Biomedical subjects

Theo J C van Berkel

Publications and source records attributed to Theo J C van Berkel.

At least 19 recordsLinked to original sources

PubMed2006

Interruption of the Tnfrsf4/Tnfsf4 (OX40/OX40L) pathway attenuates atherogenesis in low-density lipoprotein receptor-deficient mice.

OBJECTIVE: Atherosclerosis is a chronic (auto-)inflammatory disease and T cell activation is an important factor in this process. Tnfrsf4 (OX40) and Tnfsf4 (OX40 ligand) are members of the tumor necrosis factor (TNF) and TNF receptor family and OX40/OX40L mediated signaling is important in co-activation of T cells and facilitates B-T cell interaction. In this study we assessed the role of the OX40/OX40L pathway in atherosclerosis and the effect of interruption of the OX40/OX40L pathway on lesion development. METHODS AND RESULTS: We treated low-density lipoprotein receptor-deficient (LDLr-/-) mice with an anti-OX40L antibody which lead to a 53% decrease in atherosclerotic lesion formation. Treatment resulted in inhibition of Th2 mediated isotype switching by decreasing interleukin (IL)-4 secretion and subsequent low IgG1 serum levels against oxLDL, whereas protective anti-oxLDL specific IgM titers were increased in treated mice compared with control. CONCLUSIONS: We conclude that blocking the OX-40/OX40L interaction reduced atherogenesis by inhibition of IL-4 mediated Th2 induced isotype switching and subsequent increased levels of anti-oxLDL IgM.

Animals↗

PubMed2006

Vascular endothelial growth factor-A induces plaque expansion in ApoE knock-out mice by promoting de novo leukocyte recruitment.

Vascular endothelial growth factor-A is widely used in clinical trials for the treatment of cardiac ischemia. VEGF-A was recently suggested to act in a proinflammatory manner, which could aggravate adjacent atherogenesis in VEGF-A-based therapy. To assess potential bystander effects, VEGF-A was focally overexpressed in advanced atherosclerotic plaques in ApoE-/- mice. Sheer-induced carotid artery plaques were transluminally incubated with Ad.hVEGF-A leading to neointimal overexpression of VEGF-A. Ad.hVEGF-A treatment of pre-existing lesions was seen to promote plaque expansion, with a concomitant increase in macrophage and lipid content, whereas it lowered collagen content. In general, Ad.hVEGF-A-treated plaques displayed a more vulnerable phenotype. VEGF-A overexpression was not accompanied by increased microvessel development in the neointima, suggesting that VEGF-A destabilizes atherosclerotic plaques through an angiogenesis-independent mechanism. Intravital microscopy confirmed that treatment with Ad.hVEGF-A led to an increased monocyte adhesion, which was mediated by a VCAM-1/PECAM-1-dependent pathway. VEGF-A indeed induced a differential expression of VCAM-1 and PECAM-1 in endothelial cells. Our data underline the importance of regular monitoring of stenotic vessels adjacent to the site of VEGF-A application. We propose that VCAM-1/PECAM-1-directed cotherapy may be an efficient strategy to prevent bystander effects of focal VEGF-A therapy in patients suffering from cardiovascular disease.

Adenoviridae↗

PubMed2006

Therapeutic potential of a synthetic peptide inhibitor of nuclear factor of activated T cells as antirestenotic agent.

OBJECTIVE: The calcineurin/nuclear factor of activated T cells (NFAT) axis plays a pivotal role in the regulation of critical genes in vascular smooth muscle cell (vSMC) proliferation and inflammation, which makes NFAT inhibition an attractive modality in the prevention of restenosis. METHODS AND RESULTS: Synthetic peptide VIVIT potently inhibited NFAT activation in RAW 264.7 macrophages, Ea.Hy.926 endothelial cells and vSMCs, and blocked ionomycin-elicited nuclear import of NFAT. VIVIT, as well as cyclosporine A (CsA) or FK506, completely blunted platelet-derived growth factor-BB (PDGF-BB) and thrombin-induced vSMC proliferation. Moreover, it significantly inhibited PDGF-BB and thrombin-induced interleukin-6, interleukin-8, transforming growth factor-beta1, stromal cell-derived factor-1alpha, and monocyte chemotactic protein-1 expression in vSMCs. Unlike FK506 or CsA, VIVIT did not affect nuclear factor kappaB reporter gene activation and did only marginally affect endothelial wound healing in vitro. VIVIT did not intervene in phorbol 12-myristate 13-acetate-stimulated extracellular signal-regulated kinase activation, confirming its specificity for NFAT. Furthermore, our data establish that NFAT is a regulator of PDGF-BB induced vSMC proliferation. CONCLUSIONS: VIVIT appears to be a specific and potent inhibitor of NFAT activation and thus of NFAT-mediated proliferation and inflammation. Unlike FK506 or CsA, synthetic VIVIT therapy will not be accompanied by non-NFAT-mediated side effects on calcineurin signaling and constitutes a promising lead in antirestenotic therapy.

Animals↗

PubMed2006

Specific and efficient targeting of adenovirus vectors to macrophages: application of a fusion protein between an adenovirus-binding fragment and avidin, linked to a biotinylated oligonucleotide.

BACKGROUND: The application of serotype 5 adenoviruses (Ad5) in macrophages is hampered by the absence of the endogenous coxsackie adenovirus receptor (CAR). METHODS: To overcome this limitation, we first generated a linker protein consisting of the virus-binding domain of CAR and the C-terminus of avidin. Second, to target macrophages, this linker protein was equipped with the biotinylated (bio) oligonucleotide dA6G10, which was previously shown to display a high affinity for the scavenger receptor A (SR-A). RESULTS: As compared to nontargeted virus, the linker protein equipped with bio-dA6G10 showed a 500-fold increased reporter gene expression in mouse macrophage RAW264.7 cells. A linker protein equipped with a bio-dA16 control oligonucleotide was inactive. Moreover, the bio-dA6G10-equipped linker showed a 390-fold increased luciferase expression in the macrophage cell line J774 and 276- and 150-fold increased reporter gene expression in primary peritoneal and bone marrow (BM)-derived macrophages, respectively. Using BM-derived macrophages from SR-A knockout mice, it was shown that the dA6G10-mediated uptake is predominantly SR-A-mediated. CONCLUSIONS: Thus, we have developed a novel tool to link biotinylated ligands to a virus-binding fragment of CAR and have exploited this linker protein to extend the applicability of Ad5 to infect transformed and primary macrophages.

Adenoviridae↗

PubMed2006

Selective targeting of liposomes to macrophages using a ligand with high affinity for the macrophage scavenger receptor class A.

Macrophages play an important role in inflammatory processes and are crucially involved in the onset and progression of atherosclerosis and tumorigenesis. Therefore, macrophages are regarded as an excellent target for therapeutic intervention. Since the scavenger receptor class A (SRA) is highly expressed on macrophages, we developed in the present study an SRA-specific particulate drug carrier by providing phosphatidylcholine liposomes with a targeting ligand for SRA. To enable firm association with liposomes, the high-affinity SRA ligand decadeoxyguanine was covalently attached via a linker to lithocholic oleate (LCO-dA(2)dG(10)). Incorporation of LCO-dA(10)dG(2) into liposomes resulted in an increased electronegative surface charge and a dramatically enhanced serum clearance (t(1/2) < 2 min versus > 5 h). The LCO-dA(2)dG(10)-induced liposome clearance was fully dependent on SRA, as the clearance could be efficiently inhibited by the SRA competitor polyinosinic acid. LCO-dA(2)dG(10) enhanced the affinity of liposomes for SRA in vivo selectively, since introduction of overall or clustered negative charges by other modifications (e.g. oxidation, inclusion of phosphatidylserine, or exposure of glutamic acid residues) did not affect their serum clearance substantially, albeit that these modifications resulted in an at least equally high negative surface charge. LCO-dA(2)dG(10) also increased the association of liposomes with RAW264.7 cells, resulting in an enhanced intracellular delivery and bioactivity of encapsulated dexamethasone-phosphate. Therefore, the SRA-specificity of LCO-dA(2)dG(10)-liposomes may be applied for the specific delivery of drugs to macrophages, which may be of therapeutic benefit in general inflammatory disorders, atherosclerosis, and tumorigenesis.

Animals↗

PubMed2005

Stimulation of liver-directed cholesterol flux in mice by novel N-acetylgalactosamine-terminated glycolipids with high affinity for the asialoglycoprotein receptor.

OBJECTIVE: Interventions that promote liver-directed cholesterol flux can suppress atherosclerosis, as demonstrated for scavenger receptor-BI overexpression in hypercholesterolemic mice. In analogy, we speculate that increasing lipoprotein flux to the liver via the asialoglycoprotein receptor (ASGPr) may be of therapeutic value in hypercholesterolemia. METHODS AND RESULTS: A bifunctional glycolipid (LCO-Tyr-GalNAc3) with a high-nanomolar affinity for the ASGPr (inhibition constant 2.1+/-0.2 nmol/L) was synthesized that showed rapid association with lipoproteins on incubation with serum. Prior incubation of LCO-Tyr-GalNAc3 with radiolabeled low-density lipoprotein or high-density lipoprotein (0.5 microg/microg of protein) resulted in a dramatic induction of the liver uptake of these lipoproteins when injected intravenously into mice (70+/-3% and 78+/-1%, respectively, of the injected dose at 10 minutes of low-density lipoprotein and high-density lipoprotein), as mediated by the ASGPr on hepatocytes. Intravenously injected LCO-Tyr-GalNAc3 quantitatively incorporated into serum lipoproteins and evoked a strong and persistent (> or =48 hour) cholesterol-lowering effect in normolipidemic mice (37+/-2% at 6 hours) and hyperlipidemic apoE(-/-) mice (32+/-2% at 6 hours). The glycolipid was also effective on subcutaneous administration. CONCLUSIONS: LCO-Tyr-GalNAc3 is very effective in promoting cholesterol uptake by hepatocytes and, thus, may be a promising alternative for the treatment of those hyperlipidemic patients who do not respond sufficiently to conventional cholesterol-lowering therapies.

Acetylgalactosamine↗

PubMed2005

HIV entry inhibitor TAK-779 attenuates atherogenesis in low-density lipoprotein receptor-deficient mice.

OBJECTIVE: HIV combination therapy using protease inhibitors is associated with elevated plasma levels of atherogenic lipoproteins and increased risk for atherosclerosis. We investigated whether the HIV entry inhibitor TAK-779 affects lipoprotein levels and atherogenesis in low-density lipoprotein receptor-deficient mice. TAK-779 is an antagonist for the chemokine receptors CCR5 and CXCR3, which are expressed on leukocytes, especially T-helper 1 cells, and these receptors may be involved in recruitment of these cells to atherosclerotic plaques. METHODS AND RESULTS: TAK-779 treatment of low-density lipoprotein receptor-deficient mice did not elevate the levels of atherogenic lipoproteins, whereas it dramatically reduced atherosclerosis in the aortic root and in the carotid arteries. The number of T cells in the plaque was reduced by 95%, concurrently with a 98% reduction in the relative IFN-gamma area. TAK-779-treated animals showed a decreased percentage of CD4+ and CD8+ T cells in peripheral blood and in mediastinal lymph nodes compared with control-treated animals. CONCLUSIONS: TAK-779 not only suppresses HIV entry via blockade of CCR5 but also attenuates atherosclerotic lesion formation by blocking the influx of T-helper 1 cells into the plaque. TAK-779 treatment may be especially beneficial for young HIV patients as they face lifelong treatment, and this drug impairs atherogenesis.

Amides↗

PubMed2005

Increased vulnerability of pre-existing atherosclerosis in ApoE-deficient mice following adenovirus-mediated Fas ligand gene transfer.

OBJECTIVE: The death receptor Fas and Fas ligand (FasL) are present in human advanced atherosclerotic plaques. The activation of the Fas/FasL pathway of apoptosis has been implicated in plaque vulnerability. In the present study, we investigated whether overexpression of FasL in pre-existing atherosclerotic lesions can induce lesion remodelling and rupture-related events. METHODS AND RESULTS: Carotid atherogenesis was initiated in apolipoprotein E-deficient mice by placement of a perivascular silastic collar. The resulting plaques were incubated transluminally with recombinant adenovirus carrying FasL (Ad-FasL, lateral) or control beta-galactosidase (Ad-LacZ, contralateral). Transfection was restricted to the smooth muscle cell-rich cap of the plaque, and FasL expression led to a three-fold increase in apoptosis in the cap one day after gene transfer. Three days after gene transfer, FasL expression led to a 38% reduction in the number of cap cells. Two weeks after Ad-FasL transfer, non-thrombotic rupture, intra-plaque haemorrhage, buried caps and iron deposits were observed in 6 out of 17 Ad-FasL-treated carotid arteries versus 0 out of 17 controls (P=0.009), indicative of enhanced plaque vulnerability. CONCLUSIONS: These data demonstrate that advanced murine plaques are sensitive to Fas/FasL-induced apoptosis, which may indicate that stimulation of this pathway could result in plaque remodelling towards a more vulnerable phenotype.

Adenoviridae↗

PubMed2005

Gallic acid antagonizes P-selectin-mediated platelet-leukocyte interactions: implications for the French paradox.

BACKGROUND: Current paradigm attributes the low incidence of cardiovascular disorders in Mediterranean countries despite a high saturated fat intake, the "French paradox," to the antioxidant capacity of red wine polyphenols. Conceivably, other antiinflammatory pathways may contribute to at least a similar extent to the atheroprotective activity of these polyphenols. We have investigated whether gallic acid (GA), an abundant red wine polyphenol, modulates the activity of P-selectin, an adhesion molecule that is critically involved in the recruitment of inflammatory cells to the vessel wall and thus in atherosclerosis. METHODS AND RESULTS: GA potently inhibited the binding of a peptide antagonist (IC50, 7.2 micromol/L) and biotin-PAA-Le(a)-SO3H, an established high-affinity ligand, to P-selectin (IC50, 85 micromol/L). Under dynamic flow conditions, GA markedly and dose dependently attenuated the rolling of monocytic HL60 cells over P-selectin-transfected Chinese hamster ovary cells (EC50, 14.5 micromol/L) while increasing the velocity of P-selectin-dependent rolling of human blood leukocytes over a platelet monolayer. In vivo tests established that GA administration to normolipidemic C57/Bl6 and aged atherosclerotic apolipoprotein E-deficient mice impaired the baseline rolling of conjugates between activated platelets and circulating monocytes over femoral vein endothelium, as judged by online video microscopy (ED50, 1.7+/-0.3 and 1.5+/-0.4 mg x kg(-1) x h(-1), respectively). CONCLUSIONS: Our findings provide a solid mechanistic foundation through which GA intervenes in major inflammatory pathobiologies by binding and antagonizing P-selectin.

Acrylamides↗

PubMed2005

Endothelial KLF2 links local arterial shear stress levels to the expression of vascular tone-regulating genes.

Lung Krüppel-like factor (LKLF/KLF2) is an endothelial transcription factor that is crucially involved in murine vasculogenesis and is specifically regulated by flow in vitro. We now show a relation to local flow variations in the adult human vasculature: decreased LKLF expression was noted at the aorta bifurcations to the iliac and carotid arteries, coinciding with neointima formation. The direct involvement of shear stress in the in vivo expression of LKLF was determined independently by in situ hybridization and laser microbeam microdissection/reverse transcriptase-polymerase chain reaction in a murine carotid artery collar model, in which a 4- to 30-fold induction of LKLF occurred at the high-shear sites. Dissection of the biomechanics of LKLF regulation in vitro demonstrated that steady flow and pulsatile flow induced basal LKLF expression 15- and 36-fold at shear stresses greater than approximately 5 dyne/cm2, whereas cyclic stretch had no effect. Prolonged LKLF induction in the absence of flow changed the expression of angiotensin-converting enzyme, endothelin-1, adrenomedullin, and endothelial nitric oxide synthase to levels similar to those observed under prolonged flow. LKLF repression by siRNA suppressed the flow response of endothelin-1, adrenomedullin, and endothelial nitric oxide synthase (P < 0.05). Thus, we demonstrate that endothelial LKLF is regulated by flow in vivo and is a transcriptional regulator of several endothelial genes that control vascular tone in response to flow.

Adrenomedullin↗

PubMed2005

Scavenger receptors: friend or foe in atherosclerosis?

PURPOSE OF REVIEW: Scavenger receptors were originally defined by their ability to bind and internalize modified lipoproteins. Nowadays the family of scavenger receptors is composed of structurally different surface receptors which recognize a broad pattern of common ligands which include, besides modified lipoproteins, apoptotic cells and pathogens. This review focuses on the role of scavenger receptors in the development of atherosclerotic lesions. RECENT FINDINGS: Recent studies indicate that scavenger receptor A activity can be regulated by phosphorylation, glucosidases, 8-isoprostane, high glucose and nobiletin. Modulation of these regulatory components may beneficially influence scavenger receptor A's proatherogenic function. It appears that statins do lead to a reduction in CD36 transcription and could modulate in this way CD36-mediated atherosclerotic foam cell formation. Macrophage scavenger receptor BI appears to facilitate the development of small fatty streak lesions, whereas the formation of advanced atherosclerotic lesions is reduced, indicating a unique dual role for macrophage scavenger receptor BI in the pathogenesis of atherosclerosis. SUMMARY: It is proposed that the presence of scavenger receptors in macrophages is beneficial because they remove potential deleterious material from the arterial wall. Inadequate handling of the internalized material by the macrophages will lead to foam cell formation. If adequate levels of ATP-binding cassette transporters and accepting HDL are present, however, the macrophage is perfectly able to metabolize and secrete the internalized atherogenic substances whereby HDL facilitates further transport from the arterial wall to the liver, leading to release in bile.

Animals↗

PubMed2005

Low-dose FK506 blocks collar-induced atherosclerotic plaque development and stabilizes plaques in ApoE-/- mice.

Since atherosclerosis is a chronic inflammatory disease, we tested the hypothesis that the immunosuppressive drug FK506 would attenuate the development of atherosclerosis using a mouse model of collar-induced atherosclerosis. ApoE-/- mice were treated for 4 weeks with the immunosuppressive drug FK506 (0.05 mg/kg/day), yielding sustained blood levels (approximately 0.2 ng/mL) without systemic side effects. Atherosclerotic plaque development of FK506-treated mice was significantly reduced (63%) while plaque cell density was increased (52%) compared to controls. Importantly, FK506 also blocked progression of pre-existing atherosclerotic plaques. Plaque area of pre-existing plaques was 35% reduced by FK506. Cell density (35%) and collagen content (51%) were significantly increased, whereas necrotic core content was decreased (42%), indicating a more stable plaque morphology. Similar results were found during spontaneous atherosclerotic plaque development in ApoE-/- mice (treatment 17-25 weeks of age). Flow-cytometric analysis showed no peripheral effects on blood cell count or T-cell activation after FK506-treatment. In vitro, FK506 decreased vascular smooth muscle cell (VSMC) apoptosis and inhibited nuclear factor of activated T cells (NFAT)-luciferase reporter activity at concentrations in the range of the in vivo concentration. Low-dose FK506 inhibits collar-induced atherosclerotic plaque development and progression and induces more stable plaque phenotypes in ApoE-/- mice without any peripheral side effects.

Animals↗

PubMed2004

Liver Kupffer cells rapidly remove red blood cell-derived vesicles from the circulation by scavenger receptors.

Previous studies have shown that during the lifespan of red blood cells (RBCs) 20% of hemoglobin is lost by shedding of hemoglobin-containing vesicles. However, the fate of these vesicles is unknown. To study this fate we used a rat model, after having established that rat RBCs lose hemoglobin in the same way as human RBCs, and that RBC-derived vesicles are preferentially labeled by Na2(51) CrO4. Such labeled vesicles were injected into recipient rats. Within 5 minutes, 80% of the radioactivity was cleared from the circulation with a concomitant uptake by the liver of 55% of the injected dose. After 30 minutes, Kupffer cells contained considerable amounts of hemoglobin and were shown to be responsible for 92% of the liver uptake. Vesicle clearance from the blood as well as liver uptake were significantly inhibited by preinjection of the scavenger-receptor ligands polyinosinic acid and phosphatidylserine. We conclude that in rats Kupffer cells rapidly remove RBC-derived vesicles from the circulation, mainly by scavenger receptors. The same mechanism is likely to be responsible for the elimination of human RBC vesicles, thereby constituting an important pathway for the breakdown of RBCs in humans.

Animals↗

PubMed2004

Design and synthesis of novel N-acetylgalactosamine-terminated glycolipids for targeting of lipoproteins to the hepatic asialoglycoprotein receptor.

A novel glycolipid has been prepared that contains a cluster glycoside with an unusually high affinity for the asialoglycoprotein receptor (ASGPr) and a bile acid moiety that mediates stable incorporation into lipidic particles. The glycolipid spontaneously associated with low-density lipoproteins (LDL) and high-density lipoproteins (HDL) within human and murine plasma, and loading of lipoproteins with this glycolipid resulted in an efficient dose-dependent recognition and uptake of LDL and HDL by the liver (and not by spleen) upon intravenous injection into wild-type mice. Preinjection with asialoorosomucoid largely inhibited the uptake, establishing that both HDL and LDL were selectively recognized and processed by the ASGPr on liver parenchymal cells. Finally, repeated intravenous administration of the glycolipid to hyperlipidemic LDL receptor-deficient mice evoked an efficient and persistent cholesterol-lowering effect. These results indicate that the glycolipid may be a promising alternative for the treatment of hyperlipidemic patients who do not respond sufficiently to current cholesterol-lowering therapies.

Acetylgalactosamine↗

PubMed2004

Binding of low density lipoprotein to platelet apolipoprotein E receptor 2' results in phosphorylation of p38MAPK.

Binding of low density lipoprotein (LDL) to platelets enhances platelet responsiveness to various aggregation-inducing agents. However, the identity of the platelet surface receptor for LDL is unknown. We have previously reported that binding of the LDL component apolipoprotein B100 to platelets induces rapid phosphorylation of p38 mitogen-activated protein kinase (p38MAPK). Here, we show that LDL-dependent activation of this kinase is inhibited by receptor-associated protein (RAP), an inhibitor of members of the LDL receptor family. Confocal microscopy revealed a high degree of co-localization of LDL and a splice variant of the LDL receptor family member apolipoprotein E receptor-2 (apoER2') at the platelet surface, suggesting that apoER2' may contribute to LDL-induced platelet signaling. Indeed, LDL was unable to induce p38MAPK activation in platelets of apoER2-deficient mice. Furthermore, LDL bound efficiently to soluble apoER2', and the transient LDL-induced activation of p38MAPK was mimicked by an anti-apoER2 antibody. Association of LDL to platelets resulted in tyrosine phosphorylation of apoER2', a process that was inhibited in the presence of PP1, an inhibitor of Src-like tyrosine kinases. Moreover, phosphorylated but not native apoER2' co-precipitated with the Src family member Fgr. This suggests that exposure of platelets to LDL induces association of apoER2' to Fgr, a kinase that is able to activate p38MAPK. In conclusion, our data indicate that apoER2' contributes to LDL-dependent sensitization of platelets.

Animals↗

PubMed2003

Adenoviral transfer of endothelial nitric oxide synthase attenuates lesion formation in a novel murine model of postangioplasty restenosis.

OBJECTIVE: Restenosis remains a major late complication of percutaneous transluminal coronary angioplasty (PTCA), for which the development of prevention strategies has thus far been hampered by the lack of a representative and practical animal model. We have, therefore, developed a murine model of PTCA-induced restenosis. METHODS AND RESULTS: Rigid probe angioplasty of pre-existing atherosclerotic lesions in the carotid arteries of ApoE-deficient mice was found to result in an increase in lesion size (0.14+/-0.04x10(5) microm2 to 0.42+/-0.09x10(5) microm2, P=0.007) with a smooth muscle cell-rich, fibrotic lesion morphology. In an additional experiment, lesions were incubated immediately after angioplasty with adenovirus bearing an endothelial nitric oxide synthase (eNOS) transgene (Ad.APT.eNOS), or an "empty" control virus (Ad.APT.empty) at a titer of 1.5x10(9) pfu/mL. Ad.APT.eNOS treatment was seen to lead to a 73.1% reduction in plaque size (0.27+/-0.04x10(5) microm2 versus 1.02+/-0.39x10(5) microm2, P=0.07), which translated to a significantly lowered average degree of stenosis (33.6+/-4.1% versus 74.6+/-14.0%, P=0.02). Ad.APT.eNOS also decreased lesional collagen content from 29.1% to 4.8% (P<0.001). CONCLUSIONS: We believe that we have established a representative murine model of postangioplasty restenosis, which may serve to elucidate the mechanisms underlying restenosis and to evaluate potential antirestenotic therapies.

Adenoviridae↗

PubMed2003

P-selectin as a candidate target in atherosclerosis.

P-selectin is of critical importance in early atherogenesis by initiating leukocyte rolling at the site of endothelial injury. In order to validate P-selectin as a candidate target for the development of anti-atherogenic strategies, we wanted to obtain quantitative information on P-selectin expression, and identify novel peptide-based lead structures that interact with P-selectin. P-selectin mRNA expression in the aortic arch and in other tissues of apoE-deficient (apoE-/-) mice was determined by real-time PCR technology. P-selectin mRNA expression of apoE-/- mice increased steadily with age to levels 14-fold higher than that of control animals. The onset and level of P-selectin expression correlated well with the extent of lesion development, and was more specific for atherosclerotic tissue as compared with other adhesion molecules. Phage display technology was used to obtain novel P-selectin antagonists. Phage display selections resulted in the isolation of a highly P-selectin-specific phage clone. Synthetic peptide-equivalents of this clone displaced the binding of the parent phage and antagonized the binding of a sialyl Lewis(x) analogue to P-selectin. In conclusion, P-selectin expression correlates with early and advanced atherosclerotic lesion development. P-selectin ligands, like the lead structure we have developed here, can therefore be considered as promising tools to identify, target or antagonize P-selectin function within the chronically inflamed arterial wall.

Animals↗

PubMed2003

Serine protease inhibitor Serp-1 strongly impairs atherosclerotic lesion formation and induces a stable plaque phenotype in ApoE-/-mice.

The myxoma virus protein Serp-1 is a member of the serine protease inhibitor superfamily. Serp-1 potently inhibits human serum proteases including plasmin, urokinase-type plasminogen activator (uPA), and tissue-type plasminogen activator (tPA). Serp-1 also displays a high antiinflammatory activity, rendering it a promising candidate for antiatherosclerotic therapy. In this study, we have thus examined the effect of Serp-1 on de novo atherosclerotic plaque formation and on advanced lesions. Perivascular collars were placed around carotid arteries of ApoE-/- mice to induce atherosclerotic plaques and Serp-1 treatment started at week 1 and week 5 after collar placement. Effects of Serp-1 on de novo atherogenesis were characterized by a significantly lower plaque size than that of control mice (18+/-5x10(3) versus 57+/-12x10(3) microm2, respectively; P=0.007). Immunostaining showed a 50% (P=0.004) decrease in the MOMA-2-stained lesion area of Serp-1-treated mice. Treatment of advanced lesions with Serp-1 resulted in a decrease in plaque size and lumen stenosis (P=0.028). Alpha-actin staining of these lesions was significantly increased compared with the control (P=0.017). In both studies, a higher cellularity of the plaque and increased collagen content was observed in Serp-1-treated mice. In vitro studies showed that Serp-1 induces proliferation and migration of vascular smooth muscle cells. In conclusion, Serp-1 inhibits carotid artery plaque growth and progression in ApoE-/- mice. Equally relevant, it enhances cellularity of the plaque core potentially leading to improved plaque stability. The above results indicate that Serp-1 constitutes a promising lead in antiatherosclerotic therapy.

Actins↗