced monocyte adhesion, both in vitro and in human LSV ex vivo, demonstrating that this pathway is necessary for the induction of the acute arterial WSS-induced pro-inflammatory response. We have identified NF-κB as a critical regulator of acute endothelial inflammation in saphenous vein in response to acute arterial WSS. Localised endothelial-specific inhibition of the NF-κB pathway may be beneficial to prevent vein graft inflammation and consequent failure

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

Background: Peripheral 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. Methods: The 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. Results: Our 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. Conclusion: The fully coupled CFD-ABM framework described atherosclerotic morphological and compositional changes triggered by a disturbed hemodynamics

Coronary artery mechanics induces human saphenous vein remodelling via recruitment of adventitial myofibroblast-like cells mediated by Thrombospondin-1

Rationale: Despite the preferred application of arterial conduits, the greater saphenous vein (SV) remains indispensable for coronary bypass grafting (CABG), especially in multi-vessel coronary artery disease (CAD). The objective of the present work was to address the role of mechanical forces in the activation of maladaptive vein bypass remodeling, a process determining progressive occlusion and recurrence of ischemic heart disease. Methods: We employed a custom bioreactor to mimic the coronary shear and wall mechanics in human SV vascular conduits and reproduce experimentally the biomechanical conditions of coronary grafting and analyzed vein remodeling process by histology, histochemistry and immunofluorescence. We also subjected vein-derived cells to cyclic uniaxial mechanical stimulation in culture, followed by phenotypic and molecular characterization using RNA and proteomic methods. We finally validated our results in vitro and using a model of SV carotid interposition in pigs. Results: Exposure to pulsatile flow determined a remodeling process of the vascular wall involving reduction in media thickness. Smooth muscle cells (SMCs) underwent conversion from contractile to synthetic phenotype. A time-dependent increase in proliferating cells expressing mesenchymal (CD44) and early SMC (SM22\u3b1) markers, apparently recruited from the SV adventitia, was observed especially in CABG-stimulated vessels. Mechanically stimulated SMCs underwent transition from contractile to synthetic phenotype. MALDI-TOF-based secretome analysis revealed a consistent release of Thrombospondin-1 (TSP-1), a matricellular protein involved in TGF-\u3b2-dependent signaling. TSP-1 had a direct chemotactic effect on SV adventitia resident progenitors (SVPs); this effects was inhibited by blocking TSP-1 receptor CD47. The involvement of TSP-1 in adventitial progenitor cells differentiation and graft intima hyperplasia was finally contextualized in the TGF-\u3b2-dependent pathway, and validated in a saphenous vein into carotid interposition pig model. Conclusions: Our results provide the evidence of a matricellular mechanism involved in the human vein arterialization process controlled by alterations in tissue mechanics, and open the way to novel potential strategies to block VGD progression based on targeting cell mechanosensing-related effectors

Mechanisms of Vascular Disease: A Reference Book for Vascular Specialists

New updated edition first published with Cambridge University Press. This new edition includes 29 chapters on topics as diverse as pathophysiology of atherosclerosis, vascular haemodynamics, haemostasis, thrombophilia and post-amputation pain syndromes

Vascular related pathologies in cardiovascular disease and cancer

Cardiovascular disease (CVD) and cancer are the leading causes of death worldwide. A damaged endothelium is one of the factors contributing towards these diseases. This thesis focused on understanding the implications of alterations to the physiological endothelium resulting in pathologies related to vascular disease and cancer metastasis. Functional healing response occurs in the diseased vessel wall aimed at restoring the vessel after an injury. Existing studies state that vascular progenitor cells contribute to the injured vasculature and aid in the repair process. Yet the mechanisms underlying the amalgamation of the cells to the endothelium, their origin and functions have not been clear. Through Study 1 using animal models of arterial injury, we examined the role of bone marrow derived cells in arterial repair and the mechanisms behind it. We observed that bone marrow-derived cells, helped in the initial stages of arterial injury and were subsequently eliminated from the artery wall. They localized in the arterial intima and most them were of endothelial phenotype. Additionally, bone marrow-derived cells did not fuse with the intima but could differentiate into vascular cells. This helped them adjust in the vessel wall and meet the needs of their new microenvironment. Fascinatingly, local delivery of bone marrow-derived endothelial cells to the sites of arterial injury caused a 1.4-fold decrease of the intimal lesion area. These results define the role of BM derived endothelial cells in the development of intimal lesions post vascular injury and this information contributes to the existing understanding of the pathogenesis of intimal hyperplasia. Hemodynamic forces are a cause of a dysfunctional endothelium. A turbulent blood flow could result in vascular disease. Studies have shown that red blood cell distribution (RDW) width as a risk factor for death in cancer and CVD. RDW is one of the haematological parameters commonly reported as part of a complete blood count An RDW higher than normal is termed as anisocytosis. Anisocytosis has been traditionally used, in combination with the red blood cell corpuscular volume, to diagnose chronic inflammatory status in the body. It has been never studied before if anisocytosis is just factor that reflects chronic inflammation in the body, or is factor that directly affects it. Hence in Study 2, we hypothesized that anisocytosis leads to changes in blood flow affecting interaction between blood and vascular endothelium at the bifurcation of arteries. We found that a high RDW is a predictive factor for the interaction between cellular components of blood and vascular wall. These interactions can lead to increased inflammation in the vessels and initiation of thrombosis. Put together, we suggest that anisocytosis measured by RDW is a predictive factor of vascular diseases. Cancer metastasis is one of the major causes of mortality and arises also due to a damaged endothelium. In Study 3 we investigated the role of murine cytomegalovirus(MCMV) in colon cancer progression using MCMV infected and non-infected animal models. Our results indicate that MCMV did not affect tumor growth but increases the incidence of metastasis to the lungs. Additionally, using microarray analysis we found cytokeratins 1, 2 and 14 to be upregulated 100 times in the infected models compared to the non-infected. We speculate that in our case metastasis is mediated possibly through a cytokeratin mediated pathway. The mechanism for dissemination is under investigation. In Study 4, we investigated the effect of C/EBPβ on metastasis and the relationship between C/EBPβ expression and overall survival of breast cancer patients. We found that decrease in C/EBPβ expression was related to shorter overall survival of breast cancer patients. Loss of C/EBPβ also, affected tumor growth, morphology and lung metastasis in murine 4T1 breast cancer model. Furthermore, inhibition of C/EBPβ resulted in an augmented expression of MHCII and CD45+, CD3+ and CD4+ lymphocytes accumulation in the tumors. Additional experiments established the role of inflammation in C/EBPβ-mediated metastasis formation

A comprehensive accounting of lncRNA dynamics within vascular smooth muscle cell pathological transitions

Vascular smooth muscle cells (VSMCs) provide vital contractile force within blood vessel walls, yet also propagate widespread cardiovascular pathologies with high mortality rates through pathological activities. The targeting of such phenotypes in VSMCs has been a commonly-touted strategy for decades yet we still have no viable option to implement this. Recent studies have established that VSMC phenotypes are driven, in part, by the diverse effects of long non-coding RNAs (lncRNAs) on gene expression. This class of largely uncharacterised gene regulators may offer a wealth of novel targets to be used to target VSMCs. However, their characterisation in VSMCs in pathological states is hampered by incomplete lncRNA representation in reference annotation. In this thesis, we address this by assembling non-reference transcripts in RNA sequencing datasets describing saphenous vein VSMCs stimulated in vitro with cytokines and growth factors or arterial VSMCs stimulated with mechanical stress. We also utilised VSMCs isolated from atherosclerotic plaques. All transcripts were subject to a rigorous lncRNA prediction pipeline to provide an expanded VSMC transcriptome with an unprecedented level of detail on the lncRNAs associated with VSMC pathological states. We found substantially improved coverage of lncRNAs responding to pro-mitogenic stimuli, with non-reference lncRNAs contributing 21–32% per dataset. We also demonstrate non-reference lncRNAs were biased towards enriched expression within VSMCs, suggesting extra lncRNAs highlighted by our pipeline have particular relevance to VSMC-specific processes. They were also biased towards transcription from enhancer sites suggesting they coordinate the regulation of neighbouring protein-coding genes. Both VSMC-enriched and enhancer-transcribed lncRNAs were large components of lncRNAs responding to pathological stimuli, yet without novel transcript discovery 33–46% of these lncRNAs would remain hidden. In parallel to this analysis, we mined the expanded VSMC annotation to initially explore functionality in a small cohort of uncharacterised lncRNAs within the saphenous vein VSMC in vitro model. In our final round of analysis, we hypothesised that many lncRNAs may be involved in directing early transcriptional changes leading up to proliferation – and so constitute targets that may be particularly high value through acting upstream of multiple mitogenic or pathogenic pathways. We therefore used our expanded VSMC annotation as a foundation to perform a deeper analysis of lncRNA activity within RNAseq samples obtained from the first 24 hours of stimulation in the saphenous vein VSMC in vitro model, aiming to identify lncRNAs influencing initial transcriptional changes prior to observable cell division. We noted an enrichment of lncRNA induction – particularly those which were VSMC-enriched or enhancer-transcribed – within an early phase of SVSMC stimuli response prior to proliferation. Transcription factor mRNA dynamics also localised to earlier phases whilst cell cycle mRNAs were overwhelmingly induced after 8 hours. This suggests the involvement of lncRNAs in an early phase of gene regulation sets the VSMC on a path towards later proliferation. To predict lncRNAs with functional impact in the 4 hour regulatory phase, we looked for evidence of their cis-regulation of nearby genes. Genes located near differentially expressed intergenic lncRNAs were 1.51x more likely to be differentially expressed within the four hour window than those located elsewhere in the genome. This effect was also identified for transcription factors and was particularly potent for genes around enhancer-transcribed lncRNAs (2.26x) but weakened when considering over longer time periods (1.16x). Together this suggests a focus of lncRNA-dependent cis-regulation activity in the first four hours after stimulation that could lead to wider downstream impact on VSMC pathological states. We finish by identifying a cohort of uncharacterised lncRNAs regulated in the initial four hour phase that have strong correlations in expression with transcription factors or other genes that explicitly link to vasculoproliferative pathology. Overall, our comprehensive VSMC lncRNA repertoire provides much needed clarity on the activity of lncRNAs within VSMC pathological states. The approach we outline allows proper prioritisation of candidates for characterisation and exemplifies a strategy to broaden our knowledge of lncRNA across a range of disease states