Mark B. Taubman

Chemokines in the Pathogenesis of Vascular Disease

Our increasing appreciation of the importance of inflammation in vascular disease has focused attention on the molecules that direct the migration of leukocytes from the blood stream to the vessel wall. In this review, we summarize roles of the chemokines, a family of small secreted proteins that selectively recruit monocytes, neutrophils, and lymphocytes to sites of vascular injury, inflammation, and developing atherosclerosis. Chemokines induce chemotaxis through the activation of G-protein-coupled receptors, and the receptors that a given leukocyte expresses determines the chemokines to which it will respond. Monocyte chemoattractant protein 1 (MCP-1), acting through its receptor CCR2, appears to play an early and important role in the recruitment of monocytes to atherosclerotic lesions and in the formation of intimal hyperplasia after arterial injury. Acute thrombosis is an often fatal complication of atherosclerotic plaque rupture, and recent evidence suggests that MCP-1 contributes to thrombin generation and thrombus formation by generating tissue factor. Because of their critical roles in monocyte recruitment in vascular and nonvascular diseases, MCP-1 and CCR2 have become important therapeutic targets, and efforts are underway to develop potent and specific antagonists of these and related chemokines.

Rapamycin inhibits vascular smooth muscle cell migration.

Abnormal vascular smooth muscle cell (SMC) proliferation and migration contribute to the development of restenosis after percutaneous transluminal coronary angioplasty and accelerated arteriopathy after cardiac transplantation. Previously, we reported that the macrolide antibiotic rapamycin, but not the related compound FK506, inhibits both human and rat aortic SMC proliferation in vitro by inhibiting cell cycle-dependent kinases and delaying phosphorylation of retinoblastoma protein (Marx, S.O., T. Jayaraman, L.O. Go, and A.R. Marks. 1995. Circ. Res. 362:801). In the present study the effects of rapamycin on SMC migration were assayed in vitro using a modified Boyden chamber and in vivo using a porcine aortic SMC explant model. Pretreatment with rapamycin (2 ng/ml) for 48 h inhibited PDGF-induced migration (PDGF BB homodimer; 20 ng/ml) in cultured rat and human SMC (n = 10; P < 0.0001), whereas FK506 had no significant effect on migration. Rapamycin administered orally (1 mg/kg per d for 7 d) significantly inhibited porcine aortic SMC migration compared with control (n = 15; P < 0.0001). Thus, in addition to being a potent immunosuppressant and antiproliferative, rapamycin also inhibits SMC migration.

Identification of Active Tissue Factor in Human Coronary Atheroma

BACKGROUND Recent observations suggest that thrombosis in vivo is initiated via the tissue factor (TF) pathway. The TF activity of human coronary atheroma has not been reported. METHODS AND RESULTS Directional coronary atherectomy (DCA) specimens from 63 lesions were analyzed with the use of a quantitative TF-specific activity assay. The median content of TF was 10 ng/g plaque (95% CI, 6 to 13 ng/g; range, 0 to 47 ng/g). After homogenization of the specimens, TF activity was detected in 28 of 31 lesions (90%). With a polyclonal anti-human TF antibody, the use of immunohistochemistry detected TF antigen in 43 of 50 lesions (86%); TF antigen was expressed in cellular and acellular areas of the plaque. Histologically defined thrombus was present in 19 of the 43 lesions with detectable TF antigen and in none of the 7 lesions without detectable TF antigen (19 of 43 versus 0 of 7; P < .02). TF antigen was undetectable with immunohistochemistry in 4 of 13 restenotic lesions (31%) and in 3 of 37 de novo lesions (8%) (P < .05). CONCLUSIONS TF contributes to the procoagulant activity of most atherosclerotic lesions treated with DCA. The association of immunohistochemically detectable TF with plaque thrombus suggests that TF plays a role in coronary thrombosis. Diminished TF expression in restenotic lesions may in part account for the lower complication rate that has been associated with DCA of restenotic versus de novo lesions. Inhibition of TF may represent a therapeutic goal for the prevention of thrombotic complications associated with percutaneous coronary interventions.

Tissue factor expression, angiogenesis, and thrombosis in pancreatic cancer.

Purpose: Hemostatic activation is common in pancreatic cancer and may be linked to angiogenesis and venous thromboembolism. We investigated expression of tissue factor (TF), the prime initiator of coagulation, in noninvasive and invasive pancreatic neoplasia. We correlated TF expression with vascular endothelial growth factor (VEGF) expression, microvessel density, and venous thromboembolism in resected pancreatic cancer. Experimental Design: Tissue cores from a tri-institutional retrospective series of patients were used to build tissue microarrays. TF expression was graded semiquantitatively using immunohistochemistry in normal pancreas (n = 10), intraductal papillary mucinous neoplasms (n = 70), pancreatic intraepithelial neoplasia (n = 40), and resected or metastatic pancreatic adenocarcinomas (n = 130). Results: TF expression was observed in a majority of noninvasive and invasive pancreatic neoplasia, including 77% of pancreatic intraepithelial neoplasias, 91% of intraductal papillary mucinous neoplasms, and 89% of pancreatic cancers, but not in normal pancreas. Sixty-six of 122 resected pancreatic cancers (54%) were found to have high TF expression (defined as grade ≥2, the median score). Carcinomas with high TF expression were more likely to also express VEGF (80% versus 27% with low TF expression, P < 0.0001) and had a higher median MVD (8 versus 5 per tissue core with low TF expression, P = 0.01). Pancreatic cancer patients with high TF expression had a venous thromboembolism rate of 26.3% compared with 4.5% in patients with low TF expression (P = 0.04). Conclusions: TF expression occurs early in pancreatic neoplastic transformation and is associated with VEGF expression, increased microvessel density, and possibly clinical venous thromboembolism in pancreatic cancer. Prospective studies evaluating the role of TF in pancreatic cancer outcomes are warranted.

Tissue factor is rapidly induced in arterial smooth muscle after balloon injury.

Tissue factor (TF) is a major activator of the coagulation cascade and may play a role in initiating thrombosis after intravascular injury. To investigate whether medial vascular smooth muscle provides a source of TF following arterial injury, the induction of TF mRNA and protein was studied in balloon-injured rat aorta. After full length aortic injury, aortas were harvested at various times and the media and adventitia separated using collagenase digestion and microscopic dissection. In uninjured aortic media, TF mRNA was undetectable by RNA blot hybridization. 2 h after balloon injury TF mRNA levels increased markedly. Return to near baseline levels occurred at 24 h. In situ hybridization with a 35S-labeled antisense rat TF cRNA probe detected TF mRNA in the adventitia but not in the media or endothelium of uninjured aorta. 2 h after balloon dilatation, a marked induction of TF mRNA was observed in the adventitia and media. Using a functional clotting assay, TF procoagulant activity was detected at low levels in uninjured rat aortic media and rose by approximately 10-fold 2 h after balloon dilatation. Return to baseline occurred within 4 d. These data demonstrate that vascular injury rapidly induces active TF in arterial smooth muscle, providing a procoagulant that may result in thrombus initiation or propagation.

Tissue Factor Is Induced by Monocyte Chemoattractant Protein-1 in Human Aortic Smooth Muscle and THP-1 Cells

Monocyte chemoattractant protein-1 (MCP-1) is a C-C chemokine thought to play a major role in recruiting monocytes to the atherosclerotic plaque. Tissue factor (TF), the initiator of coagulation, is found in the atherosclerotic plaque, macrophages, and human aortic smooth muscle cells (SMC). The exposure of TF during plaque rupture likely induces acute thrombosis, leading to myocardial infarction and stroke. This report demonstrates that MCP-1 induces the accumulation of TF mRNA and protein in SMC and in THP-1 myelomonocytic leukemia cells. MCP-1 also induces TF activity on the surface of human SMC. The induction of TF by MCP-1 in SMC is inhibited by pertussis toxin, suggesting that the SMC MCP-1 receptor is coupled to a Gi-protein. Chelation of intracellular calcium and inhibition of protein kinase C block the induction of TF by MCP-1, suggesting that in SMC it is mediated by activation of phospholipase C. SMC bind MCP-1 with a K d similar to that previously reported for macrophages. However, mRNA encoding the macrophage MCP-1 receptors, CCR2A and B, is not present in SMC, indicating that they possess a distinct MCP-1 receptor. These data suggest that in addition to being a chemoattractant, MCP-1 may have a procoagulant function and raise the possibility of an autocrine pathway in which MCP-1, secreted by SMC and macrophages, induces TF activity in these same cells.

Plasma tissue factor may be predictive of venous thromboembolism in pancreatic cancer

Pancreatic cancer is the fourth leading cause of cancer death in the US [1]. It is frequently complicated by venous thromboembolism (VTE), with published incidence rates varying from 17% to 57% [2]. VTE can directly contribute to mortality, and has been associated with poor outcomes in pancreatic cancer [3]. Tissue factor (TF) is the principal physiologic initiator of coagulation and is commonly expressed in a variety of cancers. In pancreatic cancer, high grade TF expression is an adverse prognostic factor [4]. We recently published findings showing that TF expression occurs early in malignant transformation of the pancreas, is associated with angiogenesis and may be predictive of subsequent VTE [5]. Patients with high-grade TF expression had a symptomatic VTE rate of 26.3% compared with 4.5% in patients with low expression (P = 0.04). Similar findings have been reported in ovarian cancer [6]. Patients with metastatic pancreatic cancer have been shown to have increased levels of microparticle (MP)-associated TF activity compared with healthy controls [7]. We hypothesized that elevated circulating levels of TF would be associated with development of VTE in pancreatic cancer. We, therefore, measured TF in samples obtained from patients with locally advanced or metastatic pancreatic cancer enrolled in a prospective trial conducted by the University of Rochester Community Clinical Oncology Program. Selected sites participating in the trial also offered participation in a laboratory companion study from 2 October 2002 to 29 December 2006. The study was approved by an Institutional Review Board. All patients provided written informed consent. All patients were randomized to single-agent gemcitabine alone or with dalteparin 5000 anti-Xa units until progression. Blood collection was planned at baseline (prior to initiating chemotherapy) and every 4 weeks while on the study. Citrated plasma was processed within 1 h of collection by centrifuging at 2000 × g for 15 min at 8 °C to produce platelet-poor plasma (PPP; <10 000 platelets μL−1) and then stored at −80 °C. TF antigen (Ag) levels were determined by an in-house ELISA from citrated plasma stored at −80 °C. Briefly, 100 μL of the mouse anti-human TF antibody, hTF1 (5 μg mL−1 in PBS), was coated onto 96-well ELISA plates (Nunc No. 439454) by overnight incubation at 4 °C. All subsequent washes and incubations were done at 25 °C. Plates were washed with PBS-Tween 20 and blocked with casein (Vector Labs No. SP-5020) for 15 min. Stored plasma samples were thawed, mixed, centrifuged at 350 × g for 1 min, and diluted 1:1 in casein. Serial dilutions of a recombinant soluble TF1–219 (sTF) standard were added to the plates in duplicate. One hundred microliters of the diluted plasma samples were added to the plates in triplicate, incubated at room temperature for 2 h and then washed. One hundred microliters of an sTF immuno-purified, biotinylated, F(ab′)2-fragmented rabbit anti-sTF anti-body (0.12 μg mL−1 in casein) was added to each well. The plates were incubated for 2 h and then washed. Streptavidin (Pierce No. 21126) (Pierce, Rockford, IL, USA) was diluted 1:10 000 and 100 μL was added to each well. The plates were incubated at room temperature for 30 min and then washed. One hundred microliters of the 1-Step Ultra TMB-ELISA reagent (Pierce No. 34028) was added to each well. The plates were incubated for 8 min and the reaction was stopped with 100 μL of 2 M sulfuric acid before reading the optical density (OD) at 450 nm in a 96-well reader (Spectramax190, Molecular Devices, Sunnyvale, CA, USA) from Molecular Devices using SoftmaxPro software (Molecular Devices, Sunnyvale, CA). TF concentrations were determined from a standard plot of the sTF protein concentrations and reported as pg mL−1 of sTF. To determine a normal level for TF Ag, we measured TF in plasma from 15 healthy volunteers and from a commercially available pooled plasma control (Innovative Research, Inc., Southfield, MI, USA) repeated six times in separate assays. Mean plasma TF was 27.0 pg mL−1 (SD 12.9) in healthy volunteers, and 26.9 pg mL−1 (SD 3.94) in pooled plasma control, with an interassay coefficient of variability (CV) of 14.7%. TF was also measured using a procoagulant activity (PCA) assay in a similar manner to a recent study [7]. MPs were pelleted from 200 μL of PPP by centrifugation at 20 000 × g for 15 min at 4 °C, washed twice with HBSA (137 mM NaCl, 5.38 mM KCl, 5.55 mM glucose, 10 mM HEPES, 0.1% bovine serum albumin, pH 7.5), and re-suspended in 100 μL of HBSA. Samples were incubated with either hTF1 (4 μg mL−1; 1 μL) or a control antibody (mouse IgG: 4 μg mL−1; 1 μL) for 15 min at 25 °C and then 50 μL aliquots were added to duplicate wells of a 96-well plate. Next, 50 μL of HBSA containing 10 nM FVIIa, 300 nM FX and 10 mM CaCl2 was added to each sample and the mixture incubated for 2 h at 37 °C. FXa generation was stopped by the addition of 25 μL of 25 mM EDTA buffer and 25 μL of the chromogenic substrate S2765 (4 mM) was added and incubated at 37 °C for 15 min. Finally, absorbance at 405 nm was measured using a VERSAmax microplate reader (Molecular Devices). TF activity was calculated by reference to a standard curve generated using relipidated recombinant human TF (0–55 pg mL−1). The TF-dependent FXa generation (pg mL−1) was determined by subtracting the amount of FXa generated in the presence of hTF1 from the amount of FXa generated in the presence of the control antibody. Mean TF PCA in healthy controls was 0.21 pg mL−1 (SD 0.11) with an interassay CV of 21%. Eleven patients provided at least one blood specimen (range, 1–8; median five samples per patient). TF Ag was measured in all 11 patients and TF MP PCA in 10 patients with available specimens. The mean TF Ag level (± SD) for all timepoints was 30.9 pg mL−1 (±15.4) and mean TF PCA was 0.85 pg mL−1 (±1.26). Mean TF Ag levels and MP-PCA at baseline were 56.6 and 0.95 pg mL−1, respectively. However, there was a wide variation, with baseline TF Ag levels ranging from 0 to 269 pg mL−1 (Fig. 1A) and TF MP PCA from 0 to 3.1 pg mL−1. Nine patients had levels of plasma TF Ag (32 ± 19 pg mL−1 SD) that were similar to those seen in healthy volunteers although TF MP PCA was elevated in these patients compared with controls (0.46 ± 1.26 pg mL−1, P = 0.01). In these patients, both TF levels and PCA remained stable throughout treatment. None of these patients developed VTE. Four of these nine patients had been randomized to dalteparin. In contrast, patient 5, randomized to dalteparin, had a rapid increase in TF Ag and PCA after visit 2, and patient 10 had TF Ag and MP PCA that were high at baseline and increased progressively through 3 months of chemotherapy (Fig. 1A). Both of these patients developed VTE. Patient 5 developed an occlusive popliteal deep venous thrombosis (DVT), confirmed by ultrasound, 15 days after discontinuing both gemcitabine and dalteparin due to progression of cancer. The patient subsequently presented 12 days later with sudden-onset shortness of breath while on anticoagulation with warfarin, was admitted and died during the hospital admission. An autopsy confirmed the presence of a massive pulmonary embolism (PE). The last TF Ag and MP PCA for patient 5, taken 15 days prior to the initial episode of DVT, were 377 and 4.4 pg mL−1, respectively. Patient 10, who was randomized to no prophylaxis, developed an ultrasound-confirmed peroneal vein DVT 56 days after starting chemotherapy. The patient then had extension into the femoral vein, confirmed by ultrasound, 26 days later while on therapeutic anticoagulation with warfarin. The last TF Ag and MP PCA for patient 10 taken on the day of diagnosis of DVT were 504 and 5.5 pg mL−1, respectively. When evaluating the entire study population, there was a significant association between TF Ag levels (P = 0.036) as well as TF MP PCA levels and development of VTE (P = 0.044). There was also a significant correlation between TF Ag and MP PCA assays (linear correlation coefficient 0.89, P < 0.0001; Fig. 1B). Fig. 1 (A) Serial plasma TF Ag levels in 11 patients with locally advanced or metastatic pancreatic cancer receiving systemic chemotherapy. Patients 5 and 10, marked with an asterisk, developed VTE. TF MP PCA levels for these two patients are displayed as dashed ... This analysis suggests a role for TF in the pathogenesis of VTE in pancreatic cancer. Furthermore, these data suggest that a rise in plasma TF measured either by TF Ag or MP PCA during the course of chemotherapy may be predictive of subsequent symptomatic VTE events in patients with pancreatic cancer. The source of circulating TF is an area of controversy. Studies have identified TF-containing microparticles that also possess endothelial cell-, platelet- and macrophage-derived surface antigens [8–10]. In patients with cancer, tumor cells may be a potential source of TF as well. The findings reported here are in agreement with previous reports, including ours, suggesting an association between TF expression by tumor cells and subsequent VTE in pancreatic and ovarian cancer [5,6]. Although our findings are of statistical significance, given the small sample size and small number of events, we consider this to be a preliminary report requiring confirmation. This study was also inadequately powered to evaluate the impact of thromboprophylaxis and response to chemotherapy. Our findings suggest, however, that future prospective studies should investigate the potential role of TF as a biomarker for VTE in pancreatic and other cancers known to have high levels of TF expression by tumor cells. Both TF Ag and MP PCA assays deserve further investigation in this regard.

Mouse Model of Femoral Artery Denudation Injury Associated With the Rapid Accumulation of Adhesion Molecules on the Luminal Surface and Recruitment of Neutrophils

Techniques of arterial injury commonly used in animals to mimic endovascular procedures are not suitable for small mouse arteries. This has limited examination of the response to arterial injury in genetically modified mice. We therefore sought to develop a model of transluminal injury to the mouse femoral artery that would be reproducible and result in substantial levels of intimal hyperplasia. Mice of the C57BL/6 strain underwent bilateral femoral artery denudation by passage of an angioplasty guidewire. Intimal hyperplasia was observed in 10% of injured arteries at 1 week, in 88% at 2 weeks, and in 90% at 4 weeks. The mean intimal-to-medial area ratio reached 1.1+/-0.1 at 4 weeks. No intimal proliferation was found in control sham-operated arteries. One hour after injury, the denuded surface was covered with platelets and leukocytes, predominantly neutrophils. This was associated with the accumulation of P-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. Expression of these adhesion molecules was not seen in the underlying medial smooth muscle cells. At 24 hours, few neutrophils remained on the denuded surface. At 1 week, macrophages and platelets were present in the vessel wall, partially covered by regenerated endothelium. Transluminal wire injury to the mouse femoral artery induces abundant intimal hyperplasia formation by 2 and 4 weeks and elicits the rapid accumulation of leukocytes and adhesion molecules on the denuded luminal surface. This model will be a valuable tool to study arterial injury in genetically modified mouse models.

Epidermal growth factor, a vascular smooth muscle mitogen, induces rat aortic contraction.

Atherosclerotic arteries have enhanced reactivity to vasoconstrictors, which suggests that features of the atherosclerotic process itself may result in this abnormal responsiveness. Since vascular smooth muscle proliferation is a prominent feature of atherosclerosis, we postulated that vasoactive agonists and smooth muscle mitogens may share certain common cellular mechanisms of action which potentially contribute to this hyperreactivity. To test this hypothesis, we studied the effects of epidermal growth factor (EGF), a well-characterized mitogen, on rat aortic vascular smooth muscle, both in intact aortic strips and in culture. EGF caused contraction (EC50 = 19 nM) of rat aortic strips which maximally was equivalent to 40% of that induced by angiotensin II, a potent vasoconstrictor. EGF increased 45Ca efflux (EC50 = 3 nM) from cultured rat aortic smooth muscle cells, which was an effect shared by angiotensin II and thought to reflect increased cytosolic-free calcium concentration. EGF (7.5 nM) also stimulated growth of these cultured cells to the same extent as 10% calf serum. These results demonstrate that EGF is both a vasoconstrictor and mitogen for rat aortic smooth muscle cells. The similarities in the effects of EGF and angiotensin II suggest that certain common intracellular mechanisms of action may exist for vasoactive agonists and growth factors which may contribute to the altered vasoreactivity of atherosclerotic vessels.

Dietary Lipid Lowering Reduces Tissue Factor Expression in Rabbit Atheroma

BACKGROUND The mechanisms by which lipid lowering reduces the incidence of acute thrombotic complications of coronary atheroma in clinical trials remains unknown. Tissue factor (TF) overexpressed in atheroma may accelerate thrombus formation at the sites of plaque disruption. A cell surface cytokine CD40 ligand (CD40L) enhances TF expression in vitro. METHODS AND RESULTS To test the hypothesis that lipid lowering reduces TF expression and activity, we produced atheroma in rabbit aortas by balloon injury and cholesterol feeding for 4 months (Baseline group, n=15), followed by either a chow diet (Low group, n=10) or a continued high-cholesterol diet for 16 months (High group, n=5). Immunolocalization of TF, CD40L, and its receptor CD40 was quantified by computer-assisted color image analysis. Macrophages in atheroma of the Baseline and High groups strongly expressed TF. Intimal smooth muscle cells and endothelial cells also contained immunoreactive TF. Regions of expression of CD40L and CD40 colocalized with TF. Protein expression of TF diminished substantially in the Low group in association with reduced expression of CD40L and CD40. In situ binding of TF to factors VIIa and X, detected by digoxigenin-labeled factors VIIa and X, colocalized with TF protein in atheroma and decreased after lipid lowering. We also determined reduced TF biological activity in the Low group by use of a chromogenic assay. The level of TF mRNA detected by reverse transcription-polymerase chain reaction also decreased after lipid lowering. CONCLUSIONS These results suggest decreased expression and activity of TF as a novel mechanism of reduced incidence of thrombotic complications of atherosclerosis by lipid lowering.