Kevin D. O'Brien

Characterization of the early lesion of 'degenerative' valvular aortic stenosis. Histological and immunohistochemical studies.

BackgroundNonrheumatic stenosis of trileaflet aortic valves, often termed senile or calcific valvular aortic stenosis, is considered a “degenerative” process, but little is known about the cellular or molecular factors that mediate its development. Methods and ResultsTo characterize the developing aortic valvular lesion, we performed histological and immunohisto-chemical studies on Formalin-fixed and methanol-Carnoys-fixed parafflin-embedded aortic valve leaflets or on frozen sections obtained at autopsy from 27 adults (age, 46 to 82 years) with normal leaflets (n=6), mild macroscopic leaflet thickening (n= 15), or clinical aortic stenosis (n=6). Focal areas of thickening (“early lesions”) were characterized by (1) subendothelial thickening on the aortic side of the leaflet, between the basement membrane (PAS-positive) and elastic lamina (Verhoeff–van Gieson), (2) the presence of large amounts of intracellular and extracellular neutral lipids (oil red O) and fine, stippled mineralization (von Kossa), and (3) disruption of the basement membrane overlying the lesion. Regions of the fibrosa adjacent to these lesions were characterized by thickening and by protein, lipid, and calcium accumulation. Control valves showed none of these abnormalities. Immunohistochemical studies were performed using monoclonal antibodies directed against macrophages (anti-CD68 or HAM-56), and contractile proteins of smooth muscle cells or myofibroblasts (anti-α-actin and HHF-35) or rabbit polyclonal antiserum against T lymphocytes (anti-CD3). In normal valves, scattered macrophages were present in the fibrosa and ventricularis, and occasional muscle actin-positive cells were detected in the proximal portion of the ventricularis near the leaflet base, but no T lymphocytes were found. In contrast, early lesions were characterized by the presence of an inflammatory infiltrate composed of non-foam cell and foam cell macrophages, occasional T cells, and rare α-actin-positive cells. In stenotic aortic valves, a similar but more advanced lesion was seen. ConclusionsThe early lesion of “degenerative” aortic stenosis is an active inflammatory process with some similarities (lipid deposition, macrophage and T-cell infiltration, and basement membrane disruption) and some dissimilarities (presence of prominent mineralization and small numbers of smooth muscle cells) to atherosclerosis.

Vascular cell adhesion molecule-1 is expressed in human coronary atherosclerotic plaques. Implications for the mode of progression of advanced coronary atherosclerosis.

Endothelial attachment is the initial step in leukocyte recruitment into developing atherosclerotic lesions. To determine whether vascular cell adhesion molecule-1 (VCAM-1) expression may play a role in inflammatory cell recruitment into human atherosclerotic lesions, immunohistochemistry was performed with a polyclonal rabbit antisera, raised against recombinant human VCAM-1, on 24 atherosclerotic coronary plaques and 11 control coronary segments with nonatherosclerotic diffuse intimal thickening from 10 patients. Immunophenotyping was performed on adjacent sections to identify smooth muscle cells, macrophages, and endothelial cells. To confirm VCAM-1-expressing cell types, double immunostaining with VCAM-1 antisera and each of the cell-specific markers and in situ hybridization were performed. All atherosclerotic plaques contained some VCAM-1, compared to 45% of control segments. VCAM-1 was found infrequently on endothelial cells at the arterial lumen din both plaques (21%) and in control segments (27%), but was prevalent in areas of neovascularization and inflammatory infiltrate in the base of plaques. Double immunostaining and in situ hybridization confirmed that most VCAM-1 was expressed by subsets of plaque smooth muscle cells and macrophages. The results document the presence of VCAM-1 in human atherosclerosis, demonstrate VCAM-1 expression by human smooth muscle cells in vivo, and suggest that intimal neovasculature may be an important site of inflammatory cell recruitment into advanced coronary lesions.

Calcific Aortic Valve Disease: Not Simply a Degenerative Process A Review and Agenda for Research From the National Heart and Lung and Blood Institute Aortic Stenosis Working Group Executive Summary: Calcific Aortic Valve Disease - 2011 Update

Calcific aortic valve disease (CAVD) encompasses the range of disease from initial alterations in the cell biology of the leaflets to end-stage calcification resulting in left ventricular outflow obstruction. The first detectable macroscopic changes in the leaflets, seen as calcification, or focal leaflet thickening with normal valve function, is termed aortic valve sclerosis, but it is likely that the initiating events in the disease process occur much earlier. Disease progression is characterized by a process of thickening of the valve leaflets and the formation of calcium nodules – often including the formation of actual bone – and new blood vessels, which are concentrated near the aortic surface. End stage disease, e.g. calcific aortic stenosis, is characterized pathologically by large nodular calcific masses within the aortic cusps that protrude through the outflow surfaces into the sinuses of Valsalva, interfering with opening of the cusps. For decades, this disease was thought to be a passive process in which the valve degenerates with age in association with calcium accumulation. Moreover, although calcific aortic valve disease is more common with age, it is not an inevitable consequence of aging. Instead, CAVD appears to be an actively regulated disease process that cannot be characterized exclusively as “senile” or “degenerative.” The NHLBI convened a group of scientists from different fields of study, including cardiac imaging, molecular biology, cardiovascular pathology, epidemiology, cell biology, endocrinology, bioengineering, and clinical outcomes, to review the scientific studies from the past decade in the field of CAVD. The purpose was to develop a consensus statement on the current state of translational research related to CAVD. Herein, we summarize recent scientific studies and define future directions for research to diagnose, treat and potentially prevent this complex disease process.

Interstitial Collagenase (MMP-1) Expression in Human Carotid Atherosclerosis

BACKGROUND In human atherosclerosis, most clinical events occur when plaque integrity is compromised and hemorrhage and thrombosis result. One mechanism for this might be the release by plaque cells of matrix-degrading proteases, such as interstitial collagenase (matrix metalloproteinase-1, MMP-1), which degrades two major plaque structural proteins, types I and III collagen. This study was undertaken to determine whether MMP-1 is expressed in human atherosclerotic plaques. METHODS AND RESULTS To determine the cellular source and location of MMP-1 in human carotid atherosclerotic lesions, in situ hybridization and immunohistochemistry were performed on 20 endarterectomy specimens. Six nonatherosclerotic carotid arteries also were studied. Intense MMP-1 expression (mRNA and protein) was detected in a subset of plaque macrophages located at the borders of the lipid cores adjacent to fibrous caps and shoulder regions. Subsets of plaque smooth muscle cells and endothelial cells also expressed MMP-1. There was a strong correlation between the percentage of the lipid core occupied by hemorrhage and the percentage of the lipid core perimeter positive for MMP-1 (r = .823, P = .0001). MMP-1 was not detected in any cell type in nonatherosclerotic carotid arteries. CONCLUSIONS This study demonstrates that MMP-1 is expressed by several cell types in human carotid atherosclerosis and that there is a correlation between the expression of the protease and histopathological evidence of plaque instability. Since MMP-1 may degrade the major structural collagens of the plaque, expression of the protease by macrophages in regions critical to plaque integrity could contribute to plaque expansion, rupture, and hemorrhage.

Genetic Associations with Valvular Calcification and Aortic Stenosis

BACKGROUND Limited information is available regarding genetic contributions to valvular calcification, which is an important precursor of clinical valve disease. METHODS We determined genomewide associations with the presence of aortic-valve calcification (among 6942 participants) and mitral annular calcification (among 3795 participants), as detected by computed tomographic (CT) scanning; the study population for this analysis included persons of white European ancestry from three cohorts participating in the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium (discovery population). Findings were replicated in independent cohorts of persons with either CT-detected valvular calcification or clinical aortic stenosis. RESULTS One SNP in the lipoprotein(a) (LPA) locus (rs10455872) reached genomewide significance for the presence of aortic-valve calcification (odds ratio per allele, 2.05; P=9.0×10(-10)), a finding that was replicated in additional white European, African-American, and Hispanic-American cohorts (P<0.05 for all comparisons). Genetically determined Lp(a) levels, as predicted by LPA genotype, were also associated with aortic-valve calcification, supporting a causal role for Lp(a). In prospective analyses, LPA genotype was associated with incident aortic stenosis (hazard ratio per allele, 1.68; 95% confidence interval [CI], 1.32 to 2.15) and aortic-valve replacement (hazard ratio, 1.54; 95% CI, 1.05 to 2.27) in a large Swedish cohort; the association with incident aortic stenosis was also replicated in an independent Danish cohort. Two SNPs (rs17659543 and rs13415097) near the proinflammatory gene IL1F9 achieved genomewide significance for mitral annular calcification (P=1.5×10(-8) and P=1.8×10(-8), respectively), but the findings were not replicated consistently. CONCLUSIONS Genetic variation in the LPA locus, mediated by Lp(a) levels, is associated with aortic-valve calcification across multiple ethnic groups and with incident clinical aortic stenosis. (Funded by the National Heart, Lung, and Blood Institute and others.).

HMG CoA reductase inhibitor (statin) and aortic valve calcium

There is no known pharmacological therapy for calcific aortic valvular sclerosis or stenosis. Because leaflet calcification occurs in areas of lipoprotein deposition, we hypothesised that 3-hydroxy-3-methylglutaryl-coenzyme A (HMG COA) reductase inhibitors (statins) might slow aortic valve calcium (AVC) accumulation. We retrospectively identified 65 patients who had undergone two electron-beam computed tomography scans at a mean (SD) interval of 2.5 (1.6) years. 28 (43%) patients were receiving statins. Patients who were treated with statins had a 62-63% lower median rate of AVC accumulation (p=0.006) and 44-49% fewer statin patients had definite AVC progression (p=0.043). These findings suggest that statins may decrease AVC accumulation.

Association of angiotensin-converting enzyme with low-density lipoprotein in aortic valvular lesions and in human plasma.

Background—Recent studies have demonstrated that lesions of aortic sclerosis and stenosis share several similarities with lesions of atherosclerosis. In atherosclerosis, angiotensin-converting enzyme (ACE) is expressed by a subset of macrophages. This study was undertaken to determine whether ACE might be present in aortic sclerosis or stenosis lesions. Methods and Results—Immunohistochemistry was performed on 26 paraffin-embedded human aortic valves. Monospecific antibodies were used to identify ACE, macrophages, angiotensin II type 1 receptor (AT-1 receptor), angiotensin II, and apolipoprotein B. Human low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were isolated from plasma of normal volunteers by sequential density-gradient ultracentrifugation. ACE was not present in normal valves but was present in all valves with aortic sclerosis or stenosis lesions. ACE was detected on a subset of lesion macrophages but was present primarily in an extracellular distribution, where it colocalized with apolipoprotein B. ACE was detected by Western blotting on plasma LDL but not on HDL isolated from normal volunteers. Angiotensin II, the enzymatic product of ACE, was colocalized with ACE in valve lesions. ACE also was colocalized with apolipoprotein B in an adjacent coronary atherosclerotic plaque. Conclusions—ACE is present in aortic sclerosis and stenosis lesions, where it may participate in lesion development, as is evidenced by the presence of its enzymatic product, angiotensin II. The observation of an association of ACE with LDL in both lesions and plasma suggests that LDL may deliver ACE to lesions and has implications for the role of ACE-containing LDL in other diseases, such as atherosclerosis.

Weight Loss in Severely Obese Subjects Prevents the Progression of Impaired Glucose Tolerance to Type II Diabetes: A longitudinal interventional study

OBJECTIVE To determine if weight loss may prevent conversion of impaired glucose tolerance (IGT) to diabetes, because weight loss reduces insulin resistance. The prevalence of IGT in the U. S. population is estimated at 11.2%, more than twice that of diabetes. Furthermore, because an oral glucose tolerance test is needed for its detection, most of these patients are undiagnosed. Screening for IGT would be meaningful if progression to diabetes could be delayed or prevented. RESEARCH DESIGN AND METHODS For an average of 5.8 years (range 2–10 years), 136 individuals with IGT and clinically severe obesity (>45 kg excess body weight) were followed. The experimental group included 109 patients with IGT who underwent bariatric surgery for weight loss. The control group was made up of 27 subjects with IGT who did not have bariatric surgery. The criteria of the World Health Organization was used to detect IGT and diabetes in this population. The main outcome measure of this nonrandomized control trial is the incidence density, or number of events (development of diabetes) divided by the time of exposure to risk. RESULTS Of the 27 subjects in the control group, 6 developed diabetes during an average of 4.8 ± 2.5 years of postdiagnosis follow-up, yielding a rate of conversion to diabetes of 4.72 cases per 100 person-years. The 109 individuals of the experimental group were followed for an average of 6.2 ± 2.5 years postbariatric surgery. Based on the 95% confidence interval of the comparison group, we would expect to find that between 22 and 36 subjects in the experimental group developed diabetes over the follow-up period. Only 1 of the 109 experimental-group patients developed diabetes, resulting in a conversion rate of the experimental group of only 0.15 cases per 100 person-years, which is significantly lower (P < 0.0001) than the control group. CONCLUSIONS Weight loss in patients with clinically severe obesity prevents the progression of IGT to diabetes by >30-fold.

The neuroimmune guidance cue netrin-1 promotes atherosclerosis by inhibiting the emigration of macrophages from plaques

Atherosclerotic plaque formation is fueled by the persistence of lipid-laden macrophages in the artery wall. The mechanisms by which these cells become trapped, thereby establishing chronic inflammation, remain unknown. Here we found that netrin-1, a neuroimmune guidance cue, was secreted by macrophages in human and mouse atheroma, where it inactivated the migration of macrophages toward chemokines linked to their egress from plaques. Acting via its receptor, UNC5b, netrin-1 inhibited the migration of macrophages directed by the chemokines CCL2 and CCL19, activation of the actin-remodeling GTPase Rac1 and actin polymerization. Targeted deletion of netrin-1 in macrophages resulted in much less atherosclerosis in mice deficient in the receptor for low-density lipoprotein and promoted the emigration of macrophages from plaques. Thus, netrin-1 promoted atherosclerosis by retaining macrophages in the artery wall. Our results establish a causative role for negative regulators of leukocyte migration in chronic inflammation.

Differential effect of saturated and unsaturated free fatty acids on the generation of monocyte adhesion and chemotactic factors by adipocytes: dissociation of adipocyte hypertrophy from inflammation

OBJECTIVE Obesity is associated with monocyte-macrophage accumulation in adipose tissue. Previously, we showed that glucose-stimulated production by adipocytes of serum amyloid A (SAA), monocyte chemoattractant protein (MCP)-1, and hyaluronan (HA) facilitated monocyte accumulation. The current objective was to determine how the other major nutrient, free fatty acids (FFAs), affects these molecules and monocyte recruitment by adipocytes. RESEARCH DESIGN AND METHODS Differentiated 3T3-L1, Simpson-Golabi-Behmel syndrome adipocytes, and mouse embryonic fibroblasts were exposed to various FFAs (250 μmol/l) in either 5 or 25 mmol/l (high) glucose for evaluation of SAA, MCP-1, and HA regulation in vitro. RESULTS Saturated fatty acids (SFAs) such as laurate, myristate, and palmitate increased cellular triglyceride accumulation, SAA, and MCP-1 expression; generated reactive oxygen species (ROS); and increased nuclear factor (NF) κB translocation in both 5 and 25 mmol/l glucose. Conversely, polyunsaturated fatty acids (PUFAs) such as arachidonate, eicosapentaenate, and docosahexaenate (DHA) decreased these events. Gene expression could be dissociated from triglyceride accumulation. Although excess glucose increased HA content, SFAs, oleate, and linoleate did not. Antioxidant treatment repressed glucose- and palmitate-stimulated ROS generation and NFκB translocation and decreased SAA and MCP-1 expression and monocyte chemotaxis. Silencing toll-like receptor-4 (TLR4) markedly reduced SAA and MCP-1 expression in response to palmitate but not glucose. DHA suppressed NFκB translocation stimulated by both excess glucose and palmitate via a peroxisome prolifterator–activated receptor (PPAR) γ–dependent pathway. CONCLUSIONS Excess glucose and SFAs regulate chemotactic factor expression by a mechanism that involves ROS generation, NFκB, and PPARγ, and which is repressed by PUFAs. Certain SFAs, but not excess glucose, trigger chemotactic factor expression via a TLR4-dependent pathway.