Peter C. Harpel

The atherogenic lipoprotein Lp(a) is internalized and degraded in a process mediated by the VLDL receptor.

Lp(a) is a major inherited risk factor associated with premature heart disease and stroke. The mechanism of Lp(a) atherogenicity has not been elucidated, but likely involves both its ability to influence plasminogen activation as well as its atherogenic potential as a lipoprotein particle after receptor-mediated uptake. We demonstrate that fibroblasts expressing the human VLDL receptor can mediate endocytosis of Lp(a), leading to its degradation within lysosomes. In contrast, fibroblasts deficient in this receptor are not effective in catabolizing Lp(a). Lp(a) degradation was prevented by antibodies against the VLDL receptor, and by RAP, an antagonist of ligand binding to the VLDL receptor. Catabolism of Lp(a) was inhibited by apolipoprotein(a), but not by LDL or by monoclonal antibodies against apoB100 that block LDL binding to the LDL receptor, indicating that apolipoprotein(a) mediates Lp(a) binding to this receptor. Removal of Lp(a) antigen from the mouse circulation was delayed in mice deficient in the VLDL receptor when compared with control mice, indicating that the VLDL receptor may play an important role in Lp(a) catabolism in vivo. We also demonstrate the expression of the VLDL receptor in macrophages present in human atherosclerotic lesions. The ability of the VLDL receptor to mediate endocytosis of Lp(a) could lead to cellular accumulation of lipid within macrophages, and may represent a molecular basis for the atherogenic effects of Lp(a).

Apolipoprotein(a) Induces Monocyte Chemotactic Activity in Human Vascular Endothelial Cells

BACKGROUND Elevated levels of lipoprotein(a) [Lp(a)] are associated with premature atherosclerosis; however, the mechanisms are not known. Recruitment of monocytes to the blood vessel wall is an early event in atherogenesis. METHODS AND RESULTS This study has found that unoxidized Lp(a) induced human umbilical vein endothelial cells (HUVECs) to secrete monocyte chemotactic activity (MCA), whereas LDL under the same conditions did not. In the absence of HUVECs, Lp(a) had no direct MCA. Endotoxin was shown not to be responsible for the induction of MCA. Actinomycin D and cycloheximide inhibited the HUVEC response to Lp(a), indicating that protein and RNA synthesis were required. The apolipoprotein(a) [apo(a)] portion of Lp(a) was identified as the structural component of Lp(a) responsible for inducing MCA. Lp(a) and apo(a) also stimulated human coronary artery endothelial cells to produce MCA. Granulocyte-monocyte colony-stimulating factor (GM-CSF) antigen was not detected in the Lp(a)-conditioned medium, nor was monocyte chemoattractant protein-1 mRNA induced in HUVECs by Lp(a). CONCLUSIONS These findings suggest that Lp(a) may be involved in the recruitment of monocytes to the vessel wall and provide a novel mechanism for the participation of Lp(a) in the atherogenic process.

Lipoprotein(a) and inflammation in human coronary atheroma: association with the severity of clinical presentation

OBJECTIVES The purpose of this study was the investigation of the in vivo role of lipoprotein(a) [Lp(a)] and inflammatory infiltrates in the human coronary atherosclerotic plaque and their correlation with the clinical syndrome of presentation. BACKGROUND Lipoprotein(a) is an atherogenic and thrombogenic lipoprotein, and has been implicated in the pathogenesis of acute coronary syndromes. Lipoprotein(a) induces monocyte chemoattraction and smooth muscle cell activation in vitro. Macrophage infiltration is considered one of the mechanisms of plaque rupture. METHODS This study of atherectomy specimens investigated the in vivo role of Lp(a) at different stages of the atherogenic process, and its relationship with macrophage infiltration. We examined coronary atheroma removed from 72 patients with stable or unstable angina. Specimens were stained with antibodies specific for Lp(a), macrophages (KP-1), and smooth muscle cells (alpha-actin). Morphometric analysis was used to quantify the plaque areas occupied by each of the three antigens, and their colocalization. RESULTS All specimens had localized Lp(a) staining; the mean fractional area was 58.2%. Ninety percent of the macrophage areas colocalized with Lp(a) positive areas, whereas 31.3% of the smooth muscle cell areas colocalized with Lp(a) positive areas. Patients with unstable angina (n = 46) had specimens with larger mean plaque Lp(a) areas than specimens from stable angina patients (n = 26): 64.4% versus 47.7% (p = 0.004). Unstable angina patients with rest pain (n = 28) had greater mean plaque Lp(a) area than unstable angina patients with crescendo exertional pain (n = 18): 71.1% versus 52.4% (p < 0.001). Mean KP-1 area was 31.2% in unstable rest angina versus 18.3% in stable angina (p = 0.05); alpha-actin area was greater in stable (48.5%) and crescendo exertional angina (48.8%) than in rest angina (30.4%). The strongest correlation between plaque KP-1 and Lp(a) area was in unstable rest angina (r = 0.88, p < 0.001), and between alpha-actin and Lp(a) areas in the crescendo exertional angina (r = 0.62, p < 0.01). CONCLUSIONS Lipoprotein(a) is ubiquitous in human coronary atheroma. It is detected in larger amounts in tissue from culprit lesions in patients with unstable compared to stable syndromes, and has significant colocalization with plaque macrophages. A correlation of plaque alpha-actin and Lp(a) area suggests a role of Lp(a) in plaque growth.

CC Chemokine I-309 Is the Principal Monocyte Chemoattractant Induced by Apolipoprotein(a) in Human Vascular Endothelial Cells

BACKGROUND Lipoprotein(a) [Lp(a)] is a risk factor for atherosclerosis; however, the mechanisms are unclear. We previously reported that Lp(a) stimulated human vascular endothelial cells to produce monocyte chemotactic activity. The apolipoprotein(a) [apo(a)] portion of Lp(a) was the active moiety. METHODS AND RESULTS We now describe the identification of the chemotactic activity as being due to the CC chemokine I-309. The carboxy-terminal domain of apo(a) containing 6 type-4 kringles (types 5 to 10), kringle V, and the protease domain was demonstrated to contain the I-309-inducing portion. Polyclonal and monoclonal anti-I-309 antibodies as well as an antibody against a portion of the extracellular domain of CCR8, the I-309 receptor, inhibited the increase in monocyte chemotactic activity induced by apo(a). I-309 antisense oligonucleotides also inhibited the induction of endothelial monocyte chemotactic activity by apo(a). I-309 mRNA was identified in human umbilical vein endothelial cells. Apo(a) induced an increase in I-309 protein in the endothelial cytoplasm and in the conditioned medium. Immunohistochemical studies have identified I-309 in endothelium, macrophages, and extracellular areas of human atherosclerotic plaques and have found that I-309 colocalized with apo(a). CONCLUSIONS These data establish that I-309 is responsible for the monocyte chemotactic activity induced in human umbilical vein endothelial cells by Lp(a). The identification of the endothelial cell as a source for I-309 suggests that this chemokine may participate in vessel wall biology. Our data also suggest that I-309 may play a role in mediating the effects of Lp(a) in atherosclerosis.

Changes in circulatory indices of thrombosis and fibrinolysis during total knee arthroplasty performed under tourniquet

Deep vein thrombosis may begin during surgery with the tourniquet inflated. Arterial levels of fibrinopeptide A, thrombin-antithrombin complexes, D-dimer, tissue plasminogen activator (t-PA) activity, and t-PA antigen were measured before surgery, during surgery with the tourniquet inflated, and following deflation of the tourniquet in 12 patients undergoing total knee arthroplasty. Minimal increases in fibrinopeptide A, thrombin-antithrombin complexes, and D-dimer were noted during surgery with the tourniquet inflated, but significant increases occurred immediately following deflation of the tourniquet. In 10 patients, intravenous heparin administration significantly suppressed the rise in fibrinopeptide A, but did not significantly alter the increases in either thrombin-antithrombin complexes, D-dimer, t-PA antigen, or t-PA activity. This study provides further evidence that deep vein thrombosis begins during surgery.

The John Charnley Award. Thrombogenesis during total hip arthroplasty.

The activation of the clotting cascade leading to deep venous thrombosis begins during total hip arthroplasty, but few studies have assessed changes in coagulation during surgery. A better understanding of thrombogenesis during total hip arthroplasty may provide a more rational basis for treatment. In 3 separate studies, the following observations were made. Circulating indices of thrombosis and fibrinolysis: prothrombin F1.2, thrombin-antithrombin complexes, fibrinopeptide A, and D-dimer, did not increase during osteotomy of the neck of the femur or during insertion of the acetabular component, but rose significantly during insertion of the femoral component. Thrombin-antithrombin complexes, fibrinopeptide A, and D-dimer were higher after insertion of a cemented component than insertion of a noncemented femoral component. A significant decline in central venous oxygen tension was observed after relocation of the hip joint and after insertions of cemented and noncemented femoral components, providing evidence of femoral venous occlusion during insertion of the femoral component. In patients receiving a cemented femoral component, mean pulmonary artery pressure increased after relocation of the hip joint, indicating intraoperative pulmonary embolism. No changes in mean pulmonary artery pressure were noted with noncemented total hip arthroplasty. Administration of 1000 units of unfractionated heparin before insertion of a cemented femoral component blunted the rise of fibrinopeptide A. The results of these studies suggest that (1) the greatest risk of activation of the clotting cascade during total hip arthroplasty occurs during insertion of the femoral component; (2) femoral venous occlusion and use of cemented components are factors in thrombogenesis during total hip arthroplasty; and (3) measures to prevent deep venous thrombosis during total hip arthroplasty (such as intraoperative anticoagulation) should begin during surgery rather than during the postoperative period and be applied during insertion of the femoral component.

Dose response of intravenous heparin on markers of thrombosis during primary total hip replacement.

BACKGROUND Thrombogenesis in total hip replacement (THR) begins during surgery on the femur. This study assesses the effect of two doses of unfractionated intravenous heparin administered before femoral preparation during THR on circulating markers of thrombosis. METHODS Seventy-five patients undergoing hybrid primary THR were randomly assigned to receive blinded intravenous injection of either saline or 10 or 20 U/kg of unfractionated heparin after insertion of the acetabular component. Central venous blood samples were assayed for prothrombin F1+2 (F1+2), thrombin-antithrombin complexes (TAT), fibrinopeptide A (FPA), and D-dimer. RESULTS No changes in the markers of thrombosis were noted after insertion of the acetabular component. During surgery on the femur, significant increases in all markers were noted in the saline group (P < 0.0001). Heparin did not affect D-dimer or TAT. Twenty units per kilogram of heparin significantly reduced the increase of F1+2 after relocation of the hip joint (P < 0.001). Administration of both 10 and 20 U/kg significantly reduced the increase in FPA during implantation of the femoral component (P < 0.0001). A fourfold increase in FPA was noted in 6 of 25 patients receiving 10 U/kg of heparin but in none receiving 20 U/kg (P = 0.03). Intraoperative heparin did not affect intra- or postoperative blood loss, postoperative hematocrit, or surgeons subjective assessments of bleeding. No bleeding complications were noted. CONCLUSIONS This study demonstrates that 20 U/kg of heparin administered before surgery on the femur suppresses fibrin formation during primary THR. This finding provides the pathophysiologic basis for the clinical use of intraoperative heparin during THR.

Apolipoprotein(a) is a human vascular endothelial cell agonist: studies on the induction in endothelial cells of monocyte chemotactic factor activity

Elevated levels of lipoprotein(a), Lp(a), are associated with premature atherosclerosis; however, the mechanisms of its atherogenicity are not known. Recruitment of monocytes to the blood vessel wall is an early event in atherogenesis. Since Lp(a) is associated with macrophages in the plaque, we have examined the effect of Lp(a) on inducing monocyte chemotactic activity (MCA) in vascular endothelial cells. We report that Lp(a) and apo(a) induced human umbilical vein (HUVEC) and coronary artery endothelial cells to secrete monocyte chemotactic activity as early as 30 min of incubation. In the absence of cells, Lp(a) had no direct monocyte chemotactic activity. Actinomycin D and cycloheximide inhibited the HUVEC response, indicating that protein and RNA synthesis were required. Endotoxin was shown not to be responsible for the induction of monocyte chemotactic activity. Granulocyte monocyte‐colony stimulating factor antigen was not detected in the Lp(a)‐conditioned medium, nor was monocyte chemoattractant protein‐1 mRNA induced by Lp(a). These results suggest that Lp(a) may be involved in the recruitment of monocytes to the vessel wall, thus providing a novel mechanism for the participation of Lp(a) in the atherogenic process.

Identification of mechanisms that may modulate the role of lipoprotein(a) in thrombosis and atherogenesis

In this report, we review recent findings concerning the identification of mechanisms that may modulate the role of lipoprotein(a), or Lp(a), in thrombosis and atherogenesis. Lp(a) binds to surface-immobilized plasmin-modified fibrin, thus providing a mechanism for incorporating Lp(a) into the vessel wall. We found that homocysteine and other sulfhydryl-containing amino acids markedly increase the binding of Lp(a) to plasmin-modified fibrin. Our results suggest that homocysteine alters the structure of Lp(a) to expose lysine-binding sites on the apolipoprotein(a) portion of the molecule, and thus provide a potential biochemical link between thrombosis and atherogenesis. We also found that transglutaminases catalyze the incorporation of primary amines into Lp(a). Studies in cell culture systems have found that Lp(a) stimulates endothelial cells to synthesize and release plasminogen activator inhibitor-1. Further, Lp(a) inhibits the activation of transforming growth factor-beta in a coculture of bovine endothelial and smooth muscle cells.

Lipoprotein(a), Homocysteine, and Atherogenesis

An elevated blood level of lipoprotein(a) is an independent risk factor for coronary and carotid artery atherosclerosis leading to premature myocardial infarction and stroke. Elevated liproprotein(a) is also associated with restenosis following coronary balloon angioplasty. The mechanisms by which lipoprotein(a) promote the atherosclerotic process are not clear. The apolipoprotein(a) portion of lipoprotein(a) shares partial homology with plasminogen, the precursor of plasmin, the fibrinolytic protease. Our studies have documented that lipoprotein(a) binds to fibrin, and that partial degradation of fibrin by plasmin increases binding. These findings parallel immunohistochemical studies showing colocalization of lipoprotein(a) with fibrin in atheromatous plaques, suggesting that the lipoprotein(a)-fibrin interaction may be atherogenic. We have found that homocysteine, at concentrations as low as 8 M, increased the binding of lipoprotein(a) to fibrin. Homocysteine induced a 20-fold increase in the affinity of lipoprotein(a) for plasmin-modified fibrin as compared to the binding in the absence of homocysteins. Our data indicate that homocysteine and other agents containing free sulfhydryl groups alter the lipoprotein(a) particle so as to increase the reactivity of the plasminogen-like apolipoprotein(a) portion. These observations suggest a biochemical link between lipoprotein(a), sulfhydryl compound metabolism, thrombosis, and atherogenesis.