Temporal and topographic matrix metalloproteinase expression after vascular injury in mice.

Temporal and topographic expression of matrix metalloproteinases (MMPs) after perivascular electric injury was studied in wild-type (WT) and urokinase-deficient (u-PA-/-) mice. Neointima formation after injury of the femoral artery was significantly reduced in u-PA-/- mice as compared to WT mice (area of 0.002+/-0.0007 mm2 versus 0.008 + 0.002 mm2 at 3 weeks after injury; p <0.001), associated with impaired cellular migration (nuclear cell counts of 44+/-5 versus 82+/-9in cross-sectional areas; p <0.001). Zymographic and/or microscopic analysis indicated that MMP expression gradually increased to reach a maximum at 1 to 2 weeks after vascular injury. In general, MMP levels were lower in u-PA-/- than in WT mice. In non-injured arteries, MMP-2 (gelatinase A) and MMP-3 (stromelysin-1) were produced mainly by adventitial fibroblasts and/or non-contractile smooth muscle cells (SMC). One week after injury, MMP-2 and MMP-3 levels were enhanced due to an increased number and size of producing cells; 2 to 3 weeks after injury, MMP-2 and MMP-3 were produced also by some contractile SMC, which stained with alpha-actin antiserum. MMP-9 (gelatinase B), MMP-12 (metalloelastase) and MMP-13 (collagenase-3) were found in macrophages located mainly in the adventitia. Immunogold electron microscopic examination revealed that MMP-2 was located predominantly in association with the cell surface of fibroblasts or SMC, while MMP-9 and MMP- 12 were located in well defined storage granules within macrophages. MMP-2, MMP-3 and MMP-13, but not MMP-9 or MMP-12, were also found extracellularly, associated with elastin-containing structures (MMP-2), with the basement membrane and occasionally with collagen fibres (MMP-3), or with proteoglycans, collagen and elastin (MMP-13). The temporal and topographic expression pattern of MMPs after vascular injury, coinciding with smooth muscle cell migration and neointima formation, thus is compatible with a role in vascular remodeling.     

Unique remodeling processes after vascular injury in intracranial arteries: analysis using a novel mouse model

The effectiveness of angioplasty and stenting in intracranial atherosclerotic diseases is controversial due to high rates of delayed restenosis and hemorrhage compared with extracranial arteries. However, the mechanisms underlying these differences are still unclear, because their pathophysiology is yet to be examined. To address this issue, we established a novel vascular injury model in the intracranial internal carotid arteries (IICAs) in mice, and analyzed the remodeling process in comparison to that of the femoral arteries (FAs). In IICAs, neointimal hyperplasia was observed from day 14 and grew until day 56. Although smooth muscle cells (SMCs) emerged in the neointima from day 28, SMCs in the injured media were continuously lost with eventual extinction of the media. Re-endothelialization was started from day 7 and completed on day 28. Accumulation of macrophages was continued in the adventitia until day 56. Compared with FAs, the following points are unique in IICAs: (1) delayed continuous formation of neointima; (2) accumulation of macrophages in the media on day 14; (3) continuous loss of SMCs in the media followed by extinction of the media itself; and (4) continuously growing adventitia. These pathophysiologic differences might be associated with unfavorable outcomes in percutaneous transluminal angioplasty and stenting in intracranial arteries.     

FAK-mediated inhibition of vascular smooth muscle cell migration by the tetraspanin CD9

Migration of vascular smooth muscle cells (SMC) towards the intima is a key event in vascular proliferative diseases. We investigated a potential role for the tetraspanin CD9 in this process in a wound migration assay. Aortic SMC from CD9 knock-out mice had higher migration rates and the presumably stimulatory anti-CD9 antibody ALMA-1 inhibited migration of human SMC. The signaling pathways responsible for this inhibitory effect were investigated. In migrating CD9-/- SMC, stress fiber formation was decreased and focal adhesions were smaller and more diffusely distributed, consistent with an inhibition of integrin clustering. In migrating mouse SMC expressing CD9, focal adhesion kinase (FAK) tyrosine phosphorylation was doubled. No differences in intracellular calcium signaling were observed between CD9+/+ and CD9-/- SMC during migration. We suggest that CD9 in hibits SMC migration by a stimulation of both stress fiber formation and integrin clustering, leading to a stimulation of FAK phosphorylation.     

Differential role of components of the fibrinolytic system in the formation and removal of thrombus induced by endothelial injury

The role of fibrinolytic system components in thrombus formation and removal in vivo was investigated in groups of six mice deficient in urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), or plasminogen activator inhibitor-1 (PAI-1) (u-PA-/-, t-PA-/- or PAI-1-/-, respectively) or of their wild type controls (u-PA+/+, t-PA+/+ or PAI-1+/+). Thrombus was induced in the murine carotid artery by endothelial injury using the photochemical reaction between rose bengal and green light (540 nm). Blood flow was continuously monitored for 90 min on day 0 and for 20 min on days 1, 2 and 3. The times to occlusion after the initiation of endothelial injury in u-PA+/+, t-PA+/+ or PAI-1+/+ mice were 9.4+/-1.3, 9.8+/-1.1 or 9.7+/-1.6 min, respectively. u-PA-/- and t-PA-/- mice were indistinguishable from controls, whereas that of PAI-1-/- mice were significantly prolonged (1 8.4+/-3.7 min). Occlusion persisted for the initial 90 min observation period in 10 of 18 wild type mice and was followed by cyclic reflow and reocclusion in the remaining 8 mice. At day 1, persistent occlusion was observed in 1 wild type mouse, 8 mice had cyclic reflow and reocclusion and 9 mice had persistent reflow. At day 2, all injured arteries had persistent reflow. Persistent occlusion for 90 min on day 0 was observed in 3 u-PA-/-, in all t-PA-/- mice at day 1 and in 2 of the t-PA-/-mice at day 2 (p <0.01 versus wild type mice). Persistent patency was observed in all PAI-1-/- mice at day 1 and in 5 of the 6 u-PA-/- mice at day 2 (both p <0.05 versus wild type mice). In conclusion, t-PA increases the rate of clot lysis after endothelial injury, PAI-1 reduces the time to occlusion and delays clot lysis, whereas u-PA has little effect on thrombus formation and spontaneous lysis.     

CD26 modulates nociception in mice via its dipeptidyl-peptidase IV activity

BACKGROUND: CD26 is a multifunctional cell surface glycoprotein expressed by T and B cells. It exhibits a dipeptidyl-peptidase activity (DPP-IV) that cleaves the penultimate proline from the N-terminus of polypeptides, thereby regulating their activity and concentration. METHODS: Using CD26-/- mice resulting from targeted inactivation of the gene, we examined the consequences of a DPP-IV defect on behavioural response to nociceptive stimuli and concentration of the pain modulator peptides substance P (SP) and endomorphin 2, two DPP-IV substrates. RESULTS: CD26 inactivation induced a three-fold decrease in circulating endopeptidase activity while that found in brain extracts was normal, albeit very weak. CD26-/- mice had high SP concentrations in plasma (3.4+/-1 pg/ml versus 1.5+/-0.3 pg/ml, P<10(-3)) but not in brain extracts (35+/-12 pg/ml versus 32+/-9 pg/ml, P>0.05). Endomorphin-2 levels in the two groups were in the same range for plasma and brain extracts. CD26-/- mice displayed short latencies to nociceptive stimuli (hot plate test: 6.6+/-1.2 s versus 8.6+/-1.5 s, P<10(-4); tail pinch test: 3.1+/-0.6 s versus 4.2+/-0.8 s, P<10(-3)). Administration of an SP (NK1) receptor antagonist or DPP-IV to CD26-/- mice normalised latencies. DPP-IV inhibitors decreased latencies only in CD26+/+ mice. CONCLUSIONS: Our observations represent the first fundamental evidence showing that DPP-IV influences pain perception via modulation of the peripheral SP concentration. Our work also highlights the role of peripheral NK1 receptors in nociception.     

Adenovirus-mediated expression of a truncated PDGFbeta receptor inhibits thrombosis and neointima formation in an avian arterial injury model

Platelet-derived growth factor (PDGF) is a major mediator of neointima formation after arterial injury. We constructed a recombinant adenovirus, Ad/PDGFtr, that expresses the soluble extracellular domain of the murine PDGFbeta receptor (PDGFtr). The expressed PDGFtr was appropriately glycosylated and secreted by chicken vascular smooth muscle cells (SMCs) in vitro. The expressed PDGFtr inhibited human PDGF-BB induced receptor autophosphorylation, and also inhibited PDGF-BB induced cell proliferation without affecting PDGF-AA induced mitogenesis. In vivo transduction of balloon-injured rooster femoral arteries with Ad/PDGFtr resulted in expression and secretion of the glycosylated PDGFtr. The expressed PDGFtr significantly inhibited neointima formation compared with controls. Neointima-associated thrombus was significantly reduced in Ad/PDGFtr transduced arteries compared with controls. Thus, in addition to impacting on SMC proliferation and migration, PDGF-BB plays a role in thrombus formation in response to arterial injury. Growth factor inhibition by localized gene delivery constitutes a powerful approach to intervene in the molecular pathways involved in vascular disease.     

CD18-mediated neutrophil recruitment contributes to the pathogenesis of reperfused but not nonreperfused stroke

BACKGROUND AND PURPOSE: Neutrophil (PMN) recruitment mediated by increased expression of intercellular adhesion molecule-1 expression (ICAM-1, CD54) in the cerebral microvasculature contributes to the pathogenesis of tissue injury in stroke. However, studies using blocking antibodies against the common beta2-integrin subunit on the PMN, the counterligand for ICAM-1 (CD18), have demonstrated equivocal efficacy. The current study tested the hypothesis that mice deficient in CD18 would be protected in the setting of reperfused but not nonreperfused stroke. METHODS: Two groups of mice were studied, those whose PMNs could express CD18 (CD18 +/+) and those mice hypomorphic for the CD-18 gene (CD18 -/-). PMNs obtained from CD18 -/- or CD18 +/+ mice were fluorescently labeled and tested for binding to murine brain endothelial monolayers. Using a murine model of focal cerebral ischemia in which an occluding suture placed in the middle cerebral artery (MCA) is removed after 45 minutes (transient ischemia, reperfused stroke) or left in place (permanent ischemia, nonreperfused stroke), cerebral infarct volumes (% ipsilateral hemisphere by TTC staining), cerebral blood flow (CBF, % contralateral hemisphere by laser-Doppler flowmetry), and survival (%) were examined 24 hours after the initial ischemic event. Adoptive transfer studies used 111In-labeled PMNs (from either CD18 +/+ or CD18 -/- mice) to examine the relative accumulation of PMNs in the ischemic region. RESULTS: PMNs obtained from CD18 -/- mice exhibit reduced adhesivity (compared with CD18 +/+ PMNs) for both quiescent and cytokine-activated endothelial monolayers. CD18 -/- mice (n=14) subjected to transient focal cerebral ischemia demonstrated a 53% decrease in infarct volumes versus CD18 +/+ mice (n=26, P<0.05), improved penumbral CBF at 24 hours (1.8-fold, P=0.02), and a 3.7-fold decrease in mortality (P=0.02). However, when CD18 -/- mice (n=12) were subjected to permanent focal cerebral ischemia, no differences were noted in infarct volume, mortality, or CBF versus similarly treated CD18 +/+ mice (n=10). There was a greater accumulation of CD18 +/+ PMNs in the ischemic zone of CD18 +/+ animals than CD18 -/- animals subjected to reperfused stroke (82% increase, P=0.02), although there was no difference between groups when subjected to permanent MCA occlusion. CONCLUSIONS: Deficiency for the CD18 gene confers cerebral protection in a murine model of reperfused stroke, but this benefit does not extend to CD18-deficient animals subjected to permanent MCA occlusion. These data suggest that anti-PMN strategies should be targeted to reperfused stroke and may perhaps be used in conjunction with thrombolytic therapy that establishes reperfusion.     

Sympathetic nerve traffic and circulating norepinephrine levels in RGS2-deficient mice

Regulator of G protein signaling 2 (RGS2-/-) deficient mice feature an increased resting blood pressure and an excessive pressor response to stress. We measured renal sympathetic nerve activity (RSNA) directly to test the hypothesis that RSNA is increased in RGS2-/- mice, compared to RGS2+/+ mice. Seventeen mice (RGS2-/-, n=9; RGS2+/+, n=8) were anesthetized with isoflurane. We cannulated the left jugular vein for drug administration. Renal sympathetic nerve activity (RSNA) was recorded using bipolar electrodes. Arterial blood pressure (BP) from the femoral artery, ECG (needle electrodes), and RSNA were recorded (sample rate 10 kHz) simultaneously. RSNA was analysed off-line using a modified wavelet de-noising technique and the classical discriminator method. RSNA detected during phenylephrine bolus injections or after the animals death was subtracted from baseline values. Mean arterial blood pressure, norepinephrine plasma levels, the responsiveness to vasoactive drugs, and the sympathetic baroreflex gain were similar in anesthetized RGS2+/+ and RGS2-/- animals. RSNA was lower in RGS2-/- mice compared to wild-type controls (wavelet: spike rate in Hz: RGS2+/+ 25.5+/-5.1; RGS2-/- 17.4+/-4.0; discriminator method: RGS2+/+ 41.4+/-5.7, RGS2-/- 22.0+/-4.3, p<0.05). Thus, the expected result proved not to be the case. Our data suggest a mismatch between sympathetic nerve traffic and plasma norepinephrine concentrations. This observation may depend on altered coupling between electrical nerve activity and norepinephrine release and/or a changed norepinephrine uptake in RGS2-/- mice.     

The profile of the extracellular matrix changes in the aorta after sympathectomy in the hypercholesterolemic rats

We previously showed that sympathectomy induces thickened intima and decreases the expression of cytoskeletal proteins associated with a differentiated smooth muscle cell (SMC) phenotype in hypercholesterolemic rats. In the present study, we sought to determine the effect of sympathectomy on various components of the extracellular matrix (ECM) in the aorta from these animals, since the state of SMC differentiation depends on the nature of ECM components. Collagen types I and III, previously reported to be associated with SMC dedifferentiation, and collagen VI, elastin, laminin and elastin-laminin receptor (E/L-R), previously reported to be associated with SMC differentiation, were analyzed by western immunoblot and confocal microscopy in abdominal aortae from sham rats and hypercholesterolemic rats sympathectomized with guanethidine. Both western immunoblot and immunohistological analysis showed an increase in collagens I and III (more for collagen I), with abundant labeling in the media, adventitia and thickened intima in sympathectomized aortae. Collagen IV labeling was decreased in the media and adventitia and was weak in the thickened intima in sympathectomised aortae. The E/L-R increased and was abundantly labeled in the media and weakly in the thickened intima in sympathectomized aortae. Elastin and laminin decreased and appeared less labeled in the media in the sympathectomised aortae. In the thickened intima, laminin was slightly labeled while elastin was not obviously labeled. These data show that sympathectomy favors the ECM features reported in association with a dedifferentiated/immature SMC phenotype and intimal thickening, probably by actions on both SMCs and fibroblasts.     

Adventitial expression of recombinant endothelial nitric oxide synthase gene reverses vasoconstrictor effect of endothelin-1.

The present study was designed to determine the effect of recombinant endothelial nitric oxide synthase (eNOS) gene expression on reactivity of canine basilar arteries to endothelin-1 (ET-1). Experiments were performed ex vivo. The arteries were exposed (30 minutes at 37 degrees C) to adenoviral vectors encoding eNOS gene (AdCMVeNOS) or beta-galactosidase reporter gene (AdCMVbeta-Gal). Twenty-four hours after transduction, transgene expression was evident mainly in the vascular adventitia. Rings of control (nontransduced), AdCMVbeta-Gal- and AdCMVeNOS-transduced arteries with and without endothelium were suspended for isometric tension recording. Levels of guanosine 3',5'-cyclic monophosphate (cGMP) were measured by radioimmunoassay. During contractions to uridine 5'-triphosphate, ET-1 (10(-10) to 3x10(-9) mol/L) caused further increase in tension in control and AdCMVbeta-Gal-transduced arteries. In contrast, ET-1 caused concentration-dependent relaxations of AdCMVeNOS-transduced arteries. The relaxations to ET-1 in AdCMVeNOS-transduced arteries were endothelium-independent. They were abolished by N(G)-nitro-L-arginine methyl ester or by chemical treatment of adventitia with paraformaldehyde before gene transfer. ET-1 (10(-9) mol/L) significantly increased intracellular cGMP levels in AdCMVeNOS-transduced arteries without endothelium. In arteries transduced with AdCMVeNOS, higher concentrations (10(-9) to 3x10(-8) mol/L) of ET-2 also caused relaxations, whereas ET-3 and sarafotoxin, a selective ET(B) receptor agonist, did not produce any relaxations. The relaxations to ET-1 in AdCMVeNOS-transduced arteries were strongly reduced by BQ-123 (10(-7) mol/L), an ET(A) receptor antagonist, but were not affected by BQ-788 (3x10(-7) mol/L), an ET(B) receptor antagonist. These results suggest that genetically modified adventitia can produce nitric oxide and cause relaxations in response to ET-1 via activation of ET(A) receptors. Our findings support a novel concept that successful transfer and expression of recombinant eNOS gene can lead to a qualitative change in responsiveness to vasoconstrictor substances.     

Elevated homocysteine serum level is associated with low enrichment of homocysteine in coronary arteries of patients with coronary artery disease

In the present study, we sought to investigate whether elevated serum levels of homocysteine (Hcy), predisposing to endothelial dysfunction during progression of atherosclerosis, were paralleled by increased Hcy concentrations in human coronary arteries. Paraffin sections of coronary arteries were obtained from explanted hearts of cardiac transplant recipients suffering from coronary artery disease (CAD, n=32, mean age=56.6+/-6.8), and from heart donors where transplantation was not performed due to organization-related circumstances (Co, n=6, mean age 25.0+/-10.6), and characterized immunohistochemically for Hcy, CD68, and smooth muscle alpha-actin. Although the CAD group presented with high serum Hcy levels (27.7+/-12.8 micromol/l), the media and intimal layers containing the endothelium showed the lowest enrichment of Hcy (media: 20.8+/-4.4%; intima: 6.1+/-2.3%). Surprisingly, the control group revealed an extensive Hcy enrichment, co-localizing with vascular smooth cells (media: 32.3+/-14.0%; intima: 7.0+/-2.0%). In conclusion, we have provided evidence for a reverse relation between Hcy serum concentration and enrichment of Hcy in coronary arteries of patients with severe CAD, suggesting that Hcy is not likely to be involved directly in atheromatosis development of coronary arteries.     

Matrix metalloproteinase deficiencies do not impair cell-associated fibrinolytic activity

The matrix metalloproteinase (MMP) and fibrinolytic (plasminogen/plasmin) systems cooperate in many (patho)physiological processes requiring extracellular proteolysis. The effect of MMP-3 (stromelysin-1), MMP-7 (matrilysin), MMP-9 (gelatinase B) or MMP-12 (metalloelastase) on cellular fibrinolytic activity was studied with the use of smooth muscle cells (SMC) and fibroblasts derived from mice with specific inactivation of these genes. Activation of cell-bound plasminogen by two-chain urokinase-type plasminogen activator (tcu-PA) was not significantly different with SMC or fibroblasts from the gene-deficient mice (78% to 140% of wild-type). For all cell types, very limited conversion of plasminogen to angiostatin-like kringle-containing fragments was observed (< 3% of the total cell-bound plasminogen). Activation of plasminogen in solution by cell-associated tcu-PA was also comparable for SMC or fibroblasts of the different genotypes (54% to 160% of wild-type). In vitro SMC migration on scrape wounded collagen-coated surfaces was comparable for wild-type, MMP-7(-/-), MMP-9(-/-) and MMP-12(-/-) SMC, but was significantly reduced for MMP-3(-/-) SMC (P < .005 vs. wild-type). Serum-free conditioned medium of MMP-3(-/-) and MMP-7(-/-) SMC or fibroblasts induced similar lysis of fibrin films as wild-type cells. These findings indicate that several interactions that have been described between these MMPs and the plasminogen/plasmin system in a purified system do not significantly affect plasmin-mediated cellular fibrinolytic activity under cell culture conditions.     

Increased susceptibility to ischemic brain injury in cyclooxygenase-1-deficient mice.

Cyclooxygenase-1 (COX-1), a rate-limiting enzyme in the synthesis of prostanoids, is involved in selected vasodilatatory responses of the cerebral circulation. Cyclooxygenase-1-null mice were used to determine whether COX-1 influences cerebral ischemic damage. The middle cerebral artery was occluded in COX-1 -/- and +/+ mice (n = 9/group), and lesion volume was determined in thionin-stained sections 24 or 96 hours later. Middle cerebral artery occlusion produced larger infarcts in COX-1 -/- mice, both at 24 (35 +/- 17%; P < 0.05) and 96 hours (41 +/- 16%; P < 0.05) after ischemia. The enlargement was not due to increased susceptibility to glutamate excitotoxicity, because microinjection of N-methyl-D-aspartate or kainate in the parietal cortex produced comparable lesions in COX-1 +/+ and -/- mice ( P > 0.05; n = 8/group). To examine the contribution of hemodynamic factors to the enlargement of the infarct, cerebral blood flow was monitored by laser-Doppler flowmetry in the ischemic territory (n = 6/group). Although the reduction in cerebral blood flow was comparable in the ischemic core ( P > 0.05), at the periphery of the ischemic territory the reduction was greater in COX-1 -/- mice (-58 +/- 4%) than in COX-1 +/+ mice (-34 +/- 5%; P < 0.05). It is concluded that mice lacking COX-1 are more susceptible to focal cerebral ischemia, an effect that can be attributed to a more severe cerebral blood flow reduction in vulnerable regions at the periphery of the ischemic territory. Thus, the vascular effects of COX-1 may contribute to maintain cerebral blood flow in the postischemic brain and, as such, play a protective role in ischemic brain injury.     

p53 potentiates hippocampal neuronal death caused by global ischemia.

Although p53 controls cell death after various stresses, its role in neuronal death after brain ischemia is poorly understood. To address this issue, we subjected p53-deficient (p53-/- and p53+/-) mice (backcrossed for 12 generations with C57BL/6 mice) and wild-type mice (p53+/+) to transient global ischemia by the three-vessel occlusion method. Despite similar severity of ischemia, as shown by anoxic depolarization and cortical blood flow, neuronal death in the hippocampal cornus ammonis (CA)1 region was much more extensive in p53+/+ than in p53-/- mice (surviving neuronal count, 9.3%+/-3.0% versus 61.3%+/-34.0% of nonischemic p53+/+ controls, respectively, P<0.0037). In p53+/- mice, a similar trend was also observed, though not statistically significant (43.5% of nonischemic p53+/+ controls). In p53+/+ mice, p53-like immunoreactivity in hippocampal CA1 neurons was enhanced at 12 h after ischemia, and messenger ribonucleic acid for Bax, a direct downstream target of p53, was also increased. These results indicate that p53 potentiates ischemic neuronal death in vivo and suggest that this molecule could be a therapeutic target in neuronal death after cerebral ischemia.     

Enhanced fibrinolytic potential in mice with combined homozygous deficiency of alpha2-antiplasmin and PAI-1

Alpha2-antiplasmin (alpha2-AP) and plasminogen activator inhibitor-1 (PAI-1) are the main physiological inhibitors of the plasminogen/plasmin system in mammalian plasma. In the present study, the relative importance of both inhibitors was evaluated with the use of mice with single or combined deficiency of alpha2-AP and PAI-1 in the same genetic background. Mice with combined deficiency (alpha2-AP-/-:PAI-1-/-) are viable, develop normally and are fertile. After amputation of the tail, bleeding times are prolonged (>15 min) in alpha2-AP-/-: PAI-1-/- mice, as compared to double wild-type or single deficient mice (4.6 to 10 min). Spontaneous lysis after 4 h of intravenously injected 125I-fibrin labeled plasma clots is significantly higher in mice with alpha2-AP deficiency both in the PAI-1+/+ background (89+/-2% versus 42+/-3%; p = 0.002) and in the PAI-1-/- background (83+/-4% versus 53+/-5%; p = 0.002). PAI-1 deletion in the alpha2-AP+/+ or alpha2-AP-/- background, however, has no significant effect (p = 0.13 or 0.18, respectively). Four hours after endotoxin injection, fibrin deposition in the kidneys is not significantly affected by PAI-1 deletion in mice with alpha2-AP+/+ or alpha2-AP-/- background (p = 0.07 and 0.19, respectively). In contrast, alpha2-AP deletion causes significantly reduced fibrin deposition in the PAI-1+/+ background (p = 0.01). Endotoxin injection causes a dramatic increase in PAI-1 antigen levels in kidney extracts of PAI-1+/+ animals, without effect on alpha2-AP levels. Taken together, these data indicate that the higher endogenous fibrinolytic capacity observed in mice with combined deficiency is mainly due to the lack of alpha2-AP and suggest a less important role for PAI-1.     

Prothrombotic effect of Rofecoxib in a murine venous thrombosis model

The potential prothrombotic effect of the cyclooxygenase-2 (COX-2) inhibitor Rofecoxib (Vioxx) was investigated using murine thrombosis models. In a jugular vein thrombosis model (photochemically induced injury) in lean wild-type mice, Rofecoxib treatment for 4 weeks induced a mild prothrombotic tendency, as indicated by a shorter occlusion time as compared to placebo (median of 12 min versus 36 min; p<0.05). Thrombus size was somewhat, but not significantly, enhanced after Rofecoxib treatment. In a femoral artery thrombosis model (FeCl(3) induced injury) Rofecoxib did not cause an enhanced thrombotic tendency in mice with nutritionally induced or genetically determined (ob/ob) obesity. The occlusion time was comparable for obese wild-type mice with (8.8+/-0.7 min) or without (7.8+/-2.1 min) Rofecoxib treatment, as well as for ob/ob mice (8.5+/-0.7 min versus 6.8+/-3.0 min). Thus, an enhanced prothrombotic effect of Rofecoxib was detected when using a venous thrombosis model in lean mice, but not when using an arterial thrombosis model in obese mice.     

The CXCR4 antagonist POL5551 is equally effective as sirolimus in reducing neointima formation without impairing re-endothelialisation

Impaired endothelial recovery after the implantation of drug-eluting stents is a major concern because of the increased risk for late stent thrombosis. The disruption of the chemokine axis CXCL12/CXCR4 inhibits neointima formation by blocking the recruitment of smooth muscle progenitor cells. To directly compare a CXCR4-targeting treatment strategy with drugs that are currently used for stent coating, we studied the effects of the CXCR4 antagonist POL5551 and the drug sirolimus on neointima formation. Apolipoprotein E-deficient mice were treated with POL5551 or sirolimus continuously for 28 days after a carotid wire injury. POL5551 inhibited neointima formation by 63% (for a dosage of 2 mg/kg/day) and by 70% (for a dosage of 20 mg/kg/day). In comparison, sirolimus reduced the neointimal area by 69%. In contrast to treatment with POL5551 during the first three days after injury, injection of POL5551 (20 mg/kg) once per day for 28 days diminished neointimal hyperplasia by 53%. An analysis of the cellular composition of the neointima showed a reduction in the relative smooth muscle cell (SMC) and macrophage content in mice that had been treated with a high dose of POL5551. In contrast, the diminished SMC content after sirolimus treatment was associated with a neointimal enrichment of macrophages. Furthermore, endothelial recovery was impaired by sirolimus, but not by POL5551. Therefore, the inhibition of CXCR4 by POL5551 is equally effective in preventing neointima formation as sirolimus, but POL5551 might be more beneficial because treatment with it results in a more stable lesion phenotype and because it does not impair re-endothelialisation.     

Increased expression of protease activated receptor-2 (PAR-2) in balloon-injured rat carotid artery.

Protease-induced cell signaling is mediated by specific receptors such as the emerging family of protease activated receptors (PARs). Since proteases are involved in various aspects of vascular injury, we assessed expression of PAR-2, a protease-activated receptor closely related to the thrombin receptor (PAR-1) but activated by an unknown protease, in vascular injury. Rat carotids were subjected to balloon-catheter injury and perfusion fixed at 1, 3, 7 or 14 days after injury. Sections of injured and normal carotid arteries were immunohistochemically labeled with a polyclonal antibody raised against the N-terminal residues 37-53 of human PAR-2. Sections were also labeled with antibodies to factor VIII-related antigen, smooth muscle actin and a proliferating cell nuclear antigen (PCNA). In normal vessels, PAR-2 labeling was diffuse and patchy in medial smooth muscle and endothelium. At one and three days after injury, before appearance of neointima, PAR-2 labeling increased in cells adjacent to damaged or necrotic smooth muscle cells. In addition, proliferating adventitial myofibroblasts labeled strongly for PAR-2. At 7 and 14 days after injury, the media and neointima of injured vessels had increased PAR-2 labeling which was most intense at the luminal edge of the neointima. Double immunohistochemical labeling confirmed the greatest expression of PAR-2 in areas with the greatest density of PCNA-positive cells. In addition, PAR-2 mRNA localization using in situ hybridization paralleled PAR-2 expression. The data suggest an upregulation of PAR-2 in response to vascular injury which is associated with medial smooth muscle damage, proliferating adventitial myofibroblasts and smooth muscle cells of the neointima, particularly those at the proliferating luminal edge of the neointima. Possible functional consequences of this receptor upregulation and its role in the response to vascular injury remain to be determined.     

Obesity promotes injury induced femoral artery thrombosis in mice

An FeCl(3) induced femoral arterial thrombosis model was applied to lean (47+/-1.4 g) and obese (64+/-1.7 g) mice (Swiss genetic background) in order to study the relation between obesity and thrombotic risk. As compared to lean mice, obese mice showed a significantly shorter occlusion time (9.9+/-1.0 min versus 13+/-0.5 min; p=0.04) and lower total blood flow (37+/-7.3% versus 69+/-6.7%; p=0.008). A significant negative correlation was observed between body weight and both occlusion time (r=-0.57; p=0.014) and blood flow (r=-0.57; p=0.028). Analysis of the coagulation profile revealed significantly higher levels of plasminogen activator inhibitor-1 (PAI-1), thrombin-antithrombin complex, Factor V activity and combined Factors II/VII/X activity, and moderately elevated Factor VIII activity in obese mice. The degree of arterial damage and the thrombus extension were, however, not significantly different. A significant positive correlation was observed between body weight and either PAI-1 (r=0.63; p=0.003), Factors II/VII/X levels (r=0.80; p<0.0001) or Factor V levels (r=0.65; p=0.003). Thus, this injury induced femoral artery thrombosis model in mice establishes experimentally a correlation between obesity and prothrombotic tendency.