Balloon Catheter Deendothelialization Research Articles

Platelet and fibrinogen turnover at the exposed subendothelium measured over 1 year after a balloon catheter de-endothelializing injury to the rabbit aorta:: Thrombotic eruption at the late re-endothelialization stage

Balloon catheter de-endothelialization of the rabbit aorta in vivo causes a rapid release of thrombin and a consequent hemostatic response at the surface of the exposed subendothelium. Previously, we have compared the net fluxes of several hemostatic proteins from plasma into the exposed aorta subendothelium for up to 600 days after injury. We now report the turnover of platelets, compared to fibrinogen, at the de-endothelialized aorta for up to 390 days after injury. Anesthetized NZW rabbits received either a de-endothelializing or a sham injury (controls) to their aortas. At a predetermined time (either 10 min before or up to 390 days after injury), each rabbit was infused with known quantities of rabbit 51Cr-platelets and rabbit 125I-fibrinogen; the radiolabels were allowed to circulate for 10 min before the rabbit was rapidly exsanguinated. Radioactivity measurements and tissue analysis revealed that at 10 min after balloon injury, approximately 165,000 platelets/mm 2 were associated with the aorta surface, and platelet turnover was 840/min/mm 2. Turnover had decreased to <200/min/mm 2 at 10–21 days but, from 65 to 390 days, had increased to approximately 1500/min/mm 2. In comparison, approximately 17 pmol of fibrinogen/cm 2 saturated the ballooned surface by 10 min after injury. Fibrinogen turnover at the aorta surface at 10 min after injury amounted to 0.2 pmol/min/cm 2, increasing to 0.7 at 10 days but decreasing to 0.25 at 21 days. Between 65 and 390 days, fibrinogen turnover increased slowly to 1.3 pmol/min/cm 2. Fibrinogen turnover at the surface of the aorta paralleled that within the intima-media over 390 days. Platelet and fibrin(ogen) deposits within the aorta wall increased over the 21–390 days interval as shown by immunostaining. The results are consistent with the re-endothelializing aorta tending to support thrombosis and ulceration in the late healing stage.

Uupregulation of High Affinity Cyclic AMP Phosphodiesterase (PDE) Genes Following BALloon Catheter Deendothelialization (Bal) in Rat Aorta

P75 Inhibitors of PDE 3 and/or PDE4 block smooth muscle cell (SMC) proliferation/migration in vitro and reduce restenosis. To evaluate chronic regulation of SMC PDEs, rats were subjected to BAL. At several times after BAL (30 min to 10days/10D), medial SMC were isolated from the thoracic aortae. Tissues were fractionated for total RNA (analyzed by Northern blots/ RNAase protection) or cytosolic protein (Western blots or low Km [100 nM] cAMP PDE activity). BAL-dependent increases in PDE4B mRNA were biphasic: 2.7-fold at 1hr, which declined by 24hr below control values, followed by 2-3-fold increases by 4-7D (p<0.05 vs controls, n=3-6). PDE4B and c-myc mRNAs were selectively superinduced in cycloheximide-treated rats. Despite modest changes in PDE3 mRNAs, both 80 and 120 kDa PDE3A proteins were detected and only the 80kDa increased 7D post-BAL (10-fold increase, p<0.05, n=6). PDE4B2 (80kDa) persistently increased 2.5-3-fold (p<0.05, n=4-6) from 24 hr to 10D post-BAL, while 2-fold increases in PDE4D3 (90kDa) and PDE4B1 (104kDa) were evident at 24hr or 7D, respectively. PDE activity increased 50-60% (p<0.05; n=4) at 24 hr and 7D after BAL. Thus PDE4B resembles an immediate-early gene. Selective induction of the PDE4 family is associated with 2 waves of SMC proliferation in vivo, the latter of which is also accompanied by induction of PDE3A/80kDa. Upregulation of specific SMC PDEs following angioplasty represents an important response to injury that may be a useful therapeutic target in vascular disease.

Uupregulation of High Affinity Cyclic AMP Phosphodiesterase (PDE) Genes Following BALloon Catheter Deendothelialization (Bal) in Rat Aorta

P75 Inhibitors of PDE 3 and/or PDE4 block smooth muscle cell (SMC) proliferation/migration in vitro and reduce restenosis. To evaluate chronic regulation of SMC PDEs, rats were subjected to BAL. At several times after BAL (30 min to 10days/10D), medial SMC were isolated from the thoracic aortae. Tissues were fractionated for total RNA (analyzed by Northern blots/ RNAase protection) or cytosolic protein (Western blots or low Km [100 nM] cAMP PDE activity). BAL-dependent increases in PDE4B mRNA were biphasic: 2.7-fold at 1hr, which declined by 24hr below control values, followed by 2-3-fold increases by 4-7D (p&lt;0.05 vs controls, n=3-6). PDE4B and c-myc mRNAs were selectively superinduced in cycloheximide-treated rats. Despite modest changes in PDE3 mRNAs, both 80 and 120 kDa PDE3A proteins were detected and only the 80kDa increased 7D post-BAL (10-fold increase, p&lt;0.05, n=6). PDE4B2 (80kDa) persistently increased 2.5-3-fold (p&lt;0.05, n=4-6) from 24 hr to 10D post-BAL, while 2-fold increases in PDE4D3 (90kDa) and PDE4B1 (104kDa) were evident at 24hr or 7D, respectively. PDE activity increased 50-60% (p&lt;0.05; n=4) at 24 hr and 7D after BAL. Thus PDE4B resembles an immediate-early gene. Selective induction of the PDE4 family is associated with 2 waves of SMC proliferation in vivo, the latter of which is also accompanied by induction of PDE3A/80kDa. Upregulation of specific SMC PDEs following angioplasty represents an important response to injury that may be a useful therapeutic target in vascular disease.

Expression of Membrane-Type Matrix Metalloproteinase in Rabbit Neointimal Tissue and Its Correlation with Matrix-Metalloproteinase-2 Activation

Smooth muscle cell (SMC) phenotypic alteration, followed by migration and proliferation, is a prominent feature of atherogenesis and vascular neointimal formation. Despite extensive research, mechanism(s) responsible for this alteration remain unclear. Recently, matrix metalloproteinases (MMP), a family of potent proteinases, have been implicated in vascular diseases by way of extracellular matrix degradation. Of particular interest is that expression of a 72-kD MMP (MMP-2) is elevated in neointima, and inhibition of this MMP results in reduced SMC migration and proliferation, suggesting a role for MMP-2 in neointimal development. However, MMP-2 needs activation before digesting protein; the mechanism of this activation in the arterial wall is largely unexplored. A novel membrane-type MMP termed MT-MMP-1 has recently been identified, and its expression in tumor cells is concomitant with MMP-2 activation. Transfection of this MMP cDNA into mammalian cells results in activation of MMP-2. However, the importance of this MMP in various pathological situations is not clear. The present study was designed to explore the relationship between MT-MMP-1 expression and MMP-2 activation during rabbit neointimal development. Using polymerase chain reaction, we isolated a rabbit cDNA from arterial SMC; sequence analysis indicated that it is a rabbit form of MT-MMP-1. A segment of this cDNA was subcloned into pGEM-3 and employed to synthesize a DIG-labeled RNA probe. This probe was then used in the Northern blot analysis for MT-MMP-1 mRNA expression both in aortic tissue and in neointimal tissues developed 3, 7, 14 and 21 days after balloon catheter de-endothelialization. The results show low-level expression of MT-MMP-1 in the normal aortic wall; expression is significantly increased in the neointimal tissues, with peak expression observed in tissues 3 days after injury. Expression of active MMP-2 was also determined using gel zymography. A close temporal expression pattern was observed between MT-MMP-1 and active MMP-2. These data verify the expression of MT-MMP-1 in arterial SMC and suggest its importance in MMP-2 activation after balloon catheter de-endothelialization.

Injury-induced alterations in newly synthesized sulphated proteoglycans from rabbit arterial neointima covered by regenerated endothelium.

Proteoglycan (PG) synthesis is a highly regulated, dynamic process that is known to be altered during atherogenesis. Endothelial injury, which may be the primary event in atherosclerosis, has been reported to stimulate PG synthesis and accumulation in the arterial extracellular matrix. The objective of this investigation was to study injury-induced alterations in PG synthesis and accumulation in the neointima, developed in response to a selective balloon catheter de-endothelialization of aortas of normocholesterolaemic rabbits. One group of rabbit aortas was incubated with 35S-Na2SO4 for 8 h, to study in vitro the de novo synthesis of sulphated PG. Another group of rabbit aortas was used to study PG accumulated in aortic neointima vs. PG present in intima-media of normal aortas. Newly synthesized sulphated PG was characterized by light microscopic radioautography and size exclusion chromatography. Purified intimal-medial PG extracts from unlabelled aortas were analysed for protein and glycosaminoglycan (GAG) content and GAG distribution pattern. Results from this study revealed that the neointima of injured aortas synthesized sulphated PG at a significantly higher concentration than the intima of normal aortas. Size exclusion chromatography revealed that neointima synthesized higher molecular weight PG, and in a higher proportion, than its counterpart PG from normal aortas. PG accumulated in neointima of injured aortas showed a significantly altered GAG distribution pattern. These data confirm that neointima developed in response to injury synthesizes and accumulates PG which is altered compared to PG present in the intima-media of normal aorta.

Effects of inflation pressure of balloon catheter on vascular injuries and subsequent development of intimai hyperplasia in rabbit aorta

Balloon catheter de-endothelialization is the most popular means of arterial injury in experimental animals and has been used as the model system to investigate atherogenesis and restenosis after percutaneous transluminal coronary angioplasty (PTCA). The aim of this study was to examine the relationship between balloon inflation pressure and vascular damage and also subsequent intimai hyperplasia. Retrograde pullback balloon injury of rabbit aortas was made at three different balloon pressures (1.5, 1.75, and 2.0 atm). The medial injuries, such as necrosis of smooth muscle cells and disruption of elastic lamina, were occasionally found in the injured segment of the aorta by balloon catheter at 1.75 atm and more frequently at 2.0 atm. No prominent medial injury was observed in the aortic segment to balloon catheter injury at 1.5 atm; Intimal hyperplasia developed in each animal and increased with time, 2, 4, and 8 weeks after injury. The intimal hyperplasia followed by balloon injury at 1.75 and 2.0 atm was more prominent than that at 1.5 atm, however, the development of the intimal hyperplasia was not parallel to the degree of inflation pressure. On the other hand, decrease of DNA content of the media and reduction of norepinephrine-induced vasoconstriction were observed in a pressure-dependent manner after balloon injury. These findings indicate that intimal hyperplasia is not proportionally correlated to the severity of the vascular injury. The control, of inflation pressure is very important in order to examine vascular injuries, subsequent intimal hyperplasia and vasomotor responses in animal models of balloon catheter injury.

Mitogenic Factors Released from Smooth Muscle Cells Are Responsible for Neointimal Cell Proliferation after Balloon Catheter Deendothelialization

Smooth muscle cell (SMC) proliferation results in neointimal formation in response to selective deendothelialization. The neointimal SMC are characteristically different from normal, medial SMC. The major difference is that neointimal SMC have a higher proliferation rate. The high proliferation rate has been observed 15 weeks after endothelial injury. In this study, it is noted that neointimal SMC release some mitogenic factor(s) which is(are) responsible for inducing persistent SMC proliferation in an autocrine manner. The SMC were cultured from the media of normal rabbit aorta as well as the neointimal tissue formed 15 weeks after an endothelial injury. From the culture of the neointimal SMC, the conditioned medium was collected and growth factors, including PDGF-AB, TGF-β 1, TGF-β 2, and bFGF, were assayed. The conditioned medium was used to culture the medial SMC from normal rabbit aorta. The mitogenic effect of the conditioned medium was evaluated by the incorporation of [3H]thymidine into the SMC. Results demonstrated that PDGF-AB and TGF-β 1 were increased in neointimal SMC-conditioned medium. After incubation with the conditioned medium, medial SMC incorporated significantly higher [3H]thymidine, compared to incubation with control medium ( P ≤ 0.01). The data indicate that endothelial injury induces production of some growth factors, including PDGF and TGF-β, by the neointimal SMC. These growth factors may act in an autocrine manner to stimulate SMC proliferation for a long time following a single deendothelialization.

Enhanced incorporation of [ 14C]glucosamine into glycosaminoglycans of aortic neointima of balloon-injured and cholesterol-fed rabbits in vitro

Glycosaminoglycans (GAG), which form the elementary constituent of extracellular matrix proteoglycans (PG), are implicated in the pathogenesis of atherosclerosis, mainly due to their lipoprotein binding capability and their abundance in a developing lesion during atherogenesis. However, the reasons for the increment of GAG content are poorly understood. In the present study, the influence of two well known atherogenic factors on arterial GAG synthesis were examined by estimating the incorporation of [ 14C]glucosamine into aortic GAG in an in vitro incubation system. Radioactivity associated with GAG was taken to represent their synthesis. GAG synthesis by neointimal tissue of rabbit aortas, 12 weeks following balloon catheter deendothelialization was measured and compared in rabbits fed a normal or 0.25% cholesterol supplemented diet for the preceding 6 weeks. In normolipaemic rabbits synthesis was found to be 12 438 ± 173, 17 884 ± 1390 and 15 960 ± 1355 dpm/mg dry defatted tissue from uninjured (control), deendothelialized (DEA) and reendothelialized (REA) areas of rabbit aortas, respectively. This incorporation of radioactivity was significantly greater in hypercholesterolaemic rabbits corresponding to 13 426 ± 239, 32 670 ± 3077 and 27 496 ± 3287 in the control, DEA and REA, respectively. The results demonstrated a synergistic effect of cholesterol feeding and arterial endothelial denudation in stimulating GAG synthesis. Although GAG synthesis was found to be stimulated by either cholesterol feeding or arterial injury, the stimulation by'cholesterol feeding alone was only marginal. Further, results show a much higher retention of newly synthesized GAG by the tissue from REA. This study provided a possible explanation for increased GAG content in a developing proliferative lesion.

Effects of angiotensin converting enzyme inhibition with cilazapril on intimal hyperplasia in injured arteries and vascular grafts in the baboon.

To determine the importance of angiotensin converting enzyme (ACE) activity in the development of arterial proliferative lesions in a primate model, the response to vascular injury was studied in five baboons treated with oral cilazapril (20 mg/kg/day) and in five untreated control animals. Each animal underwent three procedures: 1) carotid artery endarterectomy, 2) balloon catheter deendothelialization of the superficial femoral artery, and 3) surgical placement of bilateral aorto-iliac expanded polytetrafluoroethylene (Gore-Tex) vascular grafts. Cilazapril therapy was initiated 1 week preoperatively and continued throughout the study interval. At 1 and 3 weeks postoperatively, plasma ACE activity was inhibited by more than 96% versus control values. After animals were killed at 3 months, injured vessel and graft segments were evaluated morphometrically. Although the response between animals was variable, average cross-sectional areas of neointima did not differ between the cilazapril-treated and control groups at sites of carotid endarterectomy (0.26 +/- 0.12 versus 0.34 +/- 0.17 mm2, respectively; p greater than 0.5), femoral artery ballooning (0.15 +/- 0.08 versus 0.11 +/- 0.01 mm2; p greater than 0.5), or at graft anastomoses (1.86 +/- 0.50 versus 1.72 +/- 0.50 mm2; p greater than 0.5). Thus, cilazapril did not reduce intimal thickening over 3 months in these primate arterial injury models. However, a possible beneficial effect of cilazapril, which might be apparent at earlier time points or with larger animal groups, cannot be excluded.

Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF.

Approximately 30 to 40 percent of atherosclerotic coronary arteries treated by angioplasty or by bypass surgery occlude as a result of restenosis. This restenosis is due principally to the accumulation of neointimal smooth muscle cells, which is also a prominent feature of the advanced lesions of atherosclerosis. The factors responsible for the accumulation of intimal smooth muscle cells have not been identified. Platelet-derived growth factor (PDGF) is a potent smooth muscle chemoattractant and mitogen. It is present in platelets and can be formed by endothelium, smooth muscle, and monocyte-derived macrophages. The development of an intimal lesion in the carotid artery of athymic nude rats induced by intraarterial balloon catheter deendothelialization was inhibited by a polyclonal antibody to PDGF. These data demonstrate that endogenous PDGF is involved in the accumulation of neointimal smooth muscle cells associated with balloon injury and may be involved in restenosis after angioplasty, and perhaps in atherogenesis as well.

Intimal hyperplasia is reduced by ornithine decarboxylase inhibition

Polyamines are intracellular cations that are thought to play a role in the regulation of cell growth and differentiation. This study was undertaken to determine if inhibition of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis, suppresses formation of intimal hyperplasia (IH) after arterial injury. Twenty New Zealand white rabbits underwent balloon catheter deendothelialization of a common carotid artery. Treated animals ( n = 10) were given α-difluoromethylornithine (DFMO), an inhibitor of ODC, ad lib in drinking water as a 2% solution. DFMO was begun 3 days prior to surgery and continued until vessel harvest. Vessels were perfusion-fixed at harvest, 2 ( n = 10) and 4 ( n = 10) weeks postoperatively. All arteries remained patent. There were no histologic differences in the IH between treated and untreated animals. The intima and media surface areas on serial arterial cross sections were determined using computer-assisted planimetry. There was a significant difference in the IH surface area of injured arteries between untreated and DFMO-treated animals at both 2 (17.6 ± 2.0 vs 3.0 ± 1.6 μm 2; P ⩽ 0.001) and 4 weeks (27.4 ± 5.6 vs 7.1 ± 1.8 μm 2; P ⩽ 0.008). No differences were seen in medial thickness. We conclude that ODC inhibition reduces early development of IH after arterial deendothelialization. These data support the hypothesis that polyamines may be cellular messengers involved in the regulation of IH formation.

Experimental determination and mathematical model of the transient incorporation of cholesterol in the arterial wall

Experimental data of the radial incorporation of labeled cholesterol [14C-4] into the artery wall is regressed against a mathematical model that predicts macromolecular transport in this biological system. Data is obtained using excised canine carotid arteries that are perfused in vitro under pulsatile hemodynamic conditions for 2 hr. Vessels are exposed to either normotensive hemodynamics, hypertensive hemodynamics, or simulations in which the rate of flow or vessel compliance is deliberately altered. Several arteries are studied under normotensive conditions following balloon catheter deendothelialization. Transmural concentration profiles of [14C-4] activity are determined by microcryotomy of longitudinal sections of perfused vessels. Nonlinear Marquardt regression on 12 experimental cases yields parameter estimates of effective diffusivity, D and solute filtration velocity, V. Results of this experimental investigation support our hypothesis that hemodynamics and the endothelial lining influence wall flux in intact vessels. Exposure to altered (vs normotensive) hemodynamics is associated with increased incorporation of labeled cholesterol. A similar observation is made for deendothelialized vessels (e.g. a greater accumulation of label and a rise in convective flux). Based upon our companion measurements of vessel wall forces and endothelial cellular morphology accompanying hemodynamic simulations, we suggest that hemodynamically induced alterations to endothelial structures lead to the increased permeability, convection and incorporation that we observe in this work.

Differential effect of nicotinic acid derivatives on smooth muscle and endothelial cell proliferation.

The pathogenesis of atherosclerosis is a multifactorial process. A possible anti-atherosclerotic drug should therefore interfere with different targets that are important during the development of an atherosclerotic lesion. Two of the early events are the activated migration and proliferation of arterial smooth muscle cells. Here we investigated in several in vivo and in vitro experiments the effect of two nicotinic acid derivatives L44 and L44-0, on smooth muscle cell migration and proliferation. Balloon catheter de-endothelialization was used as an animal model for intimal lesion formation. Migration was subsequently quantified in vitro using the explant outgrowth technique. Subcultured smooth muscle and endothelial cells were used to test the effect of the drugs on proliferation. Time-lapse video microscopy was applied to differentiate between smooth muscle cell migration and proliferation on the level of individual cells. We showed that L44 and L44-0 are very effective in decreasing smooth muscle cell proliferation and migration. Endothelial cell proliferation, important to re-establish endothelial integrity was, however, not affected.

Extraction and analysis of glycosaminoglycans from intima-media of single rabbit aortae: effect of balloon catheter de-endothelialization on the content and profile of glycosaminoglycans

A novel procedure is described for extracting, and simultaneously 3H-labelling, glycosaminoglycans from the intima-media of a single rabbit aorta. The procedure was used to compare contents and types of glycosaminoglycans isolated from uninjured (control) aortae, and partially re-endothelialized aortae at 11 weeks after de-endothelialization by a balloon catheter. Briefly, the isolated delipidated tissue was digested in 0.8 M NaOH containing NaB 3H 4 at room temperature. The neutralized digest was then degraded by a non-specific proteinase during dialysis. The 3H-labelled glycosaminoglycan fraction was recovered after a gel filtration step. The yield (10.5 μg of glycosaminoglycan/mg of dry, delipidated tissue) was within the range reported previously for rabbit aorta. Although the de-endothelialized (DEA) and re-endothelialized areas (REA) of all injured aortae contained a significantly thickened intima, the glycosaminoglycan concentration (DEA, 11.2 μg/mg; REA, 11.6 μg/mg) did not differ significantly from that of control aorta. The profile of [ 3H]glycosaminoglycan types was determined by the serial use of glycosaminoglycan-selective methods: > 86% of 3H was released as small molecular weight products by this analytical scheme. The glycosaminoglycan profile for control tissue compared well with several previous reports. Compared with control aortae, both DEA and REA contained relatively less chondroitin sulphate, whereas DEA contained more hyaluronic acid and REA contained more heparan sulphate. Future use of this procedure will improve measurement of the changes to the extracellular matrix which take place after injury to the vessel wall and which may precede atherogenesis.

Accumulation of native and methylated low density lipoproteins by healing rabbit arterial wall.

To determine whether healing arterial wall accumulation of low density lipoproteins (LDL) is mediated by the high affinity LDL receptor, normocholesterolemic rabbits were injected with 125I-LDL, 99mTc-LDL, or the reductively methylated analogs of these compounds (125I-MeLDL, 99mTc-MeLDL), 1 month after balloon catheter deendothelialization of the abdominal aorta. If the mechanism of accumulation requires interaction with the LDL receptor, reductively methylated lipoproteins which do not bind to the receptor should not accumulate in healing arterial wall. Twenty-four hours after injection of labelled lipoproteins, each animal was injected with Evans blue dye, in order to distinguish reendothelialized from deendothelialized aorta. One hour after dye injection, the aorta was fixed, removed, divided into abdominal (ballooned) and thoracic (unballooned) regions and counted. For all lipoprotein preparations, there were three to four times as many counts in the abdominal as in the thoracic aorta. En face autoradiographs were made of the aortas that had been exposed to 125I-labelled lipoproteins. In the autoradiographs, the areas of the lowest activity corresponded to the centers of healing endothelial islands. The most intense radioactivity for both lipoproteins occurred in the region of the leading edge of the endothelial islands where active endothelial regeneration was in progress. The overall distribution of native and MeLDL accumulation was the same. The results suggest that low density lipoproteins are accumulated in areas of active endothelial regeneration by a mechanism that does not involve the high affinity LDL receptor.

Kinetics of low density lipoprotein interactions with rabbit aortic wall following balloon catheter deendothelialization.

The kinetics of the interaction of circulating lipoproteins with the balloon catheter-injured aortic wall of rabbits were investigated. The fate of radioiodinated low density lipoprotein (LDL) was followed up to 48 hours after intravenous injection and compared with radioiodinated albumin injected simultaneously. The accumulation of LDL was calculated from the radioactivity in the aortic tissue divided by the average specific activity, measured from the area below the plasma radioactivity versus the time curve, and expressed as cpm/ml/hr. It was observed that the aortic area of endothelial regeneration (AER), which is relatively impermeable to Evans blue dye and shows very little albumin concentration, accumulated considerably more LDL than control (uninjured) tissues. Furthermore, removal of labeled lipoprotein from the intima during a 24-hour efflux period following the initial 48-hour loading period indicated that there was greater removal from normal and deendothelialized aortic (DEA) tissues than from neointima covered by endothelium. These findings are consistent with previous observations indicating changes in the morphological and chemical composition of AER. There is evidence that greater neointimal lipid accumulation in response to endothelial injury parallels the concentration of glycosaminoglycans (GAG). The relatively increased accumulation of label in AER, while plasma-specific activity is rapidly decreasing, indicates that there may be an ionic interaction of GAG with LDL in the AER.

Lipid composition of rabbit aortic wall following removal of endothelium by balloon catheter.

To determine the lipid content of the neointima formed following balloon catheter deendothelialization, we measured the chemical composition of intimal medial samples of the aortic wall of rabbits at 6, 12, and 24 months after the operation. The areas of endothelial regrowth and the denuded areas were outlined by intravenous injection of Evan's blue dye. Lipids were extracted by chloroform/methanol (2:1, vol/vol) separated by thin-layer chromatography, eluted, and estimated by gas-liquid chromatography. No tendency to regression was observed. By contrast we observed a progressive increase over time in the concentration of most lipid classes in the areas of endothelial regrowth. There is evidence indicating that the increase in the lipid concentration parallels the increase in the glycosaminoglycan content in the areas of endothelial regrowth. It is possible that endothelial injury causes some irreversible changes in the composition of the neointima in areas of endothelial regeneration.

Selective accumulation of low density lipoproteins in damaged arterial wall.

To determine whether damaged arterial wall selectively accumulates lipoproteins, normocholesterolemic rabbits were injected with human radiolabeled low density lipoproteins, high density lipoproteins, and/or albumin 24 hr to 12 weeks after balloon-catheter de-endothelialization of the abdominal aorta. When 125I-labeled low density lipoproteins and 99mTc-labeled albumin were injected simultaneously, the amount of 125I-low density lipoprotein present 24 hr later in abdominal aortas increased steadily, for several weeks, above the amount present at 24 hr in control animals. The increase correlated closely with the degree of re-endothelialization and correlated closely with the degree of re-endothelialization and reached an average maximum for the whole abdominal aorta of three times control when re-endothelialization was between 75 and 85% complete. By contrast, the amounts of 99mTc-albumin or 125I-labeled high density lipoprotein in balloon-damaged abdominal aortas, and the amounts of 125I-low density lipoprotein, 125I-high density lipoprotein, or 99mTc-albumin in undamaged thoracic aortas of injured animals showed no such increase. As early as 2 weeks after de-endothelialization, en face radioautographs made following injection of 125I-labeled low density lipoproteins revealed localized areas of greatest radioactivity around the leading edges of regenerating endothelial islands, broad areas of intermediate radioactivity corresponding to the de-endothelialized areas, and very like radioactivity in the re-endothelialized areas. This pattern occurred rarely with 125I-labeled high density lipoproteins and not at all with 125I-labeled albumin. The results suggest that low density lipoproteins are selectively accumulated by the healing rabbit aorta and that the accumulation is greatest in regions where the endothelium is actively regenerating.