Angiotensin II administration to atherosclerotic mice increases macrophage uptake of oxidized ldl: a possible role for interleukin-6.

The goal of the present study was to elucidate mechanisms for angiotensin II (Ang II) induction of oxidized low density lipoprotein (Ox-LDL) uptake by macrophages, the hallmark of early atherosclerosis. Compared with placebo treatment, Ang II injections (0.1 mL, 10(-7) mol/L per day) for 2 weeks to apolipoprotein E-deficient mice significantly increased Ox-LDL degradation, CD36 mRNA expression, and CD36 protein expression by their peritoneal macrophages (MPMs). These effects were abolished by treatment with losartan (5 to 50 mg/kg per day) before Ang II administration. Because no such effect was obtained in vitro, the ex vivo effect of Ang II on macrophage uptake of Ox-LDL could be mediated by a factor that is not expressed at a significant level in vitro. Because Ang II stimulates cellular production of interleukin-6 (IL-6), we analyzed the possible role of IL-6 as a mediator of Ang II-mediated cellular uptake of Ox-LDL by using several approaches. First, incubations of IL-6 with MPM or IL-6 administration in mice increased macrophage Ox-LDL degradation and CD36 mRNA expression. Second, injection of IL-6 receptor antibodies in mice during Ang II treatment reduced macrophage Ox-LDL uptake and CD36 expression compared treatment with Ang II alone. Finally, Ang II treatment of IL-6-deficient mice did not affect their MPM Ox-LDL uptake and CD36 protein levels. Thus, we conclude that a novel mechanism for Ang II atherogenicity, related to macrophage cholesterol accumulation and foam cell formation, may involve its stimulatory effect on macrophage uptake of Ox-LDL, a process mediated byIL-6.

The postnatal management of congenital cystic adenomatoid malformation.

BACKGROUND: Routine prenatal ultrasound has increased the frequency of prenatal diagnosis of congenital cystic lung malformation, such as cystic adenomatoid malformation, pulmonary sequestration, congenital lobar emphysema, and bronchogenic cyst. OBJECTIVES: To evaluate the methods of postnatal diagnosis, the optimal age for operation since surgery is always required, and the optimal extent of lung resection. METHODS: The clinical courses of 11 patients with congenital lung cysts who underwent surgical lung resection (8 lobectomies and 3 segmentectomies) were reviewed. RESULTS: The diagnosis was confirmed by computed tomography scan in all. In nine patients the diagnosis was made prenatally. Chest X-ray was normal postnatally in all patients except for two who had recurrent pneumonia. Postoperative follow-up showed excellent recovery in all operated children. One patient who underwent surgery for CCAM following episodes of severe pneumonia died from another cause 5 months later. Postoperative chest CT scan showed no residual disease in eight patients. In two who had undergone limited resection, tomography showed a small segment of residual disease in one and a suspected residual lesion in the other. CONCLUSION: With prenatal ultrasound the true frequency of congenital cystic lung anomaly appears to be higher than previously reported. Postnatal CT is mandatory to confirm or to rule out the diagnosis. The mere presence of cystic lung malformation is an indication for surgery. Complete removal of the affected lung lobe is recommended. Segmental resection may be inadequate. Early operation is tolerated well by infants and small children and we recommend that surgery be performed in children between 6 and 12 months of age.

Cecal diverticulitis: a diagnostic challenge.

BACKGROUND: Cecal diverticulitis is frequently indistinguishable from acute appendicitis preoperatively and is sometimes mistaken for carcinoma at laparotomy. The surgeon must be aware of the possibility of diverticulitis of the cecum in the operating room and choose the appropriate treatment. PURPOSE: Because there is no universal therapeutic approach to these patients, we decided to assess the presenting symptoms, clinical findings, preoperative diagnosis, operative findings determining the proper management of these patients. METHODS: A retrospective chart review of 13 patients with pathologically confirmed cecal diverticulitis, who underwent surgery in our department from 1984 to 1998, was undertaken. RESULTS: The mean age of patients was 43.5 years. Right lower quadrant pain and local tenderness were the only clinical findings in 92.3%, with preoperative diagnosis of acute appendicitis in 84.6% of patients. The operative finding in most cases was inflammatory mass of the cecum; in 6 cases it was indistinguishable from perforated cecal carcinoma. Six patients underwent right hemicolectomy, 5 had ileocecectomy, 1 patient was treated by tube cecostomy, and 1 had diverticulectomy. There were three minor postoperative complications: pneumonia, wound infection and lower limb superficial thrombophlebitis. CONCLUSIONS: Cecal diverticulitis needs a high index of suspicion for achieving a preoperative diagnosis. We suggest that the operative therapy should be ileocecectomy. The surgical specimen should be examined during surgery and only if carcinoma is found should the patient have a formal colectomy.

Losartan inhibits cellular uptake of oxidized LDL by monocyte-macrophages from hypercholesterolemic patients.

Angiotensin II (Ang II) and oxidized LDL (Ox-LDL) are risk factors for atherosclerosis, and both of them contribute to macrophage cholesterol accumulation, the hallmark of early atherosclerosis. As Ang II was shown to increase macrophage uptake of Ox-LDL, we investigated the effect of losartan, an Ang II receptor antagonist with antiatherogenic properties, on the cellular uptake of Ox-LDL by human monocyte-derived macrophages (HMDM) from hypercholesterolemic patients. Eight normotensive hypercholesterolemic patients were treated with losartan (50 mg/day) for a period of 4 weeks. Losartan therapy did not significantly affect the degradation of native LDL by the patients' HMDM. However, losartan therapy significantly reduced HMDM uptake of Ox-LDL as shown by a 78% reduction in Ox-LDL cell-association and a 21% reduction in Ox-LDL degradation. CD36 (an Ox-LDL receptor) mRNA expression in HMDM obtained after losartan treatment was decreased by 54% compared to HMDM obtained before treatment. The ability of losartan to inhibit HMDM CD36 mRNA expression and, hence, Ox-LDL uptake and macrophage foam cell formation is probably related to the blockage of Ang II binding to the cell surface and thus to the prevention of Ang II atherogenic effects.

Tumor immunity in perforin-deficient mice: a role for CD95 (Fas/APO-1).

CTL and NK cells use two distinct cytocidal pathways: 1) perforin and granzyme based and 2) CD95L/CD95 mediated. The former requires perforin expression by the effectors (CTL or NK), whereas the latter requires CD95 (Fas/APO-1) expression by the target. We have investigated how these two factors contribute to tumor immune surveillance by studying the immunity of perforin-deficient mice against the progressor C57BL/6 Lewis lung carcinoma 3LL, which expresses no CD95 when cultured in vitro. Unexpectedly, the results indicated that the perforin-independent CD95L/CD95 pathway of CTL/NK plays a role in acting against D122 and Kb39.5 (39.5) high and low metastatic sublines, respectively, derived from the 3LL tumor. Although no membrane-bound CD95 was detected on cultured D122 and 39. 5 cells, surface CD95 expression on both D122 and 39.5 was considerably up-regulated when the tumors were grown in vivo. A similarly enhanced expression of CD95 was observed with three additional tumors; LF-, BW, and P815, injected into syngeneic and allogeneic mice. The finding of up-regulated CD95 expression on tumor cells placed in vivo suggests that a CD95-based mechanism plays a role in tumor immunity at early stages of tumor growth. Consequently, the progressive down-regulation of CD95 expression during tumor progression may indeed be an escape mechanism as previously reported. Together, these results suggest a role for CD95-dependent, perforin-independent immunity against certain tumors.

Attenuation of atherosclerosis in apolipoprotein E-deficient mice by ramipril is dissociated from its antihypertensive effect and from potentiation of bradykinin.

We investigated the mechanism of the antiatherosclerotic effect of the angiotensin-converting enzyme (ACE) inhibitor, ramipril, in the apolipoprotein (apo) E-deficient mice. Mice that received a high dose (5 mg/kg/day) of ramipril supplemented in their drinking water for 10 weeks showed reduced aortic lesion size by 75% compared with placebo-treated mice. At this dosage, ramipril significantly reduced blood pressure from 95+/-5 mm Hg before treatment to 68+/-4 mm Hg at the end of the treatment period. Ramipril also increased the resistance of the mouse low-density lipoprotein (LDL) to CuSO4-induced oxidation, as shown by a prolongation of the lag time required for the initiation of LDL oxidation from 90 min in the placebo-treated mice to >180 min in the ramipril-treated mice. Similarly, a reduction in the maximal LDL-associated conjugated dienes after 180 min of oxidation by 250% in comparison with placebo-treated mice was noted. Ramipril (1 mg/kg/day) that was still adequate to reduce their plasma ACE activity and LDL propensity to lipid peroxidation was insufficient to reduce their blood pressure. This dosage also inhibited the progression of atherosclerosis in the apo E-deficient mice by 74%. The contribution of bradykinin potentiation to the ACE-inhibitor action was assessed by cotreatment of ramipril with the bradykinin B2-receptor antagonist, icatibant (HOE-140, 0.5 mg/kg given subcutaneously twice a day) for a period of 10 weeks. HOE-140 had no effects on ACE activity, LDL lipid peroxidation, blood pressure, or atherosclerosis. In combination with ramipril, no additional effect of HOE-140 on LDL oxidation or on atherosclerosis was noted in comparison with ramipril treatment alone. We thus conclude that the antiatherogenic effect of ramipril in E(0) mice is independent of blood pressure reduction and is not mediated by bradykinin. It seems, therefore, that most of its antiatherosclerotic and antioxidative effects are mediated through the inhibition of angiotensin II production.

Angiotensin II atherogenicity in apolipoprotein E deficient mice is associated with increased cellular cholesterol biosynthesis.

Angiotensin II (Ang II) was shown to be an important risk factor for accelerated atherosclerosis. Inhibition of Ang II action on the arterial wall by blocking its production with angiotensin converting enzyme (ACE) inhibitors, or by blocking binding to its receptors on cells with antagonists was shown to attenuate atherogenesis in animal model of atherosclerosis. We questioned whether Ang II atherogenicity is related to a stimulatory effect of Ang II on macrophage cholesterol biosynthesis. Angiotensin II injected intraperitoneally once a day (0.1 ml of 10(-7) M per mouse) for a period of 30 days, to the apolipoprotein E deficient mice increased the atherosclerotic lesion area by 95% (P < 0.01 vs. control), compared to placebo-injected mice, with no significant effect on blood pressure or on plasma cholesterol levels. On using mouse peritoneal macrophages (MPMs) that were harvested after intraperitoneally injection of Ang II, an increased rate of cellular cholesterol biosynthesis (measured as incorporation of [3H]acetate into cholesterol) by up to 90% (P < 0.01 vs. control) was observed. In mice treated with the ACE inhibitor, Fosinopril (25 mg/kg per day) a reduction in their MPM's cholesterol synthesis by up to 70% (P < 0.01 vs. control) was obtained. In vitro studies in human monocyte-derived macrophages (HMDM), in MPMs from control BALB/c mice, and in J-774 A.1 macrophage-like cell line demonstrated up to 44, 34 and 30% stimulation of macrophage cholesterol biosynthesis, respectively, following cell incubation with 10(-7) M Ang II for 18 h at 37 degrees C. The stimulatory effect of Ang II on macrophage cholesterol biosynthesis could be related to its interaction with the macrophage AT1 receptor, as Losartan (10(-5) M), an AT1 blocker, but not PD 123319 (10(-5) M), an AT2 blocker, prevented the stimulatory effect on macrophage cholesterol synthesis. Furthermore, in cells that lack the AT1 receptor (RAW macrophages), Ang II did not increase cellular cholesterol synthesis. Ang II increased macrophage 3-hydroxy-3-methyl glutaryl CoA (HMG CoA) reductase mRNA levels in a dose dependent manner in J-774 A.1 macrophages and in MPM. Losartan, the AT1 receptor antagonist clearly attenuated this mRNA induction. We thus conclude that Ang II stimulation of macrophage cholesterol biosynthesis is related to its interaction with the AT1 receptor, followed by stimulation of macrophage HMG CoA reductase gene expression, which leads to increased cellular cholesterol biosynthesis, and can possibly result in macrophage cholesterol accumulation and foam cell formation.

The angiotensin-converting enzyme inhibitor, fosinopril, and the angiotensin II receptor antagonist, losartan, inhibit LDL oxidation and attenuate atherosclerosis independent of lowering blood pressure in apolipoprotein E deficient mice.

OBJECTIVE: To investigate the possible mechanisms of the antiatherosclerotic effects of the angiotensin-converting enzyme (ACE) inhibitor, fosinopril, in apolipoprotein (apo) E deficient mice. METHODS: Apo E deficient (E0) mice at the age of 8 weeks received either placebo or a high dose (25 mg/kg/d) of fosinopril supplemented in their drinking water. RESULTS: After 12 weeks of treatment, fosinopril reduced the aortic lesion size by 70%, compared with the placebo group. At this dosage, fosinopril significantly reduced blood pressure from 93 +/- 2 mmHg before treatment to 70 +/- 2 mmHg at the end of the treatment period (P < 0.005). Fosinopril also increased the resistance of the mice plasma low density lipoprotein (LDL) to CuSO4-induced oxidation, as shown by a 90% reduction in the LDL content of malondialdehyde (MDA) and also by a prolongation of the lag time required for the initiation of LDL oxidation (from 100 min in the placebo-treated mice to more than 240 min in the fosinopril-treated mice; P < 0.001). In addition, fosinopril inhibited CuSO4-induced oxidation of LDL that was obtained from the aortas of the treated mice, as shown by an 18% and 37% reduction in the LDL content of lipid peroxides and hydroperoxy-cholesterol linoleate, respectively, compared with the placebo-treated mice (P < 0.01). A low dosage of fosinopril (5 mg/kg/d) that was still adequate to reduce their plasma ACE activity and LDL propensity to lipid peroxidation was insufficient to lower their blood pressure. This dosage also reduced the aortic lesion size in the apo E deficient mice by 40% (P < 0.01). CONCLUSIONS: The antiatherogenic effects of fosinopril in apo E deficient mice are due not only to blood pressure reduction but also to the direct inhibition of angiotensin II-dependent effects, which are probably also associated with the inhibition of LDL oxidation.

Angiotensin, LDL peroxidation and atherosclerosis.

Hypertension is a known risk factor for the development of atherosclerosis. However, in most of the studies, no effect of blood pressure reduction was demonstrated on the incidence of coronary artery disease, except in the SHEP study in which it was shown that in older persons, with isolated systolic hypertension, antihypertensive stepped-care drug treatment reduced the incidence of total stroke and major cardiovascular event. In hypertensive patients with elevated plasma renin activity, a 5-fold increased incidence of myocardial infarction was demonstrated. As oxidation of low density lipoprotein (LDL) was suggested to be a major risk factor for atherosclerosis, we studied the relationship between hypertension and LDL oxidation. We demonstrated increased propensity of LDL obtained from hypertensive patients to oxidative modification, in comparison with LDL obtained from normotensive subjects and suggested that angiotensin II (Ang-II) may be involved in this effect. Ang-II was shown to enhance macrophage lipid peroxidation both in vivo and in vitro. This effect was dose-dependent and involved the binding of Ang-II to its receptor on the macrophage surface. In addition, these lipid peroxidized Ang-II-treated macrophages could substantially oxidize LDL. Ang-II was shown to possess additional atherogenic properties such as increasing the activity of the macrophage oxidized LDL receptors. It also binds to LDL, thus leading to the formation of a modified lipoprotein, which is taken up by macrophages at enhanced rate through the scavenger receptor. Inhibition of Ang-II formation by angiotensin converting enzyme inhibitors reduced LDL peroxidation in hypertensive patients as well as in the atherosclerotic apo E deficient mice. The reduction in LDL peroxidation in these mice was accompanied by a 70-90% reduction in the atherosclerotic lesion area. A similar effect in these mice was demonstrated with the Ang-II receptor antagonist, Losartan. Thus, we suggest that Ang-II is involved in the development of atherogenesis in hypertensive patients and inhibition of Ang-II formation or prevention of its interaction with its receptor may attenuate the atherosclerotic process.

Antiatherosclerotic and antioxidative effects of captopril in apolipoprotein E-deficient mice.

The effect of the angiotensin-converting enzyme (ACE) inhibitor, captopril, on the development of atherosclerosis was determined in the apolipoprotein (apo) E-deficient mice. These mice develop severe hypercholesterolemia and extensive atherosclerotic lesions on chow diet, similar to those found in humans. Furthermore, in these mice, accelerated atherosclerosis is associated with increased plasma lipid peroxidation, a phenomenon that may play a crucial role in the buildup of the atherosclerotic lesions. Mice received either placebo or 50 mg/kg/day of captopril. After 12 weeks of treatment, captopril reduced the aortic-lesion area by 70% compared with that of the placebo-treated group. Captopril also increased the resistance of low-density lipoprotein (LDL) to CuSO4-induced oxidative stress, as shown by a significant reduction in the LDL content of malondialdehyde (MDA) by 30%, as well as by the prolongation of the lag time required for LDL oxidation from 55 min in the placebo-treated mice to 70 min in the captopril-treated mice, and reduction of the maximum LDL oxidation at 150 min by 35%. In vitro studies demonstrated that preincubation of LDL with captopril, inhibited the onset of CuSO4-induced LDL peroxidation up to 120 min, and reduced the LDL content of MDA by 90%. We conclude that captopril attenuates atherosclerosis in the apo E-deficient mice, and this phenomenon may be related to its inhibitory effect on the plasma LDL oxidation.

Interactions of platelets, macrophages, and lipoproteins in hypercholesterolemia: antiatherogenic effects of HMG-CoA reductase inhibitor therapy.

To assess the effect of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors on plasma cholesterol concentrations and on platelet aggregation, lovastatin or fluvastatin, 40 mg daily, was given to hypercholesterolemic patients. After 24 weeks, plasma low-density lipoprotein (LDL) cholesterol concentrations were reduced by 37% after lovastatin therapy and 29% after fluvastatin therapy. The platelet cholesterol/phospholipid ratio was reduced by 33% and 26%, respectively. Platelet aggregation was significantly reduced by 12-15% (p < 0.01) after 4 weeks of therapy with either agent. Lovastatin or fluvastatin therapy reduced platelet aggregation through an in vivo hypocholesterolemic action on the platelet cholesterol content and also through a direct effect on platelet function, as a result of drug binding to the platelets. We also studied the effect of these HMG-CoA reductase inhibitors on LDL susceptibility to oxidation. LDL oxidation (induced by copper ions) was reduced by 31% after lovastatin therapy and by 37% after fluvastatin therapy. The inhibitory effect of HMG-CoA reductase inhibitors on LDL oxidation involved their stimulatory effect on the removal of LDL from the circulation and a direct binding effect of the drugs to the lipoprotein. Because HMG-CoA reductase inhibitors can inhibit platelet aggregation, macrophage foam cell formation, and LDL oxidation, major contributors to atherogenesis, the use of these drugs can significantly attenuate the atherosclerotic process.

[Acute gastroenteritis caused by enterohemorrhagic E. coli O157:H7].

We report a 48-year-old man admitted for watery diarrhea, high fever, chills and abdominal cramps. Enterohemorrhagic E. coli O157:H7 was isolated. This new, dangerous pathogen causes dysentery and complications such as hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. These complications can cause renal failure, neurological deficit and death. Recognition of E. coli O157:H7 infection is important since it causes a rare and dangerous condition. To the best of our knowledge this is the first case reported in Israel.

Angiotensin II injection into mice increases the uptake of oxidized LDL by their macrophages via a proteoglycan-mediated pathway.

Angiotensin II (Ang-II) has been shown to possess several atherogenic properties including its ability to induce macrophage-mediated oxidation of LDL and to form Ang-II-modified LDL which is taken up by macrophages at enhanced rate. Oxidized-LDL (Ox-LDL) is also taken up by macrophages at enhanced rate via several scavenger receptors, leading to macrophage cholesterol accumulation. In the present study we examined the effect of Ang-II on the uptake of Ox-LDL by peritoneal macrophages derived from Balb/c mice (MPM). Intraperitoneal injection of Ang-II (10(-7) M, once daily for a period of 2 days) to the mice resulted in an increased Ox-LDL uptake up to 60%, in comparison to macrophages from placebo-treated mice. Similar results were obtained when Ang-II (10(-7) M) was injected to the mice twice a week for a period of three months. This Ox-LDL uptake was Ang-II dose-dependent. The cellular uptake of acetylated-LDL (Ac-LDL), another ligand for scavenger receptors, however, was not affected by Ang-II injection to the mice. Furthermore, preincubation of the MPM with the monoclonal antibody, anti CD36, reduced macrophage uptake of Ox-LDL in Ang-II-treated mice by only 11%. Ang-II administration to mice resulted in a 60% increase in the macrophage cellular proteoglycan content. Chondroitinase treatment of MPM decreased Ox-LDL cellular uptake by 20% and by 38% in placebo-treated and Ang-II-treated cells, respectively. We thus conclude that Ang-II administration to mice enhances their macrophage Ox-LDL uptake via its stimulating effect on cellular proteoglycan content and this process can lead to foam cell formation and atherosclerosis.

The angiotensin-II receptor antagonist, losartan, inhibits LDL lipid peroxidation and atherosclerosis in apolipoprotein E-deficient mice.

The potential antiatherogenic actions of the angiotensin II receptor antagonist, losartan were investigated in apolipoprotein (apo) E deficient mice, an animal model with severe hypercholesterolemia and extensive atherosclerosis. In these animals accelerated atherosclerosis is associated with increased lipid peroxidation which may play a crucial role in the build up of the atherosclerotic lesions. Administration of losartan (25mg/kg/d) to the apo E deficient mice for a 3-month period increased the plasma renin activity 3.5-fold compared to the placebo group. Losartan increased the resistance of LDL to CuSO4-induced oxidative modification as shown by a significant reduction in the LDL content of malondialdehyde by 55% compared to placebo, as well as by the prolongation of the lag time required for LDL oxidation, from 60 min in the placebo-treated mice to more than 140 min in the losartan-treated mice. Losartan reduced significantly the mean atherosclerotic lesion area by 80% compared to the placebo group. We conclude that losartan inhibits LDL lipid peroxidation in the apo E deficient mice and this effect may have an important role in the attenuation of the accelerated atherosclerosis.

Severe minocycline-induced eosinophilic pneumonia: extrapulmonary manifestations and the use of in vitro immunoassays.

OBJECTIVE: To report a severe and unusual reaction to minocycline and the use of in vitro immunologic assays. CASE SUMMARY: A 46-year-old white man developed severe respiratory distress with pulmonary infiltrates on chest X-ray and eosinophilia in blood, bronchoalveolar lavage fluid, and biopsied lung tissue during exposure to minocycline. Additional manifestations included pleuropericardial effusion, liver function abnormality, and bone marrow eosinophilia. Macrophage inhibition factor and mast cell degranulation assays were positive to minocycline. DISCUSSION: The patient's manifestations were compatible with the diagnosis of eosinophilic pneumonia. After excluding other possible etiologies, minocycline was identified as the offending agent. Generalized damage was suggested by the presence of a combination of extrapulmonary manifestations previously not reported. Results of the in vitro immunologic assays supported the hypersensitivity nature of the disease and confirmed the diagnosis. CONCLUSIONS: Minocycline-induced eosinophilic pneumonia may involve extrapulmonary sites. It is suggested that in vitro immunoassays be used for confirmation of the diagnosis rather than rechallenge or invasive procedures.

Increased uptake of LDL by oxidized macrophages is the result of an initial enhanced LDL receptor activity and of a further progressive oxidation of LDL.

Iron ions were recently shown to induce cellular lipid peroxidation in macrophages, and these oxidized cells can convert native low-density lipoprotein (LDL) to oxidized LDL (Ox-LDL). The present study demonstrates that deoxycholic acid (DCA) and angiotensin II (ANG-II) can also induce oxidative modification of macrophages via metal ions independent mechanisms. Furthermore, incubation of LDL (200 micrograms of protein/ml) for 24 h at 37 degrees C with DCA, ANG-II, as well as FeSO4-induced oxidized macrophages, resulted in oxidative modification of the lipoprotein as evidenced by increased TBARS formation in LDL (by 50, 105, and 258%, respectively), decreased TNBS reactivity (by 45, 56, and 42%, respectively), and increased cellular uptake (by 60, 166, and 230%, respectively). A positive correlation (n = .88) was found between the extent of the cellular lipid peroxidation and the increment in the cellular uptake of the LDL. The oxidative modification of LDL by oxidized macrophages was found to be a progressive process. Incubation of LDL with oxidized macrophages for increasing periods of time up to 24 h resulted in progressive increment in: (1) the electrophoretic mobility of the LDL; (2) the TBARS formation in LDL; (3) the cellular uptake of LDL by the oxidized macrophages via the Ox-LDL receptor. Upon fractionation on a heparin-sepharose column of LDL that was incubated for different periods of time with oxidized macrophages, a gradual increment in the unbound LDL fraction was obtained, up to 72% after 24 h of incubation. During the first hour of LDL incubation with the oxidized macrophages a twofold increase in the cellular uptake of LDL by these cells was detected, although no significant oxidation of the lipoprotein occurred during this short time period. This effect could be attributed to an increased number of LDL receptors on the cell surface of the oxidized macrophages. In conclusion, increased uptake of LDL by oxidized macrophages results from two routes: (1) enhanced uptake via the LDL receptor due to increased LDL receptor activity; (2) lipoprotein uptake via the Ox-LDL receptors due to cellular modification of LDL. Both of these processes lead to macrophage cholesterol accumulation and foam cell formation, and thus contribute to accelerated atherosclerosis under oxidative stress.

Reduced susceptibility of low density lipoprotein (LDL) to lipid peroxidation after fluvastatin therapy is associated with the hypocholesterolemic effect of the drug and its binding to the LDL.

Increased plasma cholesterol concentration in hypercholesterolemic patients is a major risk factor for atherosclerosis. The impaired removal of plasma low density lipoprotein (LDL) in these patients results in the presence of their LDL in the plasma for a long period of time and thus can contribute to its enhanced oxidative modification. In the present study we analyzed the effect of the hypocholesterolemic drug, fluvastatin, on plasma and LDL susceptibilities to oxidation during 24 weeks of therapy. Fluvastatin therapy (40 mg/day for 24 weeks) in 10 hypercholesterolemic patients resulted in 30%, 34% and 22% decrements in plasma levels of total cholesterol, LDL cholesterol and triglycerides, respectively. This effect has been achieved after only 4 weeks of therapy. We next studied the effect of fluvastatin therapy on LDL susceptibility to oxidation in vivo and in vitro. 2.2-Azobis, 2-amidinopropane hydrochloride (AAPH, 100 mM)-induced plasma lipid peroxidation was decreased by 70% and 77% after 12 weeks and 24 weeks of fluvastatin therapy respectively. The lag time required for the initiation of CuSO4 (10 microM)-induced LDL oxidation was prolonged by 1.2- and 2.5-fold, after 12 and 24 weeks of fluvastatin therapy respectively. We next analyzed the in vitro effect of fluvastatin on plasma and LDL susceptibilities to oxidation. Preincubation of plasma or LDLs that were obtained from normal subjects with 0.1 microgram/ml of fluvastatin, caused 20% or 57% reduction in AAPH-induced lipid peroxidation, respectively. Similarly, a 1.6- and 2.7-fold prolongation of the lag time required for CuSO4-induced LDL oxidation was found following LDL incubation with 0.1 and 1.0 microgram/ml of fluvastatin, respectively. To find out possible mechanisms that contribute to this inhibitory effect of fluvastatin on LDL oxidizability, we analyzed the antioxidative properties of fluvastatin. Fluvastatin did not scavenge free radicals and did not inhibit linoleic acid peroxidation. Fluvastatin also did not act as a chelator of copper ions. However, fluvastatin was shown to specifically bind mainly to the LDL surface phospholipids and this interaction altered the lipoprotein charge as evident from the 38% decrement in the electrophoretic mobility of fluvastatin-treated LDL, in comparison to nontreated LDL. The inhibitory effect of fluvastatin therapy on LDL oxidation probably involves both its stimulatory effect on LDL removal from the circulation, as well as a direct binding effect of the drug to the lipoprotein. We thus conclude that the antiatherogenic properties of fluvastatin may not be limited to its hypocholesterolemic effect, but could also be related to its ability to reduce LDL oxidizability.

Fosinopril reduces ADP-induced platelet aggregation in hypertensive patients.

Platelets are intimately involved in atherosclerosis, and hypertension is a known risk factor for coronary artery disease. The angiotensin-converting enzyme (ACE) inhibitors were demonstrated to reduce hypertension and attenuate atherosclerosis. Because increased platelet aggregation was shown in hypertensive patients, the effect of a new ACE inhibitor, fosinopril, on platelet aggregation was studied. Fosinopril therapy (10 mg/day for 4 weeks) in 18 male hypertensive patients showed > or = 31% reduction in ADP-induced platelet aggregation. In vitro studies showed that fosinopril had similar inhibitory effect on ADP-induced platelet aggregation. No inhibitory effect could be detected with collagen as the aggregating agent. Finally, inhibition of platelet aggregation by fosinopril was less effective in platelets derived from hypertensive patients as compared with platelets derived from normal subjects. We conclude that fosinopril possesses a significant inhibitory activity on ADP-induced platelet aggregation both in vitro and in vivo.

Angiotensin II-modified LDL is taken up by macrophages via the scavenger receptor, leading to cellular cholesterol accumulation.

The incidence of myocardial infarction is significantly higher in hypertensive patients with increased plasma concentration of angiotensin (Ang) II. Ang II was shown to bind to LDL in vitro, and in the present study we showed its binding to LDL in vivo. Ang II (10(-7) mol/L) was incubated with LDL for 3 hours at 37 degrees C, followed by reseparation of the modified lipoprotein (Ang II-LDL) and its incubation with J-774 A.1 macrophages. Binding of Ang II to LDL significantly increased the lipoprotein protein degradation (by 25%) and its cell association (by 75%) compared with nontreated LDL. Unlike Ang II-LDL, both Ang I-LDL and Ang III-LDL were taken up by macrophages similar to native LDL. The lipid composition and size of Ang II-LDL were similar to those of native LDL, and it was not aggregated. Ang II-LDL was not oxidized, as the contents of malondialdehyde and peroxides were not different from those found in native LDL. On heparin-Sepharose column chromatography, Ang II-LDL was eluted in the void volume, like acetylated LDL (Ac-LDL) and unlike native LDL, which binds to heparin. The cellular degradation of Ang II-125I-labeled LDL by J-774 A.1 macrophages of Ang II-125I-labeled LDL by J-774 A.1 macrophages was studied in the presence of a 50-fold excess of nonlabeled native LDL, Ang II-LDL, Ac-LDL, or oxidized LDL (Ox-LDL). Whereas native LDL had no effect on the degradation of Ang II-125I-LDL by the macrophages, Ac-LDL, Ox-LDL, and Ang II-LDL reduced the cellular uptake of the lipoprotein by 77%, 82%, and 87%, respectively. Similarly, fucoidin but not free Ang II reduced macrophage degradation of the labeled Ang II-LDL. We conclude that Ang II can modify LDL to a form that is not oxidized or aggregated but is still taken up at an enhanced rate by macrophages via the scavenger receptor.

Lesioned low density lipoprotein in atherosclerotic apolipoprotein E-deficient transgenic mice and in humans is oxidized and aggregated.

We analyzed lesioned LDL in both atherosclerotic humans and in the apo E deficient (E degree) mice and compared its characteristics to plasma LDL. Lesioned LDL, in comparison to plasma LDL, was minimally oxidized and aggregated. Upon incubation of E degree-aortic lesions with 125[I]-labeled LDL, a time-dependent oxidation of the lipoprotein occurred as evident by a rapid and substantial elevation in LDL-associated TBARS from 0.2 to 10.3 and 14.5 nmoles of MDA equivalents/mg LDL protein after 2 and 24 hours of incubation, respectively. Only minimal LDL aggregates could be detected after 2 hours of incubation. Extensive LDL aggregation (15%), however, occurred after 24 h of incubation. Similar results were obtained on using human lesioned aortas. We conclude that both oxidation and aggregation of lesioned LDL could be the result of aortic lesioned-induced modification of the lipoprotein, and both of these modified forms of LDL can further contribute to the acceleration of the atherosclerotic process.