Peripheral arterial disease (PAD) is a major cause of morbidity and mortality. Therapeutic neovascularization is an alternative therapeutic strategy aimed at improving blood flow to the lower... Show morePeripheral arterial disease (PAD) is a major cause of morbidity and mortality. Therapeutic neovascularization is an alternative therapeutic strategy aimed at improving blood flow to the lower extremities and promoting blood vessel growth. The major components of neovascularization are; arteriogenesis and angiogenesis. These processes differ from each other. Arteriogenesis is initiated by shear stress and is the formation of collateral arteries from the pre-existing arteriolar network, and will mainly be found in the upper limb of the mice after ligation of the arteries in the limb. The molecular mechanism for arteriogenesis is based on shear stress regulated inflammatory responses, accompanied by the influx of inflammatory cells in the perivascular compartment around the collateral that are being formed. This thesis provides an analysis of two different in vivo models, the mouse hind limb ischemia (HLI) model and Matrigel plug assay. Furthermore, in the second part of this thesis, we focus on investigating the underlying mechanism of neovascularization in order to identify potential targets for therapeutic neovascularization. Show less
Cardiovascular diseases (CVDs) remain the leading cause of death worldwide, and thus, novel therapies are required. CVDs generally result in local shortages in the blood supply, known as ischemia.... Show moreCardiovascular diseases (CVDs) remain the leading cause of death worldwide, and thus, novel therapies are required. CVDs generally result in local shortages in the blood supply, known as ischemia. Neovascularization is the body's innate response mechanism that stimulates the restoration of blood flow to ischemic tissues. During the last decade, microRNAs have emerged as critical regulators of both CVD and neovascularization. Recent studies demonstrated that microRNAs are altered in many ways; however, whether these microRNA modifications could be physiologically relevant remained unclear. We examined whether specific microRNAs with a known cardiovascular function are subject to particular microRNA-alterations and if they could be relevant in cardiovascular disease. Our experiments demonstrated that the level of specific microRNA alterations, including isomiR formation, adenosine-to-inosine editing, and N6-adenosine methylation, changed in response to cardiovascular pathology. Many of these alterations changed the microRNAs function, which had a direct effect on processes like neovascularization. For example, microRNA adenosine-to-inosine editing increased after ischemia in both mice and humans and promoted neovascularization. These findings suggest that microRNA modifications can potentially be harnessed as a biomarker for cardiovascular disease, or even a novel therapeutic target. Show less
Bot, I.; Velden, D. van der; Bouwman, M.; Kröner, M.J.; Kuiper, J.; Quax, P.H.A.; Vries, M.R. de 2020
Mast cells have been associated with arteriogenesis and collateral formation. In advancedhuman atherosclerotic plaques, mast cells have been shown to colocalize with plaque neovessels,and mast... Show moreMast cells have been associated with arteriogenesis and collateral formation. In advancedhuman atherosclerotic plaques, mast cells have been shown to colocalize with plaque neovessels,and mast cells have also been associated with tumor vascularization. Based on these associations,we hypothesize that mast cells promote angiogenesis during ischemia. In human ischemic muscletissue from patients with end-stage peripheral artery disease, we observed activated mast cells,predominantly located around capillaries. Also, in mouse ischemic muscles, mast cells were detectedduring the revascularization process and interestingly, mast cell activation status was enhanced up to10 days after ischemia induction. To determine whether mast cells contribute to both arteriogenesisand angiogenesis, mast cells were locally activated immediately upon hind limb ischemia in C57Bl/6mice. At day 9, we observed a 3-fold increase in activated mast cell numbers in the inguinal lymphnodes. This was accompanied by an increase in the amount of Ly6Chigh inflammatory monocytes.Interestingly, local mast cell activation increased blood flow through the hind limb (46% at day 9)compared to that in non-activated control mice. Histological analysis of the muscle tissue revealedthat mast cell activation did not aect the number of collaterals, but increased the collateral diameter,as well as the number of CD31+ capillaries. Together, these data illustrate that locally activated mastcell contribute to arteriogenesis and angiogenesis. Show less
Vascular remodeling is an active process of structural changes in the vasculature due to changes in the blood flow. It comprises diseases and processes such peripheral artery disease (PAD),... Show moreVascular remodeling is an active process of structural changes in the vasculature due to changes in the blood flow. It comprises diseases and processes such peripheral artery disease (PAD), coronary artery disease (CAD), neovascularization and vein graft disease (VGD), that are covered by cardiovascular diseases (CVD). The main underlying pathology of CVD is atherosclerosis, which can cause ischemia distal to atherosclerotic occlusions. Neovascularization (angiogenesis and arteriogenesis) naturally occurs in the body to form new blood vessels and restore blood flow to ischemic tissue. In case of severe atherosclerotic lesions, when the neovascularization capability of the body is not sufficient and revascularization interventions are no longer possible, bypass surgery is indicated with preferaby a vein graft. However, due to VGD, patency rates of vein grafts after 10 years are low. The aim of this thesis was to elucidate the role of the innate and adaptive immune system on vascular remodeling. We investigated the role of Toll-like receptors, Interferon regulatory factors and T cells on neovascularisation and VGD. In conclusion, new knowledge was obtained on several contributing factors in vascular remodeling. Described molecules of the innate and adaptive immune system might be used as targets to prevent VGD and stimulate neovascularization. Show less
This thesis describes the role of 14q32 microRNAs in vascular remodelling. The 14q32 microRNA cluster contains 54 microRNAs in humans and is highly conserved in mammals. In part I of this thesis,... Show moreThis thesis describes the role of 14q32 microRNAs in vascular remodelling. The 14q32 microRNA cluster contains 54 microRNAs in humans and is highly conserved in mammals. In part I of this thesis, we describe the role of 14q32 microRNAs in several processes of vascular remodelling. We have shown that inhibition of several 14q32 microRNAs, miR-329, miR-494 and miR-495, results in increased neovascularisation after hindlimb ischemia in mice. In addition, inhibition of the same microRNAs reduced atherosclerotic plaque formation and restenosis in experimental mouse models under hypercholesterolemic conditions. In part II of this thesis, we zoom in to the post-transcriptional regulation of 14q32 microRNAs through RNA binding proteins. The third and last part of this thesis studies the expression of microRNAs in subcutaneous adipose tissue of critical limb ischemia patients and discusses the potential use of microRNAs as biomarker to predict the risk of amputation in these patients. In conclusion, this thesis provides novel insights in the role of 14q32 microRNAs in processes of vascular remodelling. Experimental studies have identified 14q32 microRNAs as potential therapeutic targets for treatment and prevention of atherosclerosis, restenosis and peripheral arterial disease. Show less
The studies included in this thesis demonstrated a preclinical murine model to study neovascularization in vivo and subsequently a number of potential targets to stimulate therapeutic... Show moreThe studies included in this thesis demonstrated a preclinical murine model to study neovascularization in vivo and subsequently a number of potential targets to stimulate therapeutic neovascularization. This thesis contributes to a better insight into mechanisms underlying post-ischemic neovascularization and offers new therapeutic perspective to current treatment strategies for patients with critical limb ischemia. Whether stagnated blood flow recovery after an occlusive event is due to restricted pre-existing collateral bed or due to decreased collateral remodeling, we are now closer to a tailor made treatment available for each patient with peripheral arterial disease. Show less
An active pool of __Tissue Factor__ (TF) is required to initiate blood coagulation, but the mechanism that regulates the activation of TF is highly debated. The reduced coagulant activities of both... Show moreAn active pool of __Tissue Factor__ (TF) is required to initiate blood coagulation, but the mechanism that regulates the activation of TF is highly debated. The reduced coagulant activities of both human and mouse disulfide-mutated TF indicates that the formation of a Cys186-Cys209 or Cys190-Cys213 disulfide bond in the extracellular domain of TF controls its procoagulant function. Also the strong evolutionary conservation of this allosteric disulfide bond supports previous assumption. Procoagulant active TF is also found intravascularly on small vesicles (microparticles), and high levels of microparticle-associated TF is thought to increase the risk for thrombosis in cancer patients. Despite the fact that chemotherapy is an independent risk factor for thrombosis, chemotherapy-induced tumor destruction was not associated with an increase in microparticle-associated TF in testis cancer patients. The soluble isoform of TF, alternatively spliced TF (asTF), induces angiogenesis through interaction with integrins and independent of intracellular protease-activated receptor (PAR)-mediated signaling. PAR-2 but not PAR-1 plays a role in arteriogenesis in vivo. Furthermore, PAR-2 is involved in the anti-inflammatory response that may promote arteriogenesis. Show less
Bastiaansen, T.; Ewing, M.; Boer, H. de; Kraan, T.V.; Vries, M. de; Peters, E.; ... ; Nossent, A.Y. 2012
In peripheral arterial occlusive disease (PAOD), the formation of an atherosclerotic lesion eventually results in a significant stenosis of a major artery thereby disrupting blood flow in... Show moreIn peripheral arterial occlusive disease (PAOD), the formation of an atherosclerotic lesion eventually results in a significant stenosis of a major artery thereby disrupting blood flow in peripheral arteries towards lower limb tissue. Unfortunately, there is a substantial number of patients that suffer from severe critical limb ischemia, which is associated with a poor prognosis and high rates of amputation and mortality. Despite state-of-the-art revascularization treatment options and optimal control of co-morbidities these 'no option' patients remain at high risk for limb amputation. It is these patients that require new therapeutic applications to augment neovascularization and prevent them from limb amputation. Expansion of our current knowledge is required to gain insight in cellular and molecular mechanisms that are involved in neovascularization, to optimize revascularization therapies for patients with critical limb ischemia. This thesis provides a role for multiple leukocytes in blood flow recovery, in particular Natural Killer cells and CD4+ T lymphocytes, and shows that genetic differences in the Natural Killer gene complex induce differences in vascular remodeling. It was also shown how growth factor expression, signaling and regulation of gene expression modulate revascularization. Show less
