Hypoxia-inducible factor-1alpha (HIF1alpha) is a central regulator of the cellular response to hypoxia. Prolyl-hydroxylation of HIF1alpha by PHD enzymes is prerequisite for HIF1alpha degradation. Here, we demonstrate that the abundance of PHD1 and PHD3 are regulated via their targeting for proteasome-dependent degradation by the E3 ubiquitin ligases Siah1a/2, under hypoxia conditions. Siah2 null fibroblasts exhibit prolonged PHD3 half-life, resulting in lower levels of HIF1alpha expression during hypoxia. Significantly, hypoxia-induced HIF1alpha expression was completely inhibited in Siah1a/2 null cells, yet could be rescued upon inhibition of PHD3 by RNAi. Siah2 targeting of PHD3 for degradation increases upon exposure to even mild hypoxic conditions, which coincides with increased Siah2 transcription. Siah2 null mice subjected to hypoxia displayed an impaired hyperpneic respiratory response and reduced levels of hemoglobin. Thus, the control of PHD1/3 by Siah1a/2 constitutes another level of complexity in the regulation of HIF1alpha during hypoxia.

Single injections of proprotein convertase subtilisin/kexin type 9-encoding recombinant adeno-associated viral vectors are a rapid and versatile method to induce atherosclerosis in animals. This method should prove useful for experiments that are high-throughput or involve genetic techniques, strains, or species that do not combine well with current genetically engineered models.

Background-Intravascular ultrasound-derived virtual histology (VH IVUS) is used increasingly in clinical research to assess composition and vulnerability of coronary atherosclerotic lesions. However, the ability of VH IVUS to quantify individual plaque components, in particular the size of the destabilizing necrotic core, has never been validated. We tested for correlation between VH IVUS necrotic core size and necrotic core size by histology in porcine coronary arteries with human-like coronary disease. Methods and Results-In adult atherosclerosis-prone minipigs, 18 advanced coronary lesions were assessed by VH IVUS in vivo followed by postmortem microscopic examination (histology). We found no correlation between the size of the necrotic core determined by VH IVUS and histology. VH IVUS displayed necrotic cores in lesions lacking cores by histology. Conclusions-We found no correlation between necrotic core size determined by VH IVUS and real histology, questioning the ability of VH IVUS to detect rupture-prone plaques, so-called thin-cap fibroatheromas. (Circ Cardiovasc Imaging. 2010;3:384-391.)

Abstract. Thim T, Hagensen MK, Bentzon JF, Falk E (Aarhus University Hospital, Skejby; and University of Aarhus, Aarhus; Denmark). From vulnerable plaque to atherothrombosis (Review). J Intern Med 2008; 263: 506-516.Plaque rupture precipitates approximately 75% of all fatal coronary thrombi. Therefore, the plaque prone to rupture is the primary focus of this review. The lipidrich core and fibrous cap are pivotal in the understanding of plaque rupture. Plaque rupture is a localized process within the plaque caused by degradation of a tiny fibrous cap rather than by diffuse inflammation of the plaque. Atherosclerosis is a multifocal disease, but plaques prone to rupture seem to be oligofocal at most.

Background— It has been reported that circulating endothelial progenitor cells (EPCs) home to and differentiate into endothelial cells after various kinds of arterial injury. By inference, EPCs are also proposed to be important in the most important arterial disease, atherosclerosis, but the evidence for this theory is not clear. In the present study, we assessed the contribution of circulating EPCs to plaque endothelium in apolipoprotein E–deficient (apoE −/− ) mice. Methods and Results— To investigate whether EPCs in the circulating blood are a source of plaque endothelial cells during atherogenesis, we examined plaques in lethally irradiated apoE −/− mice reconstituted with bone marrow cells from enhanced green fluorescent protein (eGFP) transgenic apoE −/− mice and plaques induced in segments of common carotid artery transplanted from apoE −/− mice into eGFP + apoE −/− mice. Among 4232 endothelial cells identified by a cell-type–specific marker (von Willebrand factor) and analyzed by high-resolution microscopy, we found only 1 eGFP + . Using the Y chromosome to track cells after sex-mismatched transplants yielded similar results. To investigate whether circulating EPCs are involved in plaque reendothelialization after plaque disruption and superimposed thrombosis, we produced mechanical plaque disruptions in carotid bifurcation plaques in old lethally irradiated apoE −/− mice reconstituted with eGFP + apoE −/− bone marrow cells and carotid bifurcation plaques transplanted from old apoE −/− mice into eGFP + apoE −/− mice. Only 1 eGFP + endothelial cell was found among 3170 analyzed. Conclusions— Circulating EPCs rarely, if ever, contribute to plaque endothelium in apoE −/− mice. These findings bring into question the prevailing theory that circulating EPCs play an important role in atherogenesis.

Endothelial regeneration after vascular injury did not involve circulating EPCs but was mediated solely by migration of ECs from the adjacent healthy endothelium.

Objective— Atherosclerosis develops initially at branch points and in areas of high vessel curvature. Moreover, experiments in hypercholesterolemic mice have shown that the introduction of disturbed flow in straight, atherosclerosis-resistant arterial segments turns them highly atherosclerosis susceptible. Several biomechanical mechanisms have been proposed, but none has been demonstrated. In the present study, we examined whether a causal link exists between disturbed laminar flow and the ability of the arterial wall to retain lipoproteins. Approach and Results— Lipoprotein retention was detected at natural predilection sites of the murine thoracic aorta 18 hours after infusion of fluorescently labeled low-density lipoprotein. To test for causality between blood flow and the ability of these areas to retain lipoproteins, we manipulated blood flow in the straight segment of the common carotid artery using a constrictive collar. Disturbed laminar flow did not affect low-density lipoprotein influx, but increased the ability of the artery wall to bind low-density lipoprotein. Concordantly, disturbed laminar flow led to differential expression of genes associated with phenotypic modulation of vascular smooth muscle cells, increased expression of proteoglycan core proteins associated with lipoprotein retention, and of enzymes responsible for chondroitin sulfate glycosaminoglycan synthesis and sulfation. Conclusions— Blood flow regulates genes associated with vascular smooth muscle cell phenotypic modulation, as well as the expression and post-translational modification of lipoprotein-binding proteoglycan core proteins, and the introduction of disturbed laminar flow vastly augments the ability of a previously resistant, straight arterial segment to retain lipoproteins.

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