In vivo targeted molecular magnetic resonance imaging of free radicals in diabetic cardiomyopathy within mice.

Free radicals contribute to the pathogenesis of diabetic cardiomyopathy. We present a method for in vivo observation of free radical events within murine diabetic cardiomyopathy. This study reports on in vivo imaging of protein/lipid radicals using molecular MRI (mMRI) and immuno-spin trapping (IST) in diabetic cardiac muscle. To detect free radicals in diabetic cardiomyopathy, streptozotocin (STZ)-exposed mice were given 5,5-dimethyl-pyrroline-N-oxide (DMPO) and administered an anti-DMPO probe (biotin-anti-DMPO antibody-albumin-Gd-DTPA). For controls, non-diabetic mice were given DMPO (non-disease control), and administered an anti-DMPO probe; or diabetic mice were given DMPO but administered a non-specific IgG contrast agent instead of the anti-DMPO probe. DMPO administration started at 7 weeks following STZ treatment for 5 days, and the anti-DMPO probe was administered at 8 weeks for MRI detection. MRI was used to detect a significant increase (p < 0.001) in MRI signal intensity (SI) from anti-DMPO nitrone adducts in diabetic murine left-ventricular (LV) cardiac tissue, compared to controls. Regional increases in MR SI in the LV were found in the apical and upper-left areas (p < 0.01 for both), compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised cardiac tissues, which indicated elevated fluorescence only in cardiac muscle of mice administered the anti-DMPO probe. Oxidized lipids and proteins were also found to be significantly elevated (p < 0.05 for both) in diabetic cardiac muscle compared to controls. It can be concluded that diabetic mice have more heterogeneously distributed radicals in cardiac tissue than non-diabetic mice.

Plasmin-dependent proteolysis of tissue factor pathway inhibitor in a mouse model of endotoxemia.

BACKGROUND: The development of a procoagulant state in sepsis, owing to aberrant expression of tissue factor (TF) and a sharp decrease in the level of its major inhibitor, TF pathway inhibitor (TFPI), could lead to microthrombotic organ failure. The mechanism for the decline in TFPI activity in the lung could involve plasmin-mediated cleavage of the inhibitor. OBJECTIVE: To investigate the effect of plasmin generation on lung-associated TFPI activity, in normal conditions and during infusion of endotoxin (lipopolysaccharide [LPS]) in mice. METHODS: Plasmin generation and TFPI activity were assayed in the lungs of mice deficient in tissue-type plasminogen (Plg) activator (t-PA) or Plg, at 2 h after LPS or saline injection. RESULTS: The sharp loss of lung-associated TFPI activity at 2 h after LPS challenge paralleled the abrupt increase in plasmin generation. TFPI activity was significantly retained in both t-PA(-/-) and Plg(-/-) mice, which are unable to generate plasmin. CONCLUSION: The increased plasmin generation during the early stages of sepsis could cleave/inactivate TFPI and thus lead to thrombotic complications.

Innate immunity and coagulation.

Infection frequently elicits a coagulation response. Endotoxin triggers the formation of tissue factor initiating coagulation, down regulates anticoagulant mechanisms including the protein C pathway and heparin-like proteoglycans and up regulates plasminogen activator inhibitor. The overall physiological result of this is to promote coagulation through enhancing initiation, suppressing negative regulation and impairing fibrin removal. The response to infection also leads to tissue destruction. Nucleosomes and histones released from the injured cells trigger further inflammation, protection from the pathogen but further tissue injury leading to multi-organ failure. Such a complex response to infection presumably arises due to the role of coagulation in the control and clearance of the infectious agent.

Inflammation, innate immunity and blood coagulation.

Inflammation drives arterial, venous and microvascular thrombosis. Chronic inflammation contributes to arterial thrombotic complications, whereas acute inflammation drives venous thrombosis and microvascular thrombosis. Mechanistically, inflammation modulates thrombotic responses by upregulating procoagulants, downregulating anticoagulants and suppressing fibrinolysis. The inflammatory response can also result in cell apoptosis or necrosis. Products released from the dead cells, particularly histones, propagate further inflammation, tissue death and organ failure. Inhibition of histone mediated cytotoxicity appears to be a new mechanism for protecting against this deadly cascade.

In vivo detection of c-Met expression in a rat C6 glioma model.

The tyrosine kinase receptor, c-Met, and its substrate, the hepatocyte growth factor (HGF), are implicated in the malignant progression of glioblastomas. In vivo detection of c-Met expression may be helpful in the diagnosis of malignant tumours. The C6 rat glioma model is a widely used intracranial brain tumour model used to study gliomas experimentally. We used a magnetic resonance imaging (MRI) molecular targeting agent to specifically tag the cell surface receptor, c-Met, with an anti-c-Met antibody (Ab) linked to biotinylated Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-albumin in rat gliomas to detect overexpression of this antigen in vivo. The anti-c-Met probe (anti-c-Met-Gd-DTPA-albumin) was administered intravenously, and as determined by an increase in MRI signal intensity and a corresponding decrease in regional T(1) relaxation values, this probe was found to detect increased expression of c-Met protein levels in C6 gliomas. In addition, specificity for the binding of the anti-c-Met contrast agent was determined by using fluorescence microscopic imaging of the biotinylated portion of the targeting agent within neoplastic and 'normal'brain tissues following in vivo administration of the anti-c-Met probe. Controls with no Ab or with a normal rat IgG attached to the contrast agent component indicated no non-specific binding to glioma tissue. This is the first successful visualization of in vivo overexpression of c-Met in gliomas.

Hyperexcitability of convergent colon and bladder dorsal root ganglion neurons after colonic inflammation: mechanism for pelvic organ cross-talk.

Clinical studies reveal concomitant occurrence of several gastrointestinal and urologic disorders, including irritable bowel syndrome and interstitial cystitis. The purpose of this study was to determine the mechanisms underlying cross-organ sensitization at the level of dorsal root ganglion (DRG) after acute and subsided gastrointestinal inflammation. DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) and Fast Blue were injected into the distal colon and urinary bladder of male rats, respectively. Convergent DRG neurons were found in L1-L3 and L6-S2 ganglia with an average distribution of 14% +/- 2%. The resting membrane potential (RMP) of cells isolated from upper lumbar (UL) ganglia was -59.8 +/- 2.7 mV, whereas lumbosacral (LS) neurons were more depolarized (RMP = -49.4 +/- 2.1 mV, P < or = 0.05) under control conditions. Acute trinitrobenzene sulfonic acid (TNBS) colitis (3 days) decreased voltage and current thresholds for action potential firing in LS but not UL convergent capsaicin-sensitive neurons. This effect persisted for 30 days in the absence of overt colonic inflammation. The current threshold for action potential (AP) firing in UL cells was also decreased from 165.0 +/- 24.5 pA (control) to 85.0 +/- 19.1 pA at 30 days (P < or = 0.05), indicating increased excitability. The presence of a subpopulation of colon-bladder convergent DRG neurons and their persistent hyperexcitability after colonic inflammation provides a basis for pelvic organ cross-sensitization.

Plasminogen activation: a mediator of vascular smooth muscle cell apoptosis in atherosclerotic plaques.

BACKGROUND: Apoptosis of vascular cells is considered to be a major determinant of atherosclerotic plaque vulnerability and potential rupture. Plasmin can be generated in atherosclerotic plaques and recent in vitro data suggest that plasminogen activation may trigger vascular smooth muscle cell (VSMC) apoptosis. AIM: To determine whether plasminogen activation may induce aortic VSMC apoptosis ex vivo and in vivo. METHODS AND RESULTS: Mice with single or combined deficiencies of apolipoprotein E (ApoE) and plasminogen activator inhibitor-1 (PAI-1) were used. Ex vivo incubation with plasminogen of isolated aortic tunica media from PAI-1-deficient mice induced plasminogen activation and VSMC apoptosis, which was inhibited by alpha2-antiplasmin. In vivo, levels of plasmin, active caspase 3 and VSMC apoptotic index were significantly higher in atherosclerotic aortas from mice with combined ApoE and PAI-1 deficiencies than in those from littermates with single ApoE deficiency. A parallel decrease in VSMC density was observed. CONCLUSIONS: These data strongly suggest that plasminogen activation may contribute to VSMC apoptosis in atherosclerotic plaques.

Overexpressing endothelial cell protein C receptor alters the hemostatic balance and protects mice from endotoxin.

Previous studies have shown that blocking endothelial protein C receptor (EPCR)-protein C interaction results in about an 88% decrease in circulating activated protein C (APC) levels generated in response to thrombin infusion and exacerbates the response to Escherichia coli. To determine whether higher levels of EPCR expression on endothelial cells might further enhance the activation of protein C and protect the host during septicemia, we generated a transgenic mouse (Tie2-EPCR) line which placed the expression of EPCR under the control of the Tie2 promoter. The mice express abundant EPCR on endothelial cells not only on large vessels, but also on capillaries where EPCR is generally low. Tie2-EPCR mice show higher levels of circulating APC after thrombin infusion. Upon infusion with factor Xa and phospholipids, Tie2-EPCR mice generate more APC, less thrombin and are protected from fibrin/ogen deposition compared with wild type controls. The Tie2-EPCR animals also generate more APC upon lipopolysaccharide (LPS) challenge and have a survival advantage. These results reveal that overexpression of EPCR can protect animals against thrombotic or septic challenge.

Persistence of atherosclerotic plaque but reduced aneurysm formation in mice with stromelysin-1 (MMP-3) gene inactivation.

To investigate a potential role for stromelysin-1 (MMP-3) in the development and progression of atherosclerotic lesions and aneurysm formation, mice with a deficiency of apolipoprotein E (ApoE(-/-):MMP-3(+/+))) or with a combined deficiency of apoE and MMP-3 (ApoE(-/-):MMP-3(-/-)) were kept on a cholesterol-rich diet for 30 weeks. Atherosclerotic lesions throughout the thoracic aorta were significantly larger in ApoE(-/-):MMP-3(-/-) than in ApoE(-/-):MMP-3(+/+) mice (P<0.05) and contained more fibrillar collagen (P<0.01). Aneurysms in the thoracic and abdominal aortas were less frequent in ApoE(-/-):MMP-3(-/-) than in ApoE(-/-):MMP-3(+/+) mice (8.5+/-1.7% vs 14+/-2.1% of sections, mean+/-SD, P<0.01). Immunocytochemistry revealed enhanced accumulation of macrophages in atherosclerotic lesions of ApoE(-/-):MMP-3(+/+) mice (P<0.01) and expression of urokinase-type plasminogen activator (u-PA) and MMP-3 colocalizing with macrophages. Zymography confirmed the presence of u-PA and MMP-3 activity in extracts of atherosclerotic aortas. These data suggest that plasmin, generated by macrophage-secreted u-PA, activates pro-MMP-3 produced by accumulated macrophages. MMP-3 activity may then contribute to a reduction of plaque size, possibly by degradation of matrix components, and promote aneurysm formation by degradation of the elastica lamina.

Release of soluble urokinase receptor from vascular cells.

Urokinase-type plasminogen activator (uPA) and its cell surface-receptor (uPAR) regulate cellular functions linked to adhesion and migration and contribute to pericellular proteolysis in tissue remodelling processes. Soluble uPAR (suPAR) is present in the circulation, peritoneal and ascitic fluid and in the cystic fluid from ovarian cancer. We have investigated the origin and the vascular distribution of the soluble receptor, which accounts for 10-20% of the total receptor in vascular endothelial and smooth muscle cells. Phase separation analysis of the cell conditioned media with Triton X-114 indicated that suPAR associates with the aqueous phase, indicative of the absence of the glycolipid anchor. There was a polarized release of suPAR from cultured endothelial cells towards the basolateral direction, whereas the membrane-bound receptor was found preferentially on the apical surface. Both, uPAR and suPAR became upregulated 2-4 fold after activation of protein kinase C with phorbol ester, which required de-novo protein biosynthesis. Interleukin-1beta (IL-1beta), basic fibroblast growth factor (bFGF) or vascular endothelial growth factor increased suPAR release from endothelial cells, whereas platelet derived growth factor-BB, bFGF or IL-1beta stimulated suPAR release from vascular smooth muscle cells. Immune electron microscopy indicated that in atherosclerotic vessels (s)uPAR was observed on cell membranes as well as in the extracellular matrix. These findings indicate that (s)uPAR from vascular cells is upregulated by proangiogenic as well as proatherogenic growth factors and cytokines, is preferentially released towards the basolateral side of endothelial cells and accumulates in the vessel wall.

Adipose tissue expression of gelatinases in mouse models of obesity.

Following the observation by Brown et al. (Am J Physiol 1997; 272: C937-49) that primary rat adipocytes in culture secrete gelatinase A (MMP-2), we have evaluated gelatinase expression in adipose tissue with the use of mouse models of obesity. Wild-type mice were kept on a standard fat diet (SFD) or on a high fat diet (42% fat, HFD) and- genetically obese db/db mice were kept on SFD; gonadal and subcutaneous fat pads were removed and analysed ex vivo. These studies revealed that: 1) the HFD induced adipocyte hypertrophy; 2) after 32 weeks, significantly higher levels of 70 kDa (p <0.05) and 65 kDa proMMP-2 (p <0.01) were observed in extracts of gonadal fat pads of mice on HFD; 3) the contribution of active MMP-2 to the total level was comparable in SFD and HFD groups (20 to 30%); and 4) gelatinase B (MMP-9) was not consistently detected. These findings were confirmed by gelatin zymography and by mRNA determination using competitive RT-PCR. The presence of MMP-2 in the adipose tissue was confirmed immunologically and its localization in adipocytes revealed by immunogold electron microscopy. The potential functional role of MMP-2 in adipose tissue remains to be determined.

Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration.

Hypoxia stimulates angiogenesis through the binding of hypoxia-inducible factors to the hypoxia-response element in the vascular endothelial growth factor (Vegf) promotor. Here, we report that deletion of the hypoxia-response element in the Vegf promotor reduced hypoxic Vegf expression in the spinal cord and caused adult-onset progressive motor neuron degeneration, reminiscent of amyotrophic lateral sclerosis. The neurodegeneration seemed to be due to reduced neural vascular perfusion. In addition, Vegf165 promoted survival of motor neurons during hypoxia through binding to Vegf receptor 2 and neuropilin 1. Acute ischemia is known to cause nonselective neuronal death. Our results indicate that chronic vascular insufficiency and, possibly, insufficient Vegf-dependent neuroprotection lead to the select degeneration of motor neurons.

Deficiency or inhibition of Gas6 causes platelet dysfunction and protects mice against thrombosis.

The growth arrest-specific gene 6 product (Gas6) is a secreted protein related to the anticoagulant protein S but its role in hemostasis is unknown. Here we show that inactivation of the Gas6 gene prevented venous and arterial thrombosis in mice, and protected against fatal collagen/epinephrine-induced thrombo embolism. Gas6-/- mice did not, however, suffer spontaneous bleeding and had normal bleeding after tail clipping. In addition, we found that Gas6 antibodies inhibited platelet aggregation in vitro and protected mice against fatal thrombo embolism without causing bleeding in vivo. Gas6 amplified platelet aggregation and secretion in response to known agonists. Platelet dysfunction in Gas6-/- mice resembled that of patients with platelet signaling transduction defects. Thus, Gas6 is a platelet-response amplifier that plays a significant role in thrombosis. These findings warrant further evaluation of the possible therapeutic use of Gas6 inhibition for prevention of thrombosis.

Aberrant fibrin formation and cross-linking of fibrinogen Nieuwegein, a variant with a shortened Aalpha-chain, alters endothelial capillary tube formation.

A congenital dysfibrinogenemia, fibrinogen(Nieuwegein), was discovered in a young man without any thromboembolic complications or bleeding. A homozygous insertion of a single nucleotide (C) in codon Aalpha 453 (Pro) introduced a stop codon at position 454, which resulted in the deletion of the carboxyl-terminal segment Aalpha 454-610. The ensuing unpaired cysteine at Aalpha 442 generated fibrinogen-albumin complexes of different molecular weights. The molecular abnormalities of fibrinogen(Nieuwegein) led to a delayed clotting and a fibrin network with a low turbidity. Electron microscopy confirmed that thin fibrin bundles were organized in a fine network. The use of fibrinogen(Nieuwegein)-derived fibrin (fibrin(Nieuwegein)) in an in vitro angiogenesis model resulted in a strong reduction of tube formation. The ingrowth of human microvascular endothelial cells (hMVEC) was independent of alpha(v)beta(3), indicating that the reduced ingrowth is not due to the absence of the RGD-adhesion site at position Aalpha 572-574. Rather, the altered structure of fibrin(Nieuwegein) is the cause, since partial normalization of the fibrin network by lowering the pH during polymerization resulted in an increased tube formation. Whereas factor XIIIa further decreased the ingrowth of hMVEC in fibrin(Nieuwegein), tissue transglutaminase (TG), which is released in areas of vessel injury, did not. This is in line with the absence of the cross-linking site for TG in the alpha-chains of fibrinogen(Nieuwegein). In conclusion, this newly discovered congenital dysfibrinogenemia has a delayed clotting time and leads to the formation of an altered fibrin structure, which could not be cross-linked by TG and which is less supportive for ingrowth of endothelial cells.

Roles of growth hormone and insulin-like growth factor 1 in mouse postnatal growth.

To examine the relationship between growth hormone (GH) and insulin-like growth factor 1 (IGF1) in controlling postnatal growth, we performed a comparative analysis of dwarfing phenotypes manifested in mouse mutants lacking GH receptor, IGF1, or both. This genetic study has provided conclusive evidence demonstrating that GH and IGF1 promote postnatal growth by both independent and common functions, as the growth retardation of double Ghr/Igf1 nullizygotes is more severe than that observed with either class of single mutant. In fact, the body weight of these double-mutant mice is only approximately 17% of normal and, in absolute magnitude ( approximately 5 g), only twice that of the smallest known mammal. Thus, the growth control pathway in which the components of the GH/IGF1 signaling systems participate constitutes the major determinant of body size. To complement this conclusion mainly based on extensive growth curve analyses, we also present details concerning the involvement of the GH/IGF1 axis in linear growth derived by a developmental study of long bone ossification in the mutants.

Bemiparin and fluid flow modulate the expression, activity and release of tissue factor pathway inhibitor in human endothelial cells in vitro.

We investigated the localisation, gene expression, and activity of tissue factor pathway inhibitor (TFPI) in endothelial cells (EC) grown in static conditions or under shear stress, in the presence of unfractionated heparin (UFH) and two low-molecular-weight heparins (LMWHs). dalteparin and bemiparin (a second generation of LMWHs). All three preparations induced increased release, cellular redistribution, and enhanced activity of TFPI on the cell surface in static EC. In EC grown under shear stress (0.27, 4.1 and 19 dyne/cm2) and incubated with each heparin for 24 h, the release of TFPI was significantly correlated with the level of flow for bemiparin and dalteparin, but not for UFH. For all three levels of flow tested, bemiparin induced the highest secretion and increase of both cellular TFPI and cell surface activity of the inhibitor. The expression of TFPI mRNA, determined by Northern blotting, was specifically modulated by heparins. All three preparations increased the expression of TFPI by 60 to 120% in EC under minimal flow, but only bemiparin enhanced TFPI mRNA in EC under the arterial flow. Immunogold electron microscopy revealed that EC exhibited strong cellular labelling for TFPI when grown under arterial flow in the presence of bemiparin. We conclude that in EC subjected to shear stress in vitro bemiparin is more efficient than UFH or dalteparin in modulating the expression. release and activity of TFPI. We therefore suggest that bemiparin may be superior over the conventional heparins in maintaining the anticoagulant properties of the endothelium.

In situ analysis of tissue factor-dependent thrombin generation in human atherosclerotic vessels.

Tissue factor (TF) is expressed in human atherosclerotic plaques where it may contribute to the thrombogenicity of the lesions and their progression toward unstable syndromes and acute myocardial infarction. In this study we tested the hypothesis that thrombin generation takes place in the lesion. Localisation of TF, factor VII (FVII), factor X/Xa (FX/Xa), thrombin, thrombin receptor PAR-1 and FXa receptor EPR-1 was done by immunostaining, ligand binding, or immunogold electron microscopy. Quantitation of TF antigen was done using a modified ELISA on fixed tissue sections. The amount of antigen was correlated with the pattern and intensity of immunostaining as detected on consecutive sections using confocal microscopy. TF-dependent generation of FXa on cryosections was used to assess the functional activity of TF. Active thrombin was detected using hirudin as a specific probe, followed by anti-hirudin IgG. Our light microscopy and immunogold electron microscopy results showed that the factors involved in TF-dependent coagulation are localised in atherosclerotic plaques in close proximity and colocalise with active thrombin and fibrin deposits. We have detected 3 to 7-fold increase of TF antigen and TF-dependent FXa generation in atherosclerotic vessels as compared with controls. Hirudin binding proved that active thrombin is present within the lesions. In conclusion, our data show that active coagulation factors are generated within atherosclerotic lesions and co-localise with their cellular receptors. These findings may suggest possible roles of the TF-dependent coagulation pathway in the intramural fibrin deposition and the progression of the atherosclerotic lesions.

Fluid flow induces upregulation of synthesis and release of tissue factor pathway inhibitor in vitro.

Fluid flow modulates the synthesis and secretion by endothelial cells (ECs) of several proteins that control the hemostatic properties of the vessel wall. Tissue factor pathway inhibitor (TFPI), also synthesized by ECs, is the main downregulator of tissue factor-dependent procoagulant activity. In the present study, we investigated the effect of physiological shear stress on the expression, distribution, and release of TFPI in cultured ECs. The EA.hy926 cell line was grown in a hollow-fiber perfusion system and exposed for variable times to different shear values: 0.27 dyne/cm(2) (minimal flow), 4.1 dyne/cm(2) (venous flow), and 19 dyne/cm(2) (moderate arterial flow). Step increase of the shear stress from 0.27 to 19 dyne/cm(2) induced a sharp increase of TFPI released into the medium and a parallel decrease and redistribution of cell-associated TFPI, which suggests that an acute release of TFPI occurred from the cellular pools. During 24 hours of high shear stress, cell-associated TFPI antigen and mRNA increased time-dependently. Subjecting ECs to steady shear stress for 72 hours also upregulated the expression and production of TFPI, in direct correlation with the degree of the shear. The secretion of TFPI was enhanced 1.9-fold under venous flow and 2.4-fold under arterial flow compared with minimal flow. Equally, cell-associated TFPI antigen and cell surface TFPI activity increased proportionally with the shear stress. The expression of TFPI mRNA, as determined by Northern blotting, increased up to 2-fold in ECs under venous flow and up to 3-fold under arterial flow. These results suggest that shear forces regulate TFPI by modulating its release and gene expression in ECs in vitro.

Unfractionated and low molecular weight heparin affect fibrin structure and angiogenesis in vitro.

Cancer patients treated for venous thromboembolism with low molecular weight heparin (LMWH) have a better survival rate than patients treated with unfractionated heparin (UFH). Because fibrin-associated angiogenesis is an important determinant in the progression and metastasis of many solid tumors, the effects of heparins on in vitro angiogenesis were investigated. Both UFH and LMWH inhibited bFGF-induced proliferation of human microvascular endothelial cells (hMVECs) to the same the extent (36-60%). VEGF165-induced proliferation was inhibited to a to a lesser extent (19-33%). Turbidity measurements and electron microscopy showed that the presence of LMWH during polymerization of the fibrin matrix led to a more transparent rigid network with thin fibrin bundles, whereas the presence of UFH resulted in a more opaque more porous network with thick fibrin fibers. We used a human in vitro angiogenesis model, which consisted of hMVECs seeded on top of a fibrin matrix, and stimulated the cells with basic fibroblast growth factor plus tumor necrosis factor a to induce capillary-like tubular structures. The formation of capillary-like tubular structures was retarded with matrices polymerized in the presence of LMWH (46% inhibition compared with a control matrix for both 1.5 and 10 units/ml LMWH), whereas matrices polymerized in the presence of UFH facilitated tubular structure formation (72 and 36% stimulation compared with a control matrix for 1.5 and 10 units/ml UFH, respectively). Similar results were obtained for cells stimulated with vascular endothelial growth factor plus tumor necrosis factor alpha. These data demonstrate the inhibitory effect of heparins on proliferation of hMVECs and provide a novel mechanism by which LMWH may affect tumor progression, namely reduced ingrowth of microvascular structures in a fibrinous stroma matrix by rendering it less permissive for invasion.

CD9 gene deficiency does not affect smooth muscle cell migration and neointima formation after vascular injury in mice.

The hypothesis that CD9, a member of the tetraspanin family, plays a role in smooth muscle cell (SMC) migration was tested with the use of a vascular injury model in wild-type (CD9+/+) and CD9-deficient (CD9-/-) mice. Neointima formation 3 weeks after electric injury of the femoral artery was not significantly different in CD9+/+ and CD9-/- mice (area of 0.019 +/- 0.0034 mm2 versus 0.013 +/- 0.0036 mm2; mean +/- SEM, n = 6). The medial areas were also comparable, resulting in intima/media ratio's of 1.3 +/- 0.15 and 0.90 +/- 0.22, respectively. Nuclear cell counts in cross-sectional areas of the injured region were comparable in media (33 +/- 5 versus 27 +/- 2) and neointima (135 +/- 16 versus 97 +/- 17) of CD9+/+ and CD9-/- arteries. Immunocytochemical analysis revealed expression of CD9 in the endothelium, by SMC in the media and by some fibroblasts in the adventitia of non-injured femoral arteries. Three weeks after injury, there appeared to be a gradient of increased CD9 expression from the adventitia to the neointima, in which SMC are abundantly present. Immunogold labeling and electron microscopy with non-injured femoral arteries of CD9+/+ mice confirmed the presence of CD9 at the surface of adventitial fibroblasts and in SMC or pericytes, as well as in the endothelium. Thus, in this model CD9 is highly expressed by migrating SMC, but deficiency of CD9 does not affect SMC migration or neointima formation after perivascular injury.