Robin Poston

The lipid and non-lipid effects of statins.

The 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors, more commonly known as statins, are a class of drug widely used for the treatment of hypercholesterolaemia in patients with established cardiovascular disease as well as those at high risk of developing atherosclerosis. Their predominant action is to reduce circulating levels of low-density lipoprotein (LDL) cholesterol; to a smaller degree, they also increase high-density lipoprotein (HDL) cholesterol and reduce triglyceride concentrations. In recent years, however, there has been an increasing body of evidence that their effects on lipid profile cannot fully account for their cardiovascular protective actions: their beneficial effects are too rapid to be easily explained by their relatively slow effects on atherogenesis and too large to be accounted for by their relatively small effects on plaque regression. Experimental models have revealed that statins exert a variety of other cardiovascular effects, which would be predicted to be of clinical benefit: they possess anti-inflammatory properties, as evidenced by their ability to reduce the accumulation of inflammatory cells in atherosclerotic plaques; they inhibit vascular smooth muscle cell proliferation, a key event in atherogenesis; they inhibit platelet function, thereby limiting both atherosclerosis and superadded thrombosis; and they improve vascular endothelial function, largely through augmentation of nitric oxide (NO) generation. The relative importance of the lipid- and non-lipid-related effects of the statins in the clinical situation remains the subject of much continuing research.

ICAM-1 Deficiency Reduces Atherosclerotic Lesions in Double-Knockout Mice (ApoE−/−/ICAM-1−/−) Fed a Fat or a Chow Diet

Abstract —Intercellular adhesion molecule (ICAM)-1, a major adhesion molecule, plays a critical role in the homing of leukocytes to sites of atherosclerotic lesions. However, very little is known on the role of ICAM-1 in initiating and perpetuating vascular lesions in ApoE − /− mice fed a chow or a fat diet. This study has investigated the mean aortic lesions in mice (C57BL6 background) with a single-knockout (ApoE − /− ) or double-knockout (DKO; ApoE − /− , ICAM-1 − /− ) fed a chow or a fat diet over a period of 3, 6, 15, and 20 weeks. A 3-fold reduction in lesion size was observed at all time points in DKO mice fed a chow diet. However, in DKO mice fed a fat diet, a marked reduction in the aortic lesion was observed at 3 and 15 weeks, which did not reach a significant level at 6 and 20 weeks. This study shows in essence that DKO mice are protected from developing significant lesions for up to 6 weeks when fed a chow diet and from 3 to 6 weeks when fed a fat diet. After 6 weeks, the lesion size of the DKO mice follows that of the single-knockout mice when fed a chow diet and gets to the same level in mice fed a fat diet. Plasma cholesterol levels were not altered as a result of ICAM-1 deficiency. These studies show that ICAM-1 is implicated in the formation and progression of atherosclerotic lesions.

Monocyte chemotactic protein-1 (MCP-1) accelerates the organization and resolution of venous thrombi

PURPOSE Organization, recanalization, and contraction are common to wound healing and thrombus resolution. Monocytes are essential to wound healing and are also found in venous thrombi. We measured endogenous levels of the monocyte chemotactic protein-1 (MCP-1) in naturally resolving venous thrombi and determined the effect of injecting MCP-1 into newly formed thrombus. METHODS Endogenous MCP-1 levels were estimated in rat blood, thrombi, and the adjacent vessel wall after thrombus formation, in cohorts of eight animals at 1, 7, and 14 days. In another group (n = 10), 1 microgram of MCP-1 was injected into newly formed thrombi. Carrier was injected into the thrombi of control animals (n = 10). Thrombi and adjacent vein walls were obtained for histology at 7 days. Thrombi were given an arbitrary organization score based on erythrocyte and extracellular matrix content, which was assessed by means of computerized and observer analysis. Specimen weight, thrombus area, and cellular and monocyte content were measured. RESULTS Endogenous MCP-1 increased between days 1 and 7 in the thrombus (1-day median, 1.1 ng/g wet wt; 1-day range, 0.8 to 1.4 ng/g wet wt; 7-day median, 5.4 ng/g wet wt; 7-day range, 1.5 to 7.4 ng/g wet wt; P <.0001) and vein wall (1-day median, 1.5 ng/g wet wt; 1-day range, 0.8 to 4.3 ng/g wet wt; 7-day median, 3.3 ng/g wet wt; 7-day range, 2.7 to 8.3 ng/g wet wt; P <. 05). At 14 days, thrombus was incorporated in the vein wall, and total MCP-1 levels remained high (median, 3.9 ng/g wet wt; range, 1.1 to 7.4 ng/g wet wt). Less MCP-1 was found in the thrombus than the adjacent vessel wall at day 1 (P <.05), but there was no difference at day 7. MCP-1 could not be detected in the blood. MCP-1 injection into thrombus increased the computer (P =.016) and observer (P =.004) organization scores, reduced the thrombus area (from median, 3. 4 mm(2), and range, 1.5 to 5.7 mm(2), to median, 0.2 mm(2), and range, 0.02 to 2.6 mm(2); P =.048), and increased the surrounding vessel wall monocyte content (P =.008). Specimen weights of treated animals were lower than those of control animals (P <.02). CONCLUSION Venous thrombus MCP-1 levels increase during natural resolution. MCP-1 treatment increased the organization and resolution of thrombi. MCP-1 may therefore be of therapeutic use.

The role of the chemokines MCP-1, GRO-α, IL-8 and their receptors in the adhesion of monocytic cells to human atherosclerotic plaques

Monocyte adhesion to the arterial endothelium and subsequent migration into the intima are central events in the pathogenesis of atherosclerosis. Previous experimental models have shown that chemokines can enhance monocyte–endothelial adhesion by activating monocyte integrins. Our study assesses the role of chemokines IL-8, MCP-1 and GRO-α, together with their monocyte receptors CCR2 and CXCR2 in monocyte adhesion to human atherosclerotic plaques. In an adhesion assay, a suspension of monocytic U937 cells was incubated with human atherosclerotic artery sections and the levels of endothelial adhesion were quantified. Adhesion performed in the presence of a monoclonal antibody to a chemokine, chemokine receptor or of an isotype matched control immunoglobulin, shows that antibodies to all chemokines tested, as well as their receptors, inhibit adhesion compared to the control immunoglobulins. Immunohistochemistry demonstrated the expression of MCP-1, GRO-α and their receptors in the endothelial cells and intima of all atherosclerotic lesions. These results suggest that all these chemokines and their receptors can play a role in the adhesion of monocytes to human atherosclerotic plaques. Furthermore, they suggest that these chemokine interactions provide potential targets for the therapy of atherosclerosis.

Modulation of expression of endothelial intercellular adhesion molecule-1, platelet-endothelial cell adhesion molecule-1, and vascular cell adhesion molecule-1 in aortic arch lesions of apolipoprotein E-deficient compared with wild-type mice.

Human vascular adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), platelet–endothelial cell adhesion molecule-1 (PECAM-1), and vascular cell adhesion molecule-1 (VCAM-1), are thought to play a critical role in the homing of leukocytes to sites of atherosclerotic lesions. However, very little is known about the expression of adhesion molecules in the vasculature of mice models, such as apolipoprotein E knockout (apoE−/−) mice, the lesions of which closely mimic human atherosclerotic lesions. This study has first quantitatively characterized the mean expression of endothelial adhesion molecules, lining the whole vessel intimal circumference, over a period of time (0 to 20 weeks of diet) in aortic arch lesions of male apoE-deficient compared with wild-type (C57BL/6) mice. These animals were fed a chow or a cholesterol-rich diet. ApoE−/− animals showed first an increase (at 6 weeks) and then a reduction (at 16 weeks) in the mean expression of ICAM-1 (P <0.05) and PECAM-1 (P <0.05) but not VCAM-1 levels. Such modulation of the mean expression of adhesion molecules was not observed in wild-type mice. Confirmation of immunohistochemistry results on ICAM-1 was obtained by Northern blots performed on the aortic arch of apoE and C57BL6 chow-fed mice over a period of 20 weeks. Moreover, the presence of VCAM-1 was also confirmed at the RNA level, on aortas of control and apoE mice, by reverse transcription–polymerase chain reaction. In the second part of the study, we assayed the levels of adhesion molecules, in different types of histologically defined atherosclerotic lesions, in apoE−/− animals fed for 20 weeks. All 3 adhesion molecules (ICAM-1, PECAM-1, and VCAM-1) were observed to be reduced in fibrofatty and complex lesions but not in fatty streaks or in areas without lesions. These results indicate that the expression of these adhesion molecules in apoE-deficient animals varies with the evolution of the plaque from a fatty to a fibrous stage.

Normal expression of tissue factor, thrombomodulin, and annexin V in placentas from women with antiphospholipid syndrome

OBJECTIVE Placentas from pregnancies complicated by antiphospholipid syndrome often show thromboses and infarction, which may result from aberrations in placental coagulant pathways. We tested the hypothesis that alterations in tissue factor, thrombomodulin, and annexin V expressions contribute to poor pregnancy outcome associated with antiphospholipid syndrome. STUDY DESIGN Frozen sections from random biopsy samples of the basal plates of placentas from patients with primary antiphospholipid syndrome (n = 9), patients with secondary antiphospholipid syndrome (n = 3), and gestational age-matched control subjects (n = 10) were immunostained for tissue factor, thrombomodulin, and annexin V. Intensity of immunostaining was assessed by means of quantitative image analysis. Annexin V protein expression was evaluated with Western blotting techniques. RESULTS Tissue factor was expressed in the perivascular cells of the villous vasculature. Thrombomodulin and annexin V immunostaining was localized to the syncytiotrophoblast. There were no differences in the intensity of immunostaining for tissue factor, thrombomodulin, and annexin V between placentas from women with antiphospholipid syndrome and those from control subjects. Western blot analysis of annexin V expression showed no differences between study patients and control subjects. CONCLUSION Alterations in placental coagulant pathways involving tissue factor, thrombomodulin, and annexin V do not contribute to poor pregnancy outcome associated with antiphospholipid syndrome.

Expression of nitric oxide synthase isoforms in pregnant human myometrium

1 Endogenous nitric oxide has been proposed to play a role in the control of myometrial contractility in pregnancy. In this study, the expression, localisation and regulation of nitric oxide synthase (NOS) isoforms have been examined in human pregnant myometrium and cultured human myometrial smooth muscle cells, by immunoblotting, immunohistochemistry and reverse transcription‐polymerase chain reaction. 2 Immunoblotting of extracts from freshly isolated myometrial tissue, affinity‐enriched for NOS proteins by precipitation with ADP‐sepharose, revealed expression of endothelial NOS (eNOS or NOS3) in tissues from preterm, term non‐labour and active labour at term. Inducible NOS (iNOS or NOS2) and neuronal NOS (nNOS or NOS1) proteins were not detected at any stage of pregnancy. 3 Immunohistochemical detection showed that expression of eNOS protein was restricted to the endothelium of the myometrial vasculature, with no staining detected in myometrial smooth muscle cells. 4 Messenger RNA for all three NOS isoforms was detected, although iNOS and nNOS mRNAs were detectable only with high cycle number, implying a low copy number. 5 NOS isoforms were not detectable in human myometrial smooth muscle cells cultured from term non‐labour pregnancies. Cytokine stimulation of cultured myometrial cells did not induce iNOS expression or nitrite accumulation in the culture medium, although both iNOS protein and nitrite release were detected in the human pulmonary epithelial cell line A549. 6 Levels of eNOS protein and of NOS mRNA expression were not correlated with gestational stage, suggesting that endogenously produced NO is not likely to be a modulator of myometrial tone during human pregnancy.

Functional Significance of Inducible Nitric Oxide Synthase Induction and Protein Nitration in the Thermally Injured Cutaneous Microvasculature

Increased nitric oxide (NO) production after burn injury is well established. However, there is little information relating to the reactions that occur as a consequence of NO generation under such circumstances. We have investigated the synthesis and function of NO in a rat model of local cutaneous thermal injury. We show that NO levels are elevated from 3 hours after injury with a concomitant increase in protein nitration. A selective inducible nitric oxide synthase (iNOS) inhibitor (1400W) significantly attenuated NO synthesis, protein nitration, and neutrophil accumulation in this model, but had no effect on edema formation. The results also indicate that NO synthesis and protein nitration occurred independently of neutrophil accumulation because these parameters were unaffected by depletion of circulating neutrophils. 3-Chlorotyrosine, a marker of neutrophil/myeloperoxidase-mediated protein damage was significantly increased from 1 hour after burn. Our observations provide evidence for the involvement of reactive species in the inflammatory response after burn. The use of selective iNOS inhibitors may represent a novel approach for the management of human burn injuries.

Protein nitration in cutaneous inflammation in the rat: essential role of inducible nitric oxide synthase and polymorphonuclear leukocytes.

We have examined the relationship between neutrophil accumulation, NO• production and nitrated protein levels in zymosan‐mediated inflammation in rat skin in vivo. Rats were anaesthetized and cutaneous inflammation was induced by zymosan (injected intradermally, i.d.). Experiments were carried out up to 48 h, in recovery procedures as appropriate. Assays for neutrophil accumulation (measurement of myeloperoxidase), nitric oxide (assessment of NO2−/NO3−) and nitrated proteins (detected by ELISA and Western blot) were performed in skin extracts. The results demonstrate a close temporal relationship between these parameters. Samples were assayed at 1, 4, 8, 24 and 48 h after i.d. injection of zymosan. The highest levels measured of each parameter (P<0.001 compared with vehicle) were found at 4–8 h, with a reduction towards basal levels by 24 h. Selective depletion of circulating neutrophils with anti‐neutrophil antibody abolished neutrophil accumulation and protein nitration. In addition substantially decreased NO levels were found. A selective inducible nitric oxide synthase (iNOS) inhibitor, N‐3‐aminomethyl‐benzyl‐acetamidine‐dihydrochloride (1400W) also significantly reduced neutrophil levels and NO production and substantially inhibited protein nitration. We conclude that the neutrophil leukocyte plays an essential role in the formation of iNOS‐derived NO and nitrated proteins in inflammation, in a time‐dependent and reversible manner. The NO‐derived iNOS also has a role in stimulating further neutrophil accumulation into skin. This suggests a close mechanistic coupling between neutrophils, NO production and protein nitration.

Endothelial cells play an essential role in the thermal hyperalgesia induced by nerve growth factor

Nerve growth factor (NGF) is a potent mediator of inflammatory hyperalgesia, in addition to roles in the development and maintenance of neurons. We provide evidence for the novel concept that microvascular endothelial cells play a critical primary role in NGF‐mediated events that lead to inflammatory hyperalgesia in rat skin. We show that, surprisingly, neutrophils are not directly activated by NGF, although local administration of NGF mediates thermal hyperalgesia via mechanisms involving concomitant neutrophil accumulation. The co‐administration of actinomycin D with NGF negated both neutrophil accumulation and thermal hyperalgesia, indicating the dependence of NGF on local de novo protein synthesis. More significantly, an antibody against the endothelial‐derived adhesion molecule ICAM‐1 also blocked neutrophil accumulation and thermal hyperalgesia. Finally the ability of NGF to stimulate ICAM‐1 in human cultured umbilical vein endothelial cells is shown. We propose that NGF acts primarily to activate endothelial cells and that this response is essential for the ensuing neutrophil accumulation and hyperalgesia. The findings reveal a central role of the endothelial cell in initiating NGF‐dependent inflammatory hyperalgesia and emphasize the importance of further investigations aimed at examining the feasibility of new therapeutic strategies that target NGF.