Localization of Lipoprotein(a) in a Monkey Model of Rapid Neointimal Growth
Lipoprotein(a) [Lp(a)] has been proposed as a restenosis risk factor, but it is not known if Lp(a) is present in the injured arterial wall during the initial neointimal growth. The purpose of this study was to determine if Lp(a) is incorporated into the vessel wall during rapid neointimal formation after arterial injury in primates. In this model, distention of the iliac artery with an angioplasty catheter caused focal breaks in the internal elastic lamina (IEL) in 80% of the vessels and extensive IEL fragmentation with medial disruption in 20% of the vessels. Neointimal growth was noted in all injured arteries; thrombus formation was noted in 40% of the vessels. Based on morphometric measurements, injured arteries had neointimal areas of 0.41±0.05 (n=4) and 0.83±0.23 (n=6) mm2 at 14 and 28 days after injury, respectively. Control arteries had an intact IEL and a monolayer of intimal cells. Lp(a) localization was examined histologically by using a mouse monoclonal anti-Lp(a) antibody. Lp(a), found in all injured arteries, was localized primarily in the neointima in 50% of the vessels. In the subset of vessels with evidence of thrombus formation, intense Lp(a) immunostaining was associated with the thrombus. Lp(a) was specific to injured arteries as uninjured vessels did not stain. In addition, staining was not seen with a negative control, a nonspecific mouse IgG1 antibody. The presence of Lp(a) at the site of rapid neointimal growth supports a role for this lipoprotein in the response to vascular injury after balloon angioplasty.
- Received October 20, 1995.
- Revision received May 10, 1996.
Lp(a) consists of a plasminogen-like protein, apo(a), linked by a single disulfide bond to the apoB-100 of LDL.1 Five recent retrospective clinical studies have indicated that circulating levels of Lp(a) predict the incidence of restenosis in humans; two other investigators did not find such a relationship.2 3 4 5 6 7 8 While these studies attempted to define relationships between plasma Lp(a) levels and restenosis, they did not provide any information about the role of Lp(a) in the early response to vascular injury after balloon angioplasty.
Intimal thickening has been noted in baboons after removal of the endothelium by passing an inflated embolectomy catheter through the femoral artery; however, these studies focused on the importance of angiotensin-converting enzyme activity in the development of arterial proliferative lesions.9 The relationship between circulating Lp(a) and acute thrombus formation has been described in studies that used anesthetized cynomolgus monkeys.10 A positive correlation has been established between Lp(a) and increased incidence of permanent occlusive events after acute vessel injury. In addition, immunohistochemical analysis shows an intense positive stain for Lp(a) in areas of luminal thrombus formation. In independent studies, Lp(a) has been found within the diseased vessels of patients undergoing either coronary bypass or rebypass surgery.11 12 The identification of Lp(a) in diseased vessels suggests an important role for Lp(a) in the underlying pathology.
Lp(a) has biological activity that supports its proposed role in the stimulation of neointimal formation in response to vascular injury. In culture, human vascular SMC proliferation is accelerated in the presence of Lp(a) or apo(a).13 In a system that contained bovine aortic endothelial cells and aortic SMCs, Lp(a) promoted cell migration.14 However, to date no investigators have attempted to demonstrate the presence of Lp(a) in rapidly forming neointima. It is not known if Lp(a) is present in the injured arterial wall during the initial stages of neointimal growth.
In the present study, neointimal growth was induced by balloon injury in male cynomolgus monkeys with midrange Lp(a) levels. We examined the vascular lesions that developed 2 and 4 weeks after injury. Our purpose was to determine if Lp(a) is incorporated into the neointima and/or media of these injured arteries during rapid neointimal formation. The presence of Lp(a) at the site of early neointimal growth would support a role for this lipoprotein in the response to vascular injury.13
Ten male cynomolgus monkeys (Macaca fascicularis, Charles River Laboratories) weighing 2.9 to 6.1 kg were housed individually in stainless steel cages. The monkeys were maintained on a high-protein monkey diet (No. 5045, Ralston Purina) that contained 5% fat and were given fruit as a supplement. All procedures that used animals were conducted in compliance with state and federal laws and guidelines established by the Parke-Davis Animal Care and Use Committee.
Injury to the vascular wall of the iliac artery was achieved as described below. Monkeys were first concurrently anesthetized with ketamine (0.1 mg/kg IM, Aveco Co, Inc) and xylazine (0.6 mg/kg IM, Mobay Corp). Animals were then intubated, and anesthesia was maintained with inhalation of isoflurane (AErrane, Anaquest) and oxygen. Once adequate anesthesia was obtained, the aortoiliac region was surgically exposed by a lower abdominal incision. The luminal diameter of the iliac artery was measured by using angiography to allow choice of an appropriate balloon. Contrast medium (Renografin-76, diatrizoate meglumine and diatrizoate sodium, Solvay Animal Health, Inc) was bolus injected (5 to 8 mL) into the distal abdominal aorta through a butterfly catheter, and angiograms were obtained by using a portable x-ray unit (model E-10110, Dial-X Instrument Corp). The luminal diameter of the iliac artery was measured from the developed x-ray film by using handheld calipers.
The internal iliac artery was dissected free, and a long Cobra percutaneous transluminal coronary balloon angioplasty catheter (3.5 or 4.0 mm, Scimed Life Systems, Inc) was advanced in a retrograde direction through the freed segment (≈1 cm) until the distal end of the balloon was in the iliac artery. The proximal tip of the angioplasty balloon was either in the iliac artery or a few millimeters into the aorta. The exact tip location varied depending on the anatomy of the aortoiliac tree in individual monkeys.
The angioplasty balloon was inflated to a pressure (4 to 12 atm) that expanded the internal vessel diameter 1.5 to two times its initial size. The injury paradigm used three inflations of the balloon for 60 seconds each, with a 1-minute reperfusion between each inflation. After removal of the catheter the injured vessel was examined visually to insure that blood flow was restored. The internal iliac artery was then surgically stapled at its origin, the midline was sutured, and the animal was allowed to recover. Aspirin (4 mg/kg PO daily) was provided in gelatin for up to 4 days before injury and continuously after injury until the experiments were terminated. In addition, aspirin was given just before injury and at the conclusion of the angioplasty surgery in divided doses (total 4 mg/kg IV bolus). Animals were evaluated for pain daily and treated with buprenorphine (0.01 mg/kg IM, Buprenex hydrochloride, Reckitt and Colman Pharmaceuticals Inc) as needed.
Determination of Plasma Lp(a) Concentrations
Circulating Lp(a) levels were determined by using an enzyme-linked immunosorbent assay (Apo-Tek, Organon Teknika). This assay uses an Lp(a) monoclonal capture antibody and a polyclonal horseradish peroxidase–conjugated apoB detection antibody.15 Western blot analysis showed that the human Lp(a) capture antibody and apoB detection antibody could detect multiple cynomolgus monkey Lp(a) isoforms. The Lp(a) antibody did not cross-react with LDL or plasminogen. Over 100 cynomolgus monkeys were screened for plasma Lp(a) levels; values ranged between <1 and 72 mg/dL. Ten monkeys with midrange Lp(a) values (22 to 29 mg/dL) were selected for the present study.
Blood vessels were harvested for morphometric analysis at 14 (n=4) or 28 (n=6) days after injury. Monkeys were anesthetized (ketamine 0.1 mg/kg IM) and then given a lethal injection of Euthanasia-D solution (Schering-Plough Animal Health). The aortoiliac tree (the region from a few millimeters above the aortoiliac branch to a few millimeters caudad to the internal iliac bifurcation) was quickly removed and flushed with saline. The right and left iliac arteries were cut into four to six segments, each ≈2 mm in length. Segments were placed in 10% paraformaldehyde, fixed for 3 to 15 days, dehydrated in a series of alcohols, embedded in paraffin blocks, cut into 5-μm cross sections, and placed on poly-l-lysine– (Sigma) coated slides. Additional samples were frozen for future analysis. For morphological assessment, arteries were treated with a combination of Verhoeff's elastin and Masson's trichrome stain.
To determine intimal and medial areas, cross sections of six to 11 segments from each artery were analyzed by using a computerized image-analysis system (PGT Imagist, Princeton Gamma-Tech Inc). In many cases, multiple elastic laminae were observed; for morphometric measurements the neointima was defined as the region between the elastic lamina closest to the vessel lumen and the lumen. In general, segments near the internal iliac bifurcation and near the aortoiliac branch had either no or limited intimal thickening. Since the exact location of the angioplasty balloon in the vessel varied among the monkeys, the three to four consecutive segments with the largest neointimal areas were averaged to obtain a single value of intimal thickening in each vessel. All data are reported as mean±SEM.
Immunocytochemical Localization of Lp(a)
Immunohistochemical analysis was done by using a standard streptavidin–alkaline phosphatase reporter system. Arterial sections used for immunohistochemical analysis were from the same segments as arterial sections used for morphometric analysis. Sections were deparaffinized and rehydrated through a series of alcohol baths. They were then incubated 5 minutes in 1 mol/L citric acid–free acid (EM Science) to block potential endogenous phosphatases and rinsed in a Tris/HCl buffer, pH 7.4 to 7.6. The sections were then incubated at room temperature in a humidity chamber for 30 minutes in normal goat serum (Kirkegaard and Perry Lab Inc [KPL]) to block nonspecific binding sites on the secondary antibody. Anti-Lp(a) IgG1 monoclonal antibody (clone 4F3, 1 mg/mL, Organon Teknika) was diluted in Tris/HCl containing 1% bovine serum albumin (Sigma) and 0.5% Tween 20 (Sigma) to an optimal concentration of 1:400, applied to the tissue, and incubated as before for 60 minutes. Sections were then washed three times in Tris/HCl buffer containing 0.05% Tween 20 followed by sequential incubations for 30 minutes each in biotinylated secondary antibody (goat anti-mouse) and streptavidin–alkaline phosphatase conjugate (KPL) with washing between. After a final series of washes, the alkaline phosphatase was developed for 10 minutes by using new fuschin stain (Histomark Red, KPL) to yield a red reaction product; the reaction was stopped by a water wash, after which tissues were counterstained a contrasting blue with hematoxylin. The tissue sections were dehydrated, cleared with Americlear (Baxter Diagnostics), mounted with Accumount (Baxter Scientific Products), and placed on coverslips. On serial sections, substitution of the primary antibody with mouse IgG1 at the same protein content as the primary monoclonal antibody served as a negative control. Additional controls included the use of a nonimmune mouse serum (BioGenex) and buffer alone.
To further characterize the specificity of the Lp(a) staining, the reactivity of the primary mouse monoclonal anti-human Lp(a) antibody used (4F3 clone) was evaluated against rat, rabbit, monkey, and human plasma by using a dot blot assay. In this assay a positive stain for Lp(a) occurred against plasma from both monkey and human but not rabbit, a species considered to be a negative control. An unexpected result was a positive stain for rat plasma that also served as a negative control. A Western blot performed with the 4F3 clone and two additional Lp(a) antibodies revealed a very weak cross-reactivity with a low-molecular-weight (<70 kD) protein unique to the rat and different from Lp(a). All three antibodies recognized Lp(a) in both human and monkey plasma, but the antibodies did not recognize purified plasminogen. The isoforms of Lp(a) in the monkeys used in this study had molecular weights that ranged from 7×105 to 1×106 kD as determined by using Lp(a) reference standards. Thus, the results of the Western blot suggest that the antibody we employed recognizes both human and nonhuman primate forms of Lp(a) and that in these species cross-reactivity with other proteins is negligible.
Vascular injury was induced by balloon dilation in 10 normal cynomolgus monkey iliac arteries. All contralateral uninjured control iliac arteries (Fig 1⇓) had an intact IEL and a monolayer of endothelial cells lining the vessel lumen. After balloon dilation, intimal thickening was noted in all injured vessels with no damage to the EEL. Medial disruption and IEL fragmentation were variable. Focal breaks in the IEL with no medial disruption were noted in eight of 10 arteries (Figs 2A⇓, 2B, 3A, and 3B). The two remaining arteries showed extensive medial disruption with large gaps in the IEL. The neointima extended halfway into the media in one artery that had distinct IEL gaps. In a second artery, the neointima extended to the EEL, and IEL fracture was associated with an organized thrombus (Fig 4A⇓ and 4B). Nonocclusive thrombi were found in four of the 10 injured arteries.
Morphometric measurements were made on vessels harvested 14 and 28 days after injury. Lp(a) circulating levels were 24.3±1.7 (n=4) and 24.2±0.9 (n=6) mg/dL in the 14- and 28-day groups, respectively. The two groups had similar baseline luminal diameters (2.4±0.1 and 2.2±0.1 mm) as measured from radiographic film. Balloon injury resulted in the formation of a neointima in all 10 monkeys. Based on morphometric measurements, injured arteries had neointimal areas of 0.41±0.05 (n=4) and 0.83±0.23 (n=6) mm2 at 14 and 28 days, respectively. Eight of the 10 injured arteries had neointimal areas that ranged from 0.25 to 0.57 mm2. The other two animals, both of which were from the 28-day group, had much larger proliferative responses (1.41 and 1.71 mm2) and nonocclusive thrombi. One of the two arteries with a large lesion appeared to have only focal breaks in the IEL. However, balloon dilation likely caused far greater damage to the second artery, as there was extensive IEL fragmentation and ruptured media that allowed neointima to extend to the EEL (Fig 4A and 4B⇓⇓). The intimal area averaged <0.001 mm2 in contralateral uninjured control arteries. The medial areas of injured vessels were 1.92±0.31 and 1.62±0.14 mm2 at 14 and 28 days, respectively. The medial area in the uninjured control vessels was about one half the size of injured vessels.
Tissue Lp(a) localization was examined histologically in injured and uninjured iliac arteries by using a mouse monoclonal anti-Lp(a) antibody and a biotinylated secondary antibody (goat anti-mouse) linked to a streptavidin–alkaline phosphatase conjugate. All 10 arteries showed positive staining for Lp(a) at the site of injury; no positive Lp(a) staining was noted in any of the contralateral control vessels. Positive Lp(a) staining was localized primarily in the neointima (Fig 2C⇑) in five arteries and the media in two others (Fig 3C⇓). Three vessels had an equal distribution between the neointima and media. Intense Lp(a) staining was associated with thrombus formation (Fig 4C and 4D⇓⇓), but Lp(a) staining was also observed some distance from the thrombus in both the media and neointima (Fig 4D and 4E⇓⇓). The pattern of staining and intensity was similar between the 14- and 28-day groups. The intense positive staining for Lp(a) was highly specific, as staining was not seen with either nonspecific mouse IgG1 antibody (Fig 4F⇓) or nonimmune mouse serum (data not shown), which served as negative controls.
Lp(a) has been identified only in humans, old world monkeys, and European hedgehogs.16 Human apo(a) has been expressed in mice that lack apo(a).17 These apo(a) transgenic mice develop fatty streak lesions when placed on a high-fat diet that are similar to those found in early human atherosclerosis. Thus, transgenic mice represent a useful animal model to elicit the role of apo(a) in atherogenesis. Transgenic mice have been made that produce an Lp(a) particle,18 19 but the small caliber of mouse vessels does not permit the use of the balloon-catheter techniques commonly employed to induce vascular injury in larger rodent and nonrodent models. Thus a more relevant model for determining the involvement of Lp(a) in response to balloon-induced vascular injury is an animal that normally expresses Lp(a), such as the cynomolgus monkey.
The present study showed an accumulation of Lp(a) in the blood vessel wall of cynomolgus monkeys during a phase of neointimal growth after iliac artery injury induced by balloon dilation. The intense positive staining for Lp(a) was highly specific, as staining was not noted in uninjured arteries or arteries treated with a nonspecific mouse antibody. Additionally, Lp(a) immunostaining appeared to depend on circulating Lp(a); in a preliminary experiment Lp(a) could not be detected at the site of injury in an animal with very low plasma Lp(a) (<2 mg/dL, data not shown).
Our results support a role for Lp(a) in the arterial injury response, as this lipoprotein was localized in regions of ongoing neointimal formation. Lp(a) has been proposed as a possible restenosis risk factor in humans after percutaneous transluminal coronary angioplasty. The Lp(a)-restenosis hypothesis is based on retrospective epidemiological studies and the finding that Lp(a) shares structural homology with plasminogen, the inactive form of plasmin.20 Plasmin is an important component both of the fibrinolytic pathway and transforming growth factor-β activation.21 In vitro, transforming growth factor-β inhibits vascular SMC proliferation.13 Lp(a), by interfering with plasmin activation, may promote SMC proliferation and thus contribute to the vascular pathology that follows angioplasty.
Like Williams and coworkers,10 we also identified Lp(a)-positive immunostaining in mural thrombi after vascular injury. However, in the present study we probed for Lp(a) several days after the initial vascular injury, during the phase of rapid neointimal formation. Lp(a)-positive staining was identified in neointimal tissue, and most Lp(a)-positive regions contained no obvious mural thrombus, suggesting that Lp(a) promotes neointimal growth as well as thrombus formation. In Lp(a)-positive regions, immunostaining was limited to the neointima and media, whereas Williams and coworkers10 also identified considerable staining in the adventitia. The method of injury used in the two studies may account for the difference in staining pattern. The pinch-induced injury with needle holders used by Williams et al10 would have resulted in adventitial tissue injury, whereas vessel distention by balloon inflation would more likely injure the media. Differences in the immunocytochemical methods used to localize Lp(a) might also account for differences in the staining pattern. In the present study a streptavidin–alkaline phosphatase reporter system was used, whereas Williams and coworkers10 used a streptavidin–horseradish peroxidase system. Failure to block high levels of endogenous peroxidase in the adventitia could account for the adventitial staining they observed.
The present findings suggest that moderate iliac artery injury produces significant neointimal growth within 14 days in cynomolgus monkeys. Intimal thickening was noted in the arterial segments from all 10 monkeys, despite what appeared to be modest injury (ie, small focal breaks in the IEL) in eight of them. Thrombus formation was noted in only four monkeys. It is unclear why two of the arteries displayed much larger neointimal lesions than the others. It is possible that more extensive vessel-wall damage occurred in these two arteries due to an underestimation of baseline vessel diameter as measured with handheld calipers and x-ray film angiograms. In injured arteries the medial area was consistently larger than that of the contralateral controls, and medial areas were larger at 14 than at 28 days after injury. Clowes and coworkers22 suggest that the greater medial area noted in the rat carotid artery model may be the result of edema in response to balloon injury.
We did not allow for a correlation between circulating Lp(a) levels and extent of intimal thickening, as only monkeys with moderate circulating Lp(a) levels were selected for study. We chose to control Lp(a) levels since it was unknown if in our hands neointima would be consistently formed in response to balloon injury in the primate iliac artery. Our data suggest that if there is a relationship between circulating Lp(a) and neointimal formation, it may be possible to demonstrate this relationship in cynomolgus monkeys, as a moderate iliac artery injury consistently produced significant intimal thickening. Experiments are now underway to define the relationship between circulating Lp(a) and extent of intimal thickening as well as other markers of vascular injury in this model.
In the present study we showed that Lp(a) is deposited only at the site of vascular injury. The mechanism by which Lp(a) selectively penetrates and accumulates at the injury site is unknown. One explanation is that Lp(a) is passively taken up and thus is merely a permeability marker. However, at least two lines of evidence support the theory that Lp(a) uptake is specific. Lp(a) competes directly with plasminogen-binding sites on human platelets and endothelial cells.23 24 This binding is time dependent, specific, saturable, divalent ion independent, and temperature sensitive and is thought to represent the plasminogen receptor. Binding of plasminogen to its receptor greatly enhances the conversion of plasminogen to its active form, plasmin. Under these conditions Lp(a) competition with plasminogen would result in less plasmin generation, causing decreased levels of active transforming growth factor-β, an inhibitor of SMC proliferation. A second possibility is that Lp(a) is associated with macrophages that may have invaded the injury site. Lp(a) binds to macrophages via a specific high-affinity receptor that is not related to the plasminogen receptor.25 26
The data from the in vitro experiments outlined above suggest specific Lp(a) uptake; however, specific uptake into rapidly forming neointimal tissue has not yet been demonstrated. A potential limitation of our study is that after injury there may have been a generalized influx of plasma proteins into the vessel wall. This issue could be addressed by comparing the localization of Lp(a) with another plasma protein of similar size; however, such an experiment would not be definitive. If Lp(a) were colocalized with a biologically inactive plasma protein (ie, nonspecific influx), one might inappropriately rule out a role for Lp(a) in promoting neointimal formation. Similarly, the inability to identify a plasma protein marker in the vessel wall after injury might be the result of failure to detect the protein and not be a demonstration of selective Lp(a) uptake. For example, paraformaldehyde fixation and subsequent tissue processing could destroy the antigenic properties on a marker protein with no effect on the antibody recognition site for Lp(a). We have localized Lp(a) to the site of injury but have not identified the exact mechanistic binding site at which Lp(a) plays a proliferative role in the injury response. Additional experiments are needed to determine if in vivo Lp(a) is bound to plasminogen receptors associated with platelets or fibrin or if Lp(a) is associated with specific cell types such as macrophages. Logically these experiments can be undertaken only after it has first been demonstrated that Lp(a) is consistently found in intimal lesions after injury. The finding that Lp(a) accumulates in rapidly growing neointima represents a significant new scientific finding and indicates that additional studies are warranted.
In summary, we have shown intimal growth within 14 days after injury of the iliac artery by balloon dilation in cynomolgus monkeys, a species which, like humans, normally expresses Lp(a). In addition, we have identified Lp(a) by immunocytochemistry at the lesion site in both the neointima and media. The presence of Lp(a) at the site of early neointimal formation supports a role for this lipoprotein in the response to vascular injury after balloon angioplasty. As cynomolgus monkeys can be screened to determine circulating levels of Lp(a) before induction of vascular injury, the model described provides an opportunity to evaluate the influence of circulating Lp(a) on intimal growth; ultimately this may lead to a better understanding of the role of Lp(a) in the vascular biology of restenosis.
Selected Abbreviations and Acronyms
|EEL||=||external elastic lamina|
|IEL||=||internal elastic lamina|
|SMC||=||smooth muscle cell|
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