Mary Jo Mulligan-Kehoe

ERK1/2-Akt1 crosstalk regulates arteriogenesis in mice and zebrafish

Arterial morphogenesis is an important and poorly understood process. In particular, the signaling events controlling arterial formation have not been established. We evaluated whether alterations in the balance between ERK1/2 and PI3K signaling pathways could stimulate arterial formation in the setting of defective arterial morphogenesis in mice and zebrafish. Increased ERK1/2 activity in mouse ECs with reduced VEGF responsiveness was achieved in vitro and in vivo by downregulating PI3K activity, suppressing Akt1 but not Akt2 expression, or introducing a constitutively active ERK1/2 construct. Such restoration of ERK1/2 activation was sufficient to restore impaired arterial development and branching morphogenesis in synectin-deficient mice and synectin-knockdown zebrafish. The same approach effectively stimulated arterial growth in adult mice, restoring arteriogenesis in mice lacking synectin and in atherosclerotic mice lacking both LDL-R and ApoB48. We therefore conclude that PI3K-ERK1/2 crosstalk plays a key role in the regulation of arterial growth and that the augmentation of ERK signaling via suppression of the PI3K signaling pathway can effectively stimulate arteriogenesis.

The vasa vasorum in diseased and nondiseased arteries

The vasa vasorum form a network of microvasculature that originate primarily in the adventitial layer of large arteries. These vessels supply oxygen and nutrients to the outer layers of the arterial wall. The expansion of the vasa vasorum to the second order is associated with neovascularization related to progression of atherosclerosis. Immunohistological analysis of human plaques from autopsied aortas have defined plaque progression and show a significant correlation with vasa vasorum neovascularization. Recent technological advances in microcomputed tomography have enabled investigation of vasa vasorum structure and function in nondiseased large arteries from pigs and dogs. Smaller mammals, particularly mice with genetic modifications that enable disease development, have been used extensively to study the vasa vasorum in diseased vessels. Despite the fact that most mouse models that are used to study atherosclerosis are unable to develop plaque to the extent found in humans, studies in both humans and mice underscore the importance of angiogenic vasa vasorum in progression of atherosclerosis. Those who have examined the vasa vasorum in occluded vessels of nondiseased pigs and dogs find that inhibition of the vasa vasorum makes the animals atheroprone. Atherosclerosis is a multifactorial disease. There is increasing evidence that factors, produced in response to changes in the arterial wall, collaborate with the vasa vasorum to enhance the disease process.

Micro computed tomography for vascular exploration.

Vascular exploration of small animals requires imaging hardware with a very high spatial resolution, capable of differentiating large as well as small vessels, in both in vivo and ex vivo studies. Micro Computed Tomography (micro-CT) has emerged in recent years as the preferred modality for this purpose, providing high resolution 3D volumetric data suitable for analysis, quantification, validation, and visualization of results. The usefulness of micro-CT, however, can be adversely affected by a range of factors including physical animal preparation, numerical quantification, visualization of results, and quantification software with limited possibilities. Exacerbating these inherent difficulties is the lack of a unified standard for micro-CT imaging. Most micro-CT today is aimed at particular applications and the software tools needed for quantification, developed mainly by imaging hardware manufacturers, lack the level of detail needed to address more specific aims. This review highlights the capabilities of micro-CT for vascular exploration, describes the current state of imaging protocols, and offers guidelines and suggestions aimed at making micro-CT more accurate, replicable, and robust.

The antiangiogenic activity of rPAI-1(23) inhibits vasa vasorum and growth of atherosclerotic plaque.

Plaque vascularity has been implicated in its growth and stability. However, there is a paucity of information regarding the origin of plaque vasculature and the role of vasa vasorum in plaque growth. To inhibit growth of vasa vasorum in atherogenic mice and assess its effect on plaque growth, we used a truncated plasminogen activator inhibitor (PAI)-1 protein, rPAI-123, that has significant antiangiogenic activity. Female LDLR−/−ApoB-48–deficient mice fed Paigen’s diet without cholate for 20 weeks received rPAI-123 treatment (n=21) for the last 6 weeks. Plaque size and vasa vasorum density were compared to 2 controls: mice fed Paigen’s diet and treated with saline for the last 6 weeks (n=16) and mice fed Paigen’s diet until the onset of treatment (n=14). The rPAI-123 treatment significantly reduced plaque area and plaque cholesterol in the descending aorta and plaque area in the innominate artery. Measurements of reconstructed confocal microscopy images of vasa vasorum demonstrate that rPAI-123 treatment decreased vasa vasorum area and length, which was supported by microCT images. Confocal images provide evidence for vascularized plaque in the saline-treated group but not in rPAI-123–treated mice. The increased vessel density in saline-treated mice is attributable, in part, to upregulated fibroblast growth factor-2 expression, which is inhibited by rPAI-123. In conclusion, rPAI-123 inhibits growth of vasa vasorum, as well as vessels within the adjacent plaque and vessel wall, through inhibition of fibroblast growth factor-2, leading to reduced plaque growth in atherogenic female LDLR−/−ApoB-48–deficient mice.

Vasa Vasorum in Normal and Diseased Arteries

The vasa vasorum are a specialized microvasculature that play a major role in normal vessel wall biology and pathology. Under physiological conditions, the adventitial vasa vasorum take up molecules that are transmitted from the blood to the adventitia by mass transport through the arterial wall. The adventitia is the primary early site for vessel wall response to arterial injury that occurs on the luminal side. The vasa vasorum expand in response to the injury, which alters vessel homeostasis. This review focuses on the impact the vasa vasorum expansion has on development of vascular pathology, particularly neointima development and growth of atherosclerotic plaque. The adventitia, the outermost layer of the vessel wall, has received considerable attention in recent years. It contains a heterogeneous population of cells, including macrophages,1,2 T cells,3 B cells,1,2 dendritic cells,1,2 progenitor cells,4,5 and fibroblasts that can differentiate into myofibroblasts.6 It also contains an adrenergic nervous system,7 a lymphatic network,8 and vasa vasorum, a specialized microvasculature that plays a major role in normal vessel wall biology and pathology that is the subject of the present review. Under physiological conditions, the adventitial vasa vasorum and lymphatic vessels take up molecules that are transmitted from the blood to the adventitia by mass transport through the arterial wall.9–11 Vascular injury at the luminal side of the vessel wall significantly impacts the adventitia by convection of soluble factors, microparticles and macroparticles, mediators such as products of oxidation, tissue cytolysis, and proteolysis from the intima to the adventitia by hydraulic conductance.12 As a result, the adventitia becomes the primary early site for the vessel wall response to arterial injury, which includes myofibroblast migration into the vessel wall,13,14 inflammatory cell accumulation, …

Syndecan 4 is required for endothelial alignment in flow and atheroprotective signaling

Significance Atherosclerosis, the major cause of death and illness in industrialized nations, develops in regions of arteries in which fluid flow patterns are disturbed and endothelial cells fail to align in the direction of flow. In contrast, regions of laminar flow in which cells are aligned are protected. The current work shows that the transmembrane proteoglycan syndecan 4 is required for endothelial cell alignment in the direction of flow and for the protective effect of high laminar flow, yet other flow responses are intact. The data therefore identify a role for syndecan 4 in flow direction sensing, show that sensing flow direction is separable from sensing flow magnitude, and provide new support for the key role of cell alignment in atheroprotection. Atherosclerotic plaque localization correlates with regions of disturbed flow in which endothelial cells (ECs) align poorly, whereas sustained laminar flow correlates with cell alignment in the direction of flow and resistance to atherosclerosis. We now report that in hypercholesterolemic mice, deletion of syndecan 4 (S4−/−) drastically increased atherosclerotic plaque burden with the appearance of plaque in normally resistant locations. Strikingly, ECs from the thoracic aortas of S4−/− mice were poorly aligned in the direction of the flow. Depletion of S4 in human umbilical vein endothelial cells (HUVECs) using shRNA also inhibited flow-induced alignment in vitro, which was rescued by re-expression of S4. This effect was highly specific, as flow activation of VEGF receptor 2 and NF-κB was normal. S4-depleted ECs aligned in cyclic stretch and even elongated under flow, although nondirectionally. EC alignment was previously found to have a causal role in modulating activation of inflammatory versus antiinflammatory pathways by flow. Consistent with these results, S4-depleted HUVECs in long-term laminar flow showed increased activation of proinflammatory NF-κB and decreased induction of antiinflammatory kruppel-like factor (KLF) 2 and KLF4. Thus, S4 plays a critical role in sensing flow direction to promote cell alignment and inhibit atherosclerosis.

A Truncated Plasminogen Activator Inhibitor-1 Protein Induces and Inhibits Angiostatin (Kringles 1–3), a Plasminogen Cleavage Product

Plasminogen activator inhibitor-1 (PAI-1) is a serpin protease inhibitor that binds plasminogen activators (uPA and tPA) at a reactive center loop located at the carboxyl-terminal amino acid residues 320–351. The loop is stretched across the top of the active PAI-1 protein maintaining the molecule in a rigid conformation. In the latent PAI-1 conformation, the reactive center loop is inserted into one of the beta sheets, thus making the reactive center loop unavailable for interaction with the plasminogen activators. We truncated porcine PAI-1 at the amino and carboxyl termini to eliminate the reactive center loop, part of a heparin binding site, and a vitronectin binding site. The region we maintained corresponds to amino acids 80–265 of mature human PAI-1 containing binding sites for vitronectin, heparin (partial), uPA, tPA, fibrin, thrombin, and the helix F region. The interaction of “inactive” PAI-1, rPAI-123, with plasminogen and uPA induces the formation of a proteolytic protein with angiostatin properties. Increasing amounts of rPAI-123 inhibit the proteolytic angiostatin fragment. Endothelial cells exposed to exogenous rPAI-123 exhibit reduced proliferation, reduced tube formation, and 47% apoptotic cells within 48 h. Transfected endothelial cells secreting rPAI-123 have a 30% reduction in proliferation, vastly reduced tube formation, and a 50% reduction in cell migration in the presence of VEGF. These two studies show that rPAI-123 interactions with uPA and plasminogen can inhibit plasmin by two mechanisms. In one mechanism, rPAI-123cleaves plasmin to form a proteolytic angiostatin-like protein. In a second mechanism, rPAI-123 can bind uPA and/or plasminogen to reduce the number of uPA and plasminogen interactions, hence reducing the amount of plasmin that is produced.

Molecular imaging of vessels in mouse models of disease

Vascular imaging of angiogenesis in mouse models of disease requires multi modal imaging hardware capable of targeting both structure and function at different physical scales. The three dimensional (3D) structure and function vascular information allows for accurate differentiation between biological processes. For example, image analysis of vessel development in angiogenesis vs. arteriogenesis enables more accurate detection of biological variation between subjects and more robust and reliable diagnosis of disease. In the recent years a number of micro imaging modalities have emerged in the field as preferred means for this purpose. They provide 3D volumetric data suitable for analysis, quantification, validation, and visualization of results in animal models. This review highlights the capabilities of microCT, ultrasound and microPET for multimodal imaging of angiogenesis and molecular vascular targets in a mouse model of tumor angiogenesis. The basic principles of the imaging modalities are described and experimental results are presented.

The anti-angiogenic activity of rPAI-1(23) inhibits fibroblast growth factor-2 functions.

Many angiogenesis inhibitors are breakdown products of endogenous extracellular matrix proteins. Plasmin and matrix metalloproteinase-3 generate breakdown products of matrix-bound plasminogen activator inhibitor-1 (PAI-1). We produced a truncated form of PAI-1, rPAI-123, that possesses significant anti-angiogenic activity and stimulates high levels of apoptosis in quiescent arterial endothelial cells. Quiescent endothelial cells are less susceptible to apoptosis than angiogenic endothelial cells. The present study was designed to determine the mechanism of the rPAI-123 effects in bovine aortic endothelial cells. Apoptosis was measured in annexin V and caspase 3 assays. Expression of death and survival signaling molecules were examined by Western blot and kinase activity. Fibroblast growth factor 2 (FGF2) functions were analyzed in angiogenesis assays. The early response to rPAI-123 was an increase in annexin V-positive cells and phosphorylated (p) JNK isoform expression followed by an increase in p-Akt and p-c-Jun expression. Caspase 3 was activated at 4 h, whereas p-Akt was reduced to control levels. By 6 h of rPAI-123 treatment cell number was reduced by 35%, and p-c-Jun and p-JNK were degraded by proteasomes. Confocal microscopic images showed increased amounts of FGF2 in the extracellular matrix. However, rPAI-123 blocked FGF2 signaling through FGF receptor 1 and syndecan-4, inhibiting cell migration, tubulogenesis, and proliferation. Exogenous FGF2 stimulation could not reverse these effects. We conclude that rPAI-123 stimulation of apoptosis in BAEC triggers a cascade of death versus survival events that includes release of FGF2. The rPAI-123 anti-angiogenic activity inhibits FGF2 pro-angiogenic functions by blocking FGF2 signaling through FGF receptor 1 and syndecan-4 and downstream effectors p-Akt, p-JNK, and p-c-Jun.

Antiangiogenic Activity of rPAI-123 Promotes Vasa Vasorum Regression in Hypercholesterolemic Mice Through a Plasmin-Dependent Mechanism

Rationale: The antiangiogenic activity of rPAI-123, a truncated plasminogen activator inhibitor-1 (PAI-1) protein, induces vasa vasorum collapse and significantly reduces plaque area and plaque cholesterol in hypercholesterolemic low-density lipoprotein receptor–deficient/apolipoprotein B48–deficient mice. Objective: The objective of this study was to examine rPAI-123–stimulated mechanisms that cause vasa vasorum collapse. Methods and Results: The rPAI-123 protein opposed PAI-1 antiproteolytic function by stimulating a 1.6-fold increase in plasmin activity compared with the saline-treated counterpart. The increased proteolytic activity corresponded to increased activity of matrix metalloproteinase-3 and degradation of fibrin(ogen), nidogen, and perlecan in the adventitia of descending aortas. PAI-1 activity was reduced by 48% in response to rPAI-123; however, PAI-1 protein expression levels were similar in the rPAI-123– and saline-treated hypercholesterolemic mice. Coimmunoprecipitation assays demonstrated a novel PAI-1–plasminogen complex in protein from the descending aorta of rPAI-123– and saline-treated mice, but complexed PAI-1 was 1.6-fold greater in rPAI-123–treated mice. Biochemical analyses demonstrated that rPAI-123 and PAI-1 binding interactions with plasminogen increased plasmin activity and reduced PAI-1 antiproteolytic activity. Conclusions: We conclude that rPAI-123 causes regression or collapse of adventitial vasa vasorum in hypercholesterolemic mice by stimulating an increase in plasmin activity. The rPAI-123–enhanced plasmin activity was achieved through a novel mechanism by which rPAI-123 and PAI-1 bound plasminogen in a cooperative manner to increase plasmin activity and reduce PAI-1 activity.