Carriers of COL3A1 pathogenic variants in Denmark: Interfamilial variability in severity and outcome of elective surgical procedures.

The study describes all patients in Denmark with vascular Ehlers-Danlos syndrome (vEDS). Carriers of pathogenic or likely pathogenic COL3A1 variants were retrospectively identified through registries and specialized clinics. Medical records were reviewed for vascular- or organ ruptures and invasive procedures performed. Identified families were divided by variant type (null, splice, and missense) and familial phenotypes (severe or attenuated). Families in which at least one carrier has suffered a major event before the age of 30 were classified as severe, whereas families in which at least three carriers had reached the age of 40 without a major event were classified as attenuated. Eighty-seven persons (59 still alive) from 25 families were included with a mean observation time of 44 years. Sixty-seven percent of patients could be subclassified in a familial phenotype. Thirty-one major events were observed. Eleven complications in 172 invasive procedures were recorded. No fatal complications to elective surgery were observed. The type of COL3A1 variant did not reliably predict phenotype, but a pattern of intrafamilial consistency emerged with some families showing an attenuated form of vEDS. Elective medical procedures appear to be safer than previously thought, although data only allow for conclusions regarding individuals from families with the attenuated form of vEDS.

The role of N-terminal pro-brain natriuretic peptide, chromogranin A, and 5-hydroxyindoleacetic acid in screening for carcinoid heart disease.

Carcinoid heart disease (CHD) is a serious complication for patients with neuroendocrine tumors (NETs), and early detection is crucial. We aimed to investigate N-terminal pro-brain natriuretic peptide (NT-proBNP), chromogranin A (CgA), and plasma 5-hydroxyindoleacetic acid (P-5-HIAA) as a screening tool for detection of CHD. We prospectively included patients with disseminated small intestinal NETs (SI-NETs) and performed transthoracic echocardiography (TTE), questionnaires, and biochemical assessment of NT-proBNP, CgA, and P-5-HIAA. The presence and severity of CHD was assessed using a scoring system based on echocardiographic characteristics. A total of 93 patients were included in the final analysis. Fifteen (16%) were diagnosed with CHD. The median NT-proBNP (219 ng/L vs. 124 ng/L, p = .05), CgA (3930 pmol/L vs. 256 pmoL/L, p < .0001), and P-5-HIAA (1160 nmol/L vs. 210 nmoL/L, p < .0001) were significantly higher in patients with CHD compared to non-CHD patients. For NT-proBNP, the area under the receiver operating characteristic (AUROC) curve for detection of CHD was 0.67 (95% CI: 0.50-0.84), and at a 260 ng/L cutoff level, the sensitivity and specificity were 46% and 79%. For CgA, the AUROC was 0.91 (95% CI: 0.84-0.97), and at a cutoff level of 598 pmol/L, the sensitivity and specificity were 100% and 69%. For P-5-HIAA, the AUROC was 0.89 (95% CI: 0.80-0.98), and at a cutoff level of 752 nmol/L, the sensitivity and specificity were 92% and 85%. In conclusion, CgA and P-5-HIAA proved excellent markers of CHD while NT-proBNP lacked the required diagnostic accuracy to be used as a screening tool.

Characteristics and Outcomes in Patients With Acute Aortic Dissection: A Nationwide Registry Study.

BACKGROUND: We aimed to describe characteristics and outcomes in a nationwide population of patients with acute type A and type B aortic dissection. METHODS: All patients in Denmark with a first-time diagnosis of acute aortic dissection between 2006 and 2015 were identified by national registries. The main outcomes were in-hospital mortality and long-term survival in hospital survivors. RESULTS: The study population comprised 1157 (68%) patients with type A aortic dissection and 556 (32%) patients with type B aortic dissection, median age of 66 (57-74) years and 70 (61-79) years, respectively. Men accounted for 64%. Median follow-up was 8.9 (6.8-11.5) years. Of patients with type A aortic dissection, 74% were managed surgically, whereas 22% of the patients with type B aortic dissection were managed with surgery or endovascular technique. In-hospital mortality was 27% for type A aortic dissection overall (surgery, 18%; no surgery, 52%) and 16% for type B aortic dissection (surgery or endovascular treatment, 13%; conservative treatment, 17%; P < .001, type A vs type B). Of patients discharged alive, survival was persistently better for type A aortic dissection than for type B aortic dissection (P < .001). Unadjusted 1- and 3-year survival of patients with type A aortic dissection discharged alive was 96% and 91%, respectively, for surgically managed and 88% and 78% without surgery. For type B aortic dissection, the numbers were 89% and 83% for endovascular/surgically managed and 89% and 77% for conservatively managed. CONCLUSIONS: We found higher in-hospital mortality for type A and type B aortic dissection than is reported from referral center registries. Type A aortic dissection had the highest mortality rate during the acute phase, whereas for patients who were discharged alive, the mortality rate was higher for patients with type B aortic dissection.

Complement regulation in human atherosclerotic coronary lesions. Immunohistochemical evidence that C4b-binding protein negatively regulates the classical complement pathway, and that C5b-9 is formed via the alternative complement pathway.

OBJECTIVE: The complement system is activated in human atherosclerotic lesions and may hence aggravate local inflammation. We studied the presence and localization of C4b-binding protein (C4bp), the major inhibitor of the classical complement pathway, in human atherosclerotic lesions in relation to complement activation products and protein S, which circulates in complex with C4bp. METHODS AND RESULTS: Immunohistochemistry of human coronary arteries showed C4bp to be virtually absent in normal arteries but present in early and advanced atherosclerotic lesions. In the lesions, C4bp is associated with proteoglycans, and affinity chromatography showed that C4bp interacts with human arterial proteoglycans. Areas containing C4bp also contained IgM and C4 suggesting that C4bp is involved in the regulation of the classical complement pathway. However, C5b-9 was virtually absent in these areas but, instead, colocalized with properdin deeper in the intima, suggesting that C5b-9 is formed by the alternative complement pathway. A fraction of C4bp was associated with protein S and apoptotic cells. CONCLUSIONS: The results indicate that C4bp regulates the classical complement pathway in human atherosclerotic lesions. Thus, unlike the alternative pathway, the classical complement pathway does not generate C5b-9, but is likely to be involved in the clean-up of apoptotic cells and cell debris in the arterial intima.

Function and regulation of the complement system in cardiovascular diseases.

The complement system plays a central role in innate immunity and also regulates adaptive immunity. The complement system has been demonstrated to contribute to various diseases, including cardiovascular diseases. Complement is extensively activated in atherosclerotic lesions, in arterial aneurysms, and in the myocardium of ischemic and failing hearts. Accumulating evidence shows that limitation of excessive complement activation under these conditions may hold therapeutic value. On the other hand, defects in the classical complement pathway predispose to vasculitis and atherosclerosis, possibly due to ineffective clearance of apoptotic/necrotic cells and abnormal processing of immune complexes. Here, we describe complement activation and regulation in cardiovascular diseases and discuss the evidence derived mainly from experimental animals suggesting that modulation of complement activation may alter the course of these disorders.

OxLDL-IgG immune complexes induce survival of human monocytes.

OBJECTIVE: Immune complexes containing oxidatively modified low-density lipoprotein (oxLDL) particles are deposited in human atherosclerotic lesions during atherogenesis. Here we studied whether OxLDL-IgG immune complexes (OxLDL-IgG ICs) affect survival of human monocytes. METHODS AND RESULTS: As demonstrated by light microscopy, and analysis of cell proliferation, caspase-3 activity, and DNA fragmentation, OxLDL-IgG ICs promoted survival of cultured human monocytes by decreasing their spontaneous apoptosis. OxLDL-IgG ICs induced a concentration-dependent production of the major monocyte growth factor, monocyte colony-stimulating factor (M-CSF), by the monocytes, but its inhibition was without effect on OxLDL-IgG IC-induced monocyte survival. Rather, OxLDL-IgG ICs induced rapid phosphorylation of Akt, suggesting a direct anti-apoptotic effect mediated by cross-linking of Fcgamma receptors. Experiments with receptor blocking antibodies revealed that the OxLDL-IgG IC-induced monocyte survival was mediated by Fcgamma receptor I. CONCLUSIONS: The results show that OxLDL-IgG ICs promote survival of monocytes by cross-linking Fcgamma receptor I and activating Akt-dependent survival signaling. The results reveal a novel mechanism by which an immune reaction toward oxLDL can play a role in the accumulation of macrophages in human atherosclerotic lesions.

Lipoprotein lipase in the arterial wall: linking LDL to the arterial extracellular matrix and much more.

For low density lipoprotein (LDL) particles to be atherogenic, increasing evidence indicates that their residence time in the arterial intima must be sufficient to allow their modification into forms capable of triggering extracellular and intracellular lipid accumulation. Recent reports have confirmed the longstanding hypothesis that the major determinant(s) of initial LDL retention in the preatherosclerotic arterial intima is the proteoglycans. However, once the initial atherosclerotic lesions have formed, a shift to retention facilitated by macrophage-derived lipoprotein lipase (LPL) appears, leading to the progression of the lesions. Here, we review recent findings on the mechanisms enabling LPL to promote LDL retention and extracellular lipid accumulation in the arterial intima, and we describe the structures in the extracellular matrix that are held to be important in this process. Finally, the potentially harmful consequences of LDL linking by LPL and of other LPL actions in the arterial intima are briefly reviewed.

Association between complement factor H and proteoglycans in early human coronary atherosclerotic lesions: implications for local regulation of complement activation.

OBJECTIVE: Complement activation has been suggested to play a role in atherogenesis. To study the regulation of complement activation in human coronary atherosclerotic lesions, we examined the spatial relationships between the major complement inhibitor, factor H, and the complement activation products C3d and C5b-9. METHODS AND RESULTS: In early lesions (American Heart Association types II and III), factor H was immunohistochemically found in the superficial proteoglycan-rich layer in association with numerous macrophages and C3d, whereas C5b-9 was found deeper in the intima, where factor H was virtually absent. In vitro experiments involving surface plasmon resonance and affinity chromatography analyses demonstrated that isolated human arterial proteoglycans bind factor H, and functional complement assays showed that glycosaminoglycans inhibit the complement activation induced by modified low density lipoprotein or by a foreign surface. CONCLUSIONS: The present observations raise the possibility that proteoglycans, because of their ability to bind the major complement inhibitor factor H, may inhibit complement activation in the superficial layer of the arterial intima. In contrast, deeper in the intima, where factor H and proteoglycans are absent, complement may be activated and proceed to C5b-9. Thus, the superficial and the deep layers of the human coronary artery appear to differ in their ability to regulate complement activation.

Lysosomal enzymes are released from cultured human macrophages, hydrolyze LDL in vitro, and are present extracellularly in human atherosclerotic lesions.

OBJECTIVE: Human atherosclerotic lesions have been shown to contain lipid droplets and vesicles resembling those of in vitro enzymatically modified LDL. However, little is known about the hydrolytic enzymes in the arterial intima that induce fusion of LDL particles and so produce lipid droplets or that induce foam cell formation. METHODS AND RESULTS: Human coronary atherosclerotic lesions obtained at surgery and at autopsy were stained for lysosomal acid lipase and cathepsin D. The extracellular areas of macrophage-rich intimal regions of the atherosclerotic lesions stained positively for both cathepsin D and lysosomal acid lipase, whereas normal arteries were negative. When monocyte-derived macrophages were incubated with opsonized zymosan to stimulate the release of lysosomal enzymes from the cells and LDL was incubated with the macrophage-conditioned media, the apolipoprotein B-100, cholesteryl esters, and triacylglycerols of LDL were hydrolyzed. These hydrolytic modifications rendered the LDL particles unstable and induced their fusion. Cultured macrophages and smooth muscle cells took up the hydrolase-modified LDL particles avidly and were transformed into foam cells. CONCLUSIONS: Our in vivo and in vitro results suggest that lysosomal enzymes released from macrophages may induce hydrolytic modification of LDL and foam cell formation in the human arterial intima.

Potential pathological roles for oxidized low-density lipoprotein and scavenger receptors SR-AI, CD36, and LOX-1 in aortic valve stenosis.

OBJECTIVE: To clarify the potential mechanisms by which oxidized low-density lipoprotein (oxLDL) could contribute to the progression of aortic valve stenosis (AVS). METHODS: We investigated a total of 46 stenotic and 20 control human aortic valves. The mRNA expression levels of scavenger receptor class A type 1 (SR-A1), CD36, Lectin-like oxidized LDL receptor-1 (LOX-1), and scavenger receptor class B type 1 (SR-B1) were studied using qPCR. Their cellular distribution in the valves was assessed by immunohistochemistry, and the potential effects of oxLDL were studied in cultured myofibroblasts isolated from the aortic valves. RESULTS: In AVS, the proinflammatory SR-A1 and the angiogenic LOX-1 were upregulated (p = 0.003 and p = 0.002), whereas the antiangiogenic CD36 was downregulated (p = 0.02). The expression of the atheroprotective SR-B1 remained unchanged. Immunohistochemistry revealed that SR-A1 was expressed by macrophages, whereas the expression of CD36 and LOX-1 was confined to myofibroblasts and endothelial cells in the diseased valves. In cultured valvular myofibroblasts, mast cell-derived components and TNF-α induced LOX-1 expression (p = 0.05 and p < 0.001), whereas oxLDL promoted the expression of proinflammatory cytokines. Furthermore, the expression of osteoprotegerin, an inhibitor of valvular calcification, decreased in response to oxLDL. Finally, myofibroblasts derived from stenotic valves accumulated more DiI-labeled oxLDL than myofibroblasts derived from macroscopically healthy valves (p = 0.035), so revealing enhanced foam cell-forming potential of myofibroblasts in the diseased valves. CONCLUSION: This study unveils the presence of SR-A1, CD36, and LOX-1 in aortic valves and suggests potential mechanisms by which they may contribute to the pathological angiogenesis, inflammation, calcification, and lipid accumulation in AVS.

OxLDL-IgG immune complexes induce expression and secretion of proatherogenic cytokines by cultured human mast cells.

OBJECTIVE: Human atherosclerotic lesions contain mast cells and immunoglobulin G immune complexes containing oxidized low-density lipoproteins (oxLDL-IgG ICs). Here we studied whether such oxLDL-IgG ICs can activate human mast cells and induce them to express and secrete pro-inflammatory cytokines that are potentially capable of inducing and amplifying atherogenic processes. METHODS AND RESULTS: Incubation of cultured human mast cells in the presence of oxLDL-IgG ICs led to a significant dose-dependent upregulation of the expression and secretion of tumor necrosis factor-alpha (TNF-a) and interleukin-8 (IL-8), and the chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1). The secretory responses were dose-dependent and associated with moderate release of histamine and tryptase, which are preformed mast cell mediators contained in the cytoplasmic secretory granules of the cells. Also native LDL-IgG ICs induced similar pro-inflammatory cytokine response, suggesting that ICs per se are important for the IgG IC-induced mast cell activation. CONCLUSION: Mast cells in atherosclerotic lesions which also contain oxLDL-IgG ICs may become activated by the ICs and secrete many pro-inflammatory cytokines. Our results suggest that intimal mast cells act as a cellular link between oxLDL-IgG ICs and the inflammatory response in atherosclerosis.

Complement system is activated in stenotic aortic valves.

OBJECTIVE: To examine the role of the complement system, a source of powerful proinflammatory mediators, in aortic valve stenosis (AS). METHODS AND RESULTS: Stenotic aortic valves (n=24) were obtained at valve replacement surgery, and non-stenotic (n=12) and early sclerotic (n=4) valves at cardiac transplantations. The terminal complement complex C5b-9 was stained by immunohistochemistry. Expression of the anaphylatoxin receptors C3aR and C5aR was studied in the valves by immunohistochemistry and RT-PCR, and in isolated valve myofibroblasts after stimulation with potential AS-accelerating factors (TNF-alpha and cigarette smoke) by RT-PCR. Cultured myofibroblasts were exposed to C3a, and their secretion of proinflammatory cytokines was assessed by ELISA. C5b-9 was found already in early aortic valve lesions, and its deposition was augmented in advanced stenotic valves. In stenotic valves, expression of C3aR mRNA was upregulated (p<0.05) and strong staining of C3aR and C5aR was detected. Myofibroblasts in stenotic, but not in control valves, expressed C3aR, and, in isolated myofibroblasts, TNF-alpha and cigarette smoke induced C3aR mRNA expression (p<0.05 for both). Stimulation of myofibroblasts with C3a resulted in enhanced secretion of MCP-1 (p<0.001), IL-6 (p=0.003), and IL-8 (p=0.03). CONCLUSIONS: In stenotic aortic valves, complement is activated leading to generation of the anaphylatoxins C3a and C5a. Upregulation of C3aR in the valves as a result of inflammation and external risk factors, such as cigarette smoke, leads to an inflammatory response in aortic valve myofibroblasts. Complement activation in stenotic valves emerges as a novel pathogenic component of AS and may serve as a therapeutic target in this disease.

Receptors for the anaphylatoxins C3a and C5a are expressed in human atherosclerotic coronary plaques.

The anaphylatoxins C3a and C5a are potent chemotactic and pro-inflammatory peptides that are released during complement activation, and recent clinical work have suggested them a role in acute coronary events. Here we studied whether human coronary plaques express anaphylatoxin receptors C3aR and C5aR, i.e. whether they have the potential to respond to anaphylatoxins. For this purpose, both normal (n=14) and atherosclerotic (n=20) human coronary artery samples were collected for histological and PCR analyses. Immunohistochemistry demonstrated that in atherosclerotic, but not in normal intimas, C3aR and C5aR were present. Consistently, PCR analysis showed that the expression of both receptors was >5-fold increased in the atherosclerotic plaques (p<0.01). Double immunofluorescence stainings revealed that in the plaques the principal cells expressing both C3aR and C5aR were macrophages. Moreover, T cells expressed C5aR and a small fraction of them also expressed C3aR, the mast cells expressed C5aR, whereas endothelial cells and subendothelial smooth muscle cells expressed both C3aR and C5aR. In conclusion, the presence of receptors for anaphylatoxins in human coronary plaques suggests that anaphylatoxins activate coronary plaques, and points the complement system as a potential therapeutic target in attempts to stabilize them.

Role of complement activation in atherosclerosis.

PURPOSE OF REVIEW: Atherosclerosis is characterized by a strong inflammatory component. One factor contributing to inflammation in the arterial intima is the complement system. Here we summarize recent progress in the field of complement research on atherogenesis. RECENT FINDINGS: The complement system is activated in human atherosclerotic lesions and is actively regulated by the local synthesis of complement components and of complement regulatory proteins. Potential triggers of complement activation in the arterial intima include immunocomplexes, C-reactive protein, modified lipoproteins, apoptotic cells, and cholesterol crystals. Complement activation releases anaphylatoxins, and anaphylatoxin receptors have been identified in human atherosclerotic lesions. However, experiments on genetically engineered mice with severe hyperlipidemia have been unable to show a major role for complement in experimental atherogenesis. SUMMARY: In humans there is extensive circumstantial evidence for a role of complement in atherosclerosis, which is somewhat contradictory to recent modest or negative findings in atherosclerosis-prone genetically engineered hyperlipidemic mice.

Activator protein-1 in human male germ cell apoptosis.

Apoptosis limits germ cell number in the testis, and its dysregulation is associated with male infertility. Here, we evaluated the role of the transcription factor activator protein 1 (AP-1) in male germ cell apoptosis in a culture of human seminiferous tubules. AP-1 DNA-binding activity increased in cultured tubules within 2.5 h, which was earlier than the onset of apoptosis as detected by caspase 3 activation and apoptotic DNA fragmentation. The c-Jun, c-Fos and JunD proteins were detected in the Sertoli cell nuclei, whereas apoptosis occurred in the germ cells. Follicle-stimulating hormone (FSH), whose receptors are expressed in the Sertoli cells, inhibited germ cell apoptosis and concomitantly suppressed AP-1 DNA-binding activity, but had no effect on nuclear factor kappaB (NF-kappaB) activation. These results suggest that AP-1 transcription factors are involved in the Sertoli cell-mediated control of germ cell apoptosis, and that inhibition of germ cell apoptosis by FSH appears to involve suppression of AP-1 activation.