Jacob F. Bentzon

Mechanisms of Plaque Formation and Rupture

Atherosclerosis causes clinical disease through luminal narrowing or by precipitating thrombi that obstruct blood flow to the heart (coronary heart disease), brain (ischemic stroke), or lower extremities (peripheral vascular disease). The most common of these manifestations is coronary heart disease, including stable angina pectoris and the acute coronary syndromes. Atherosclerosis is a lipoprotein-driven disease that leads to plaque formation at specific sites of the arterial tree through intimal inflammation, necrosis, fibrosis, and calcification. After decades of indolent progression, such plaques may suddenly cause life-threatening coronary thrombosis presenting as an acute coronary syndrome. Most often, the culprit morphology is plaque rupture with exposure of highly thrombogenic, red cell-rich necrotic core material. The permissive structural requirement for this to occur is an extremely thin fibrous cap, and thus, ruptures occur mainly among lesions defined as thin-cap fibroatheromas. Also common are thrombi forming on lesions without rupture (plaque erosion), most often on pathological intimal thickening or fibroatheromas. However, the mechanisms involved in plaque erosion remain largely unknown, although coronary spasm is suspected. The calcified nodule has been suggested as a rare cause of coronary thrombosis in highly calcified and tortious arteries in older individuals. To characterize the severity and prognosis of plaques, several terms are used. Plaque burden denotes the extent of disease, whereas plaque activity is an ambiguous term, which may refer to one of several processes that characterize progression. Plaque vulnerability describes the short-term risk of precipitating symptomatic thrombosis. In this review, we discuss mechanisms of atherosclerotic plaque initiation and progression; how plaques suddenly precipitate life-threatening thrombi; and the concepts of plaque burden, activity, and vulnerability.

Update on acute coronary syndromes: the pathologists' view

Although mortality rates from coronary heart disease in the western countries have declined in the last few decades, morbidity caused by this disease is increasing and a substantial number of patients still suffer acute coronary syndrome (ACS) and sudden cardiac death. Acute coronary syndrome occurs as a result of myocardial ischaemia and its manifestations include acute myocardial infarction and unstable angina. Culprit plaque morphology in these patients varies from thrombosis with or without coronary occlusion to sudden narrowing of the lumen from intraplaque haemorrhage. The coronary artery plaque morphologies primarily responsible for thrombosis are plaque rupture, and plaque erosion, with plaque rupture being the most common cause of acute myocardial infarction, especially in men. Autopsy data demonstrate that women <50 years of age more frequently have erosion, whereas in older women, the frequency of rupture increases with each decade. Ruptured plaques are associated with positive (expansive) remodelling and characterized by a large necrotic core and a thin fibrous cap that is disrupted and infiltrated by foamy macrophages. Plaque erosion lesions are often negatively remodelled with the plaque itself being rich in smooth muscle cells and proteoglycans with minimal to absence of inflammation. Plaque haemorrhage may expand the plaque rapidly, leading to the development of unstable angina. Plaque haemorrhage may occur from plaque rupture (fissure) or from neovascularization (angiogenesis). Atherosclerosis is now recognized as an inflammatory disease with macrophages and T-lymphocytes playing a dominant role. Recently at least two subtypes of macrophages have been identified. M1 is a pro-inflammatory macrophage while M2 seems to play a role in dampening inflammation and promoting tissue repair. A third type of macrophage, termed by us as haemoglobin associated macrophage or M(Hb) which is observed at site of haemorrhage also can be demonstrated in human atherosclerosis. In order to further our understanding of the specific biological events which trigger plaque instability and as well as to monitor the effects of novel anti-atherosclerotic therapies newer imaging modalities in vivo are needed.

Smooth Muscle Cells in Atherosclerosis Originate From the Local Vessel Wall and Not Circulating Progenitor Cells in ApoE Knockout Mice

Objective—Recent studies of bone marrow (BM)-transplanted apoE knockout (apoE−/−) mice have concluded that a substantial fraction of smooth muscle cells (SMCs) in atherosclerosis arise from circulating progenitor cells of hematopoietic origin. This pathway, however, remains controversial. In the present study, we reexamined the origin of plaque SMCs in apoE−/− mice by a series of BM transplantations and in a novel model of atherosclerosis induced in surgically transferred arterial segments. Methods and Results—We analyzed plaques in lethally irradiated apoE−/− mice reconstituted with sex-mismatched BM cells from eGFP+apoE−/− mice, which ubiquitously express enhanced green fluorescent protein (eGFP), but did not find a single SMC of donor BM origin among ≈10 000 SMC profiles analyzed. We then transplanted arterial segments between eGFP+apoE−/− and apoE−/− mice (isotransplantation except for the eGFP transgene) and induced atherosclerosis focally within the graft by a recently invented collar technique. No eGFP+ SMCs were found in plaques that developed in apoE−/− artery segments grafted into eGFP+apoE−/− mice. Concordantly, 96% of SMCs were eGFP+ in plaques induced in eGFP+apoE−/− artery segments grafted into apoE−/− mice. Conclusions—These experiments show that SMCs in atherosclerotic plaques are exclusively derived from the local vessel wall in apoE−/− mice.

Chronic Renal Failure Accelerates Atherogenesis in Apolipoprotein E–Deficient Mice

Cardiovascular mortality is 10 to 20 times increased in patients with chronic renal failure (CRF). Risk factors for atherosclerosis are abundant in patients with CRF. However, the pathogenesis of cardiovascular disease in CRF remains to be elucidated. The effect of CRF on the development of atherosclerosis in apolipoprotein E-deficient male mice was examined. Seven-week-old mice underwent 5/6 nephrectomy (CRF, n = 28), unilateral nephrectomy (UNX, n = 24), or no surgery (n = 23). Twenty-two weeks later, CRF mice showed increased aortic plaque area fraction (0.266 +/- 0.033 versus 0.045 +/- 0.006; P < 0.001), aortic cholesterol content (535 +/- 62 versus 100 +/- 9 nmol/cm(2) intimal surface area; P < 0.001), and aortic root plaque area (205,296 +/- 22,098 versus 143,662 +/- 13,302 micro m(2); P < 0.05) as compared with no-surgery mice; UNX mice showed intermediate values. The plaques from uremic mice contained CD11b-positive macrophages and showed strong staining for nitrotyrosine. Systolic BP and plasma homocysteine concentrations were similar in uremic and nonuremic mice. Plasma urea and cholesterol concentrations were elevated 2.6-fold (P < 0.001) and 1.5-fold (P < 0.001) in CRF compared with no-surgery mice. Both variables correlated with aortic plaque area fraction (r(2) = 0.5, P < 0.001 and r(2) = 0.3, P < 0.001, respectively) and with each other (r(2) = 0.5, P < 0.001). On multiple linear regression analysis, only plasma urea was a significant predictor of aortic plaque area fraction. In conclusion, the present findings suggest that uremia markedly accelerates atherogenesis in apolipoprotein E-deficient mice. This effect could not be fully explained by changes in BP, plasma homocysteine levels, or total plasma cholesterol concentrations. Thus, the CRF apolipoprotein E-deficient mouse is a new model for studying the pathogenesis of accelerated atherosclerosis in uremia.

From vulnerable plaque to atherothrombosis

Plaque rupture precipitates approximately 75% of all fatal coronary thrombi. Therefore, the plaque prone to rupture is the primary focus of this review. The lipid‐rich 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.

Familial Hypercholesterolemia and Atherosclerosis in Cloned Minipigs Created by DNA Transposition of a Human PCSK9 Gain-of-Function Mutant

A transgenic pig model of familial hypercholesterolemia can be used for translational atherosclerosis research. A Model of We hope to inherit our parents’ good features, like blue eyes or musical talent, but not their high cholesterol. Familial hypercholesterolemia, which is passed down in families, results in high levels of “bad” cholesterol [low-density lipoprotein (LDL)] and early onset of cardiovascular disease. To further translational research in this area, Al-Mashhadi and coauthors created a large-animal model of this genetic disease, showing that these pigs develop hypercholesterolemia and atherosclerosis much like people do. The D374Y gain-of-function mutation in the PCSK9 gene (which is conserved between pig and human) causes a severe form of hypercholesterolemia and, ultimately, atherosclerosis. Al-Mashhadi and colleagues engineered transposon-based vectors to express D374Y-PCSK9. After confirming function in human liver cancer cells, the authors cloned minipigs that expressed the mutant gene. On a low-fat diet, these pigs had higher total and LDL cholesterol than their wild-type counterparts. Breeding the male transgenic pigs with wild-type sows produced offspring that also had higher plasma LDL levels compared with normal, healthy pigs. A high-fat, high-cholesterol diet induced severe hypercholesterolemia in these animals as well as accelerated development of atherosclerosis that has human-like lesions. Other large-animal models only develop hypercholesterolemia when placed on the right diet, and small-animal models cannot recapitulate human-like pathology. The PCSK9 transgenic pigs created by Al-Mashhadi et al. develop hypercholesterolemia even on low-fat diets, and thus reflect the inherited human disease. This large-animal model will be important for better understanding the pathogenesis of familial hypercholesterolemia and for testing new therapeutics and imaging modalities before moving into human trials. Lack of animal models with human-like size and pathology hampers translational research in atherosclerosis. Mouse models are missing central features of human atherosclerosis and are too small for intravascular procedures and imaging. Modeling the disease in minipigs may overcome these limitations, but it has proven difficult to induce rapid atherosclerosis in normal pigs by high-fat feeding alone, and genetically modified models similar to those created in mice are not available. D374Y gain-of-function mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene cause severe autosomal dominant hypercholesterolemia and accelerates atherosclerosis in humans. Using Sleeping Beauty DNA transposition and cloning by somatic cell nuclear transfer, we created Yucatan minipigs with liver-specific expression of human D374Y-PCSK9. D374Y-PCSK9 transgenic pigs displayed reduced hepatic low-density lipoprotein (LDL) receptor levels, impaired LDL clearance, severe hypercholesterolemia, and spontaneous development of progressive atherosclerotic lesions that could be visualized by noninvasive imaging. This model should prove useful for several types of translational research in atherosclerosis.

Stabilisation of atherosclerotic plaques position paper of the european society of cardiology (ESC) working group on atherosclerosis and vascular biology

Plaque rupture and subsequent thrombotic occlusion of the coronary artery account for as many as three quarters of myocardial infarctions. The concept of plaque stabilisation emerged about 20 years ago to explain the discrepancy between the reduction of cardiovascular events in patients receiving lipid lowering therapy and the small decrease seen in angiographic evaluation of atherosclerosis. Since then, the concept of a vulnerable plaque has received a lot of attention in basic and clinical research leading to a better understanding of the pathophysiology of the vulnerable plaque and acute coronary syndromes. From pathological and clinical observations, plaques that have recently ruptured have thin fibrous caps, large lipid cores, exhibit outward remodelling and invasion by vasa vasorum. Ruptured plaques are also focally inflamed and this may be a common denominator of the other pathological features. Plaques with similar characteristics, but which have not yet ruptured, are believed to be vulnerable to rupture. Experimental studies strongly support the validity of anti-inflammatory approaches to promote plaque stability. Unfortunately, reliable non-invasive methods for imaging and detection of such plaques are not yet readily available. There is a strong biological basis and supportive clinical evidence that low-density lipoprotein lowering with statins is useful for the stabilisation of vulnerable plaques. There is also some clinical evidence for the usefulness of antiplatelet agents, beta blockers and renin-angiotensin-aldosterone system inhibitors for plaque stabilisation. Determining the causes of plaque rupture and designing diagnostics and interventions to prevent them are urgent priorities for current basic and clinical research in cardiovascular area.

Smooth Muscle Cells Healing Atherosclerotic Plaque Disruptions Are of Local, Not Blood, Origin in Apolipoprotein E Knockout Mice

Background— Signs of preceding episodes of plaque rupture and smooth muscle cell (SMC)–mediated healing are common in atherosclerotic plaques, but the source of the healing SMCs is unknown. Recent studies suggest that activated platelets adhering to sites of injury recruit neointimal SMCs from circulating bone marrow–derived progenitor cells. Here, we analyzed the contribution of this mechanism to plaque healing after spontaneous and mechanical plaque disruption in apolipoprotein E knockout (apoE−/−) mice. Methods and Results— To determine the origin of SMCs after spontaneous plaque disruption, irradiated 18-month-old apoE−/− mice were reconstituted with bone marrow cells from enhanced green fluorescent protein (eGFP) transgenic apoE−/− mice and examined when they died up to 9 months later. Plaque hemorrhage, indicating previous plaque disruption, was widely present, but no bone marrow–derived eGFP+ SMCs were detected. To examine the origin of healing SMCs in a model that recapitulates more features of human plaque rupture and healing, we developed a mechanical technique that produced consistent plaque disruption, superimposed thrombosis, and SMC-mediated plaque healing in apoE−/− mice. Mechanical plaque disruption was produced in irradiated apoE−/− mice reconstituted with eGFP+apoE−/− bone marrow cells and in carotid bifurcations cross-grafted between apoE−/− and eGFP+apoE−/− mice. Apart from few non–graft-derived SMCs near the anastomosis site in 1 transplanted carotid bifurcation, no SMCs originating from outside the local arterial segment were detected in healed plaques. Conclusions— Healing SMCs after atherosclerotic plaque disruption are derived entirely from the local arterial wall and not circulating progenitor cells in apoE−/− mice.

Demonstration of the presence of independent pre-osteoblastic and pre-adipocytic cell populations in bone marrow-derived mesenchymal stem cells.

Mesenchymal stem cells (MSC) are defined as plastic-adherent, clonal cells that are common progenitors for osteoblasts and adipocytes. An inverse relationship between bone and fat has been observed in several clinical conditions and has been suggested to be caused by re-directing MSC differentiation into one particular lineage. However, this inverse relationship between bone and fat is not consistent and under certain in vivo conditions, bone and fat can change independently suggesting separate precursor cell populations. In order to test for this hypothesis, we extensively characterized two plastic-adherent clonal MSC lines (mMSC1 and mMSC2) derived from murine bone marrow. The two cell lines grew readily in culture and have undergone more than 100 population doublings with no apparent differences in their growth rates. Both cell lines were positive for the murine MSC marker Sca-1 and mMSC1 was also positive for CD13. Both cell lines were exposed to in vitro culture induction of osteogenesis and adipogenesis. mMSC1 and not mMSC2 were only able to differentiate to adipocytes evidenced by the expression of adipocyte markers (aP2, adiponectin, adipsin, PPARgamma2 and C/EBPa) and the presence of mature adipocytes visualized by Oil Red O staining. On the other hand, mMSC2 and not mMSC1 differentiated to osteoblast lineage as demonstrated by up-regulation of osteoblastic makers (CBFA1/RUNX2, Osterix, alkaline phosphatase, bone sialoprotein and osteopontin) and formation of alizarin red stained mineralized matrix in vitro. Consistent with the in vitro results, mMSC2 and not mMSC1, were able to form bone in vivo after subcutaneous implantation in immune-deficient (NOD/SCID) mice. Our data suggest that contrary to the current belief, bone marrow contains clonal subpopulations of cells that are committed to either osteoblast or adipocyte lineage. These cell populations may undergo independent changes during aging and in bone diseases and thus represent important targets for therapy.

Atherosclerotic lesions in mouse and man: is it the same disease?

Purpose of review Genetically-engineered mice with hyperlipidemia are the most widely used atherosclerosis models today, but recent advances in transgenesis open the possibility to create new models in alternative species, such as the rat and pig. It seems relevant at this point in time to review some of the strengths and weaknesses of the mouse. Recent findings The histology of lesion development in mouse and man has more similarities than differences, and comparative genetics show that many mechanisms of murine and human atherogenesis are shared. Unfortunately, the most feared complication of human atherosclerosis, that is, plaque rupture and thrombosis, occur extremely rarely in mice. This is a major problem. Most patients today are not treated before symptoms ensue, and at this late stage of the disease, mechanisms identified during plaque development in the mouse may not be very important. Summary Murine atherosclerosis models are highly valuable for identifying atherogenic mechanisms that can be targeted by preventive medicine. However, models with thrombotic complications and large animal models suitable for interventional procedures and imaging would be more supportive for current clinical practice and are highly wanted.