INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

EXTERNAL PNEUMATIC COMPRESSION (EPC) is an effective means of prophylaxis against deep venous thrombosis (DVT). EPC devices compress the leg for a few seconds of each minute. The compression collapses the veins and increases venous blood flow during the period of the pulse, thereby eliminating venous stasis and reducing the risk of thrombosis. Many studies have sought to optimize or improve EPC performance from a hemodynamic perspective (4, 18, 19, 23, 35); however, the biological correlates of various hemodynamic factors are still relatively unknown. Understanding the biological consequences of EPC can potentially optimize the performance of the EPC-generating device and provide guidance for clinical use.

Numerous clinical studies have documented the efficacy of EPC (8, 14), and improvement of local hemodynamic flow is thought to be the primary beneficial effect. Several studies demonstrated, however, that the hemodynamic effects induced by EPC are not limited to changes in venous blood flow in the area of compression but, rather, exert a broader influence in the systemic circulation. For example, clinical studies revealed that EPC caused changes in central venous and pulmonary arterial pressures (41) and enhanced popliteal artery blood flow (6, 24, 42).

Although these hemodynamic effects are thought to be the primary mechanisms of EPC, the exact nature of the biological response is not well understood. Several mechanisms have been proposed to explain the biological effects of EPC. 1) Many investigations demonstrated the ability of EPC to increase fibrinolytic activity in systemic blood (1, 35, 39) and found that the degree of fibrinolytic enhancement depended on the mode and timing of compression (35). It has been hypothesized that hemodynamic action stimulates fibrinolytic activity via production of tissue-type plasminogen activator (tPA) by the vascular endothelium (16). 2) Chouhan et al. (3) showed that EPC results in an increase in plasma tissue factor pathway inhibitor (TFPI) and a decline in factor VIIa. This result suggests that inhibition of the tissue factor pathway, the major physiological initiating mechanism of blood coagulation, might be an important mechanism for the antithrombotic effect of EPC. 3) Recently, Liu et al. (26) showed in an animal model that there is significant vasodilation in arterial and venous vessels during the application of EPC and further demonstrated that the vasodilation could be completely blocked by nitric oxide synthase (NOS) inhibitor. They also found that the inflation rate is a dominant factor in determining the degree of vasodilation by EPC (27). These findings demonstrated that the production of nitric oxide (NO) may be involved in the positive influence of EPC on the circulation. On the basis of these findings, we postulated that the rapid increase in venous velocity induced by EPC produces strong shear stress on the vascular endothelium, which stimulates an increased release of NO and thereby causes systemic vasodilation.

NO, as the primary endothelium-derived relaxing factor (EDRF), not only is a potent vasodilator but also plays an important role in preventing thrombosis formation. Its primary mode of action is to inhibit platelet activation and aggregation (31, 38). In addition, NO has also been shown to inhibit tissue factor expression, synthesis, and activity (11, 43) as well as increasing tPA release (23). All of these effects contribute to the antithrombotic properties of NO. It is likely that hemodynamic changes caused by EPC increase NO production by endothelial cells (EC), and there is reason to believe that this may be one of the factors in prevention of DVT.

On the basis of this evidence, we hypothesize that shear stress, vessel compression, or a combination of the two causes increased NO production, which contributes to the antithrombotic effect of EPC. We further hypothesize that the amount of NO production and endothelial NOS (eNOS) mRNA expression can be optimally stimulated by choosing appropriate combinations of peak shear stress, duration of flow, and compression timing cycles. To test these hypotheses, we previously developed (5) a new in vitro cell culture system (venous flow simulator, VFS) that simulates the hemodynamic shear stress and vessel wall strain associated with EPC. Here we extend that work and examine the response of human umbilical vein EC (HUVEC) to intermittent flow, vessel collapse, or a combination of the two with respect to NO production and eNOS mRNA expression. We also examine NO production in the presence of various NOS antagonists and consider different modes of EPC to explore how this might influence NO production and eNOS mRNA expression.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Experimental system. An in vitro cell culture system was built to simulate the hemodynamic shear stress and vessel collapse conditions associated with EPC of the lower leg, as described in more detail in previous studies (5) (Fig. 1A). Briefly, the system is composed of four Silastic tubes (diameter 5 mm, thickness 0.5 mm, length 12 cm) mounted inside two Plexiglas chambers. One of the chambers (Fig. 1B) has a pusher plate attached to a bellows that can be periodically inflated by an air pump to push the plate downward and compress the tubes. The other chamber is identical to the first except that it lacks the pusher plate and bellows so that the two tubes in it are not compressed. Cell culture medium is driven by either the air pump for F (pulsatile flow only) and FC (pulsatile flow and tube compression) circuits or the steady-flow perfusion pump for Control and C (tube compression only) circuits. The air pump (adapted from Aircast Venaflow, Aircast, Summit, NJ) operates so as to produce a short period (4 s) of pressure in the air space above the medium contained in a reservoir upstream of the test chambers, followed by a rest period of 56 s. This produces a short period of high flow through the tubes containing the cultured cells alternating with a much longer period of zero flow, intended to mimic the application of external compression to the lower leg. Time-varying flow rates are continuously monitored with a transit time ultrasonic flowmeter (model T109R; Transonic Systems, Ithaca, NY). The combination of tube compression and flow type leads to the four test groups. Another group in which there is no flow and no vessel compression was set aside as an additional control (Static Control group). Conditions of the five test groups are indicated in Table 1. Each group is in its own independent flow loop, which provides a sterile nourishing environment and also allows sampling of the medium for biochemical assay.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   A: schematic drawing of venous flow simulator (VFS). F, pulsatile flow; C, tube compression; FC, pulsatile flow + tube compression. B: test chamber used to produce tube compression. Air inflates the bellows, driving down the pusher plate to a position determined by small pegs inserted through the bottom to the desired level.

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View larger version (20K): [in this window] [in a new window]   Fig. 2.   Ultrasonic flowmeter-measured flow rate (A) and calculated shear stress (B) in the F group. Shear stress is calculated using the Womersley theory (29) of unsteady, pulsatile flow based on the measured time-varying flow rate.

Establishment of EC cultures. EC from human umbilical veins [obtained from umbilical cords provided by the obstetrical unit at the Massachusetts General Hospital (MGH) under strict National Institutes of Health guidelines regulated by the MGH Subcommittee on Human Studies] were isolated as previously described (17) by enzymatic treatment [0.1% collagenase (CLS II; Worthington, Freehold, NJ), 5% human serum albumin (fraction V; Sigma, St. Louis, MO)]. Primary cultures were established, maintained, and passaged on 1% gelatin-coated tissue culture plastic. HUVEC were cultured in M199 (GIBCO, Rockville, MD) supplemented with L-glutamine (2 mM, GIBCO), 10% fetal calf serum (Hyclone Laboratories, Logan, UT), heparin (150 µg/ml, Sigma), penicillin and streptomycin (100 U/ml and 100 mg/ml, respectively; GIBCO) and EC growth supplement (50 µg/ml; Becton Dickinson, Franklin Lakes, NJ). Cells were confirmed to be endothelial by their standard morphological appearance, the presence of factor VIII-related antigen (von Willebrand factor), and the specific uptake of DiI-acetylated low-density lipoprotein (Biomedical Technologies, Stoughton, MA). These criteria have also been used to assess whether HUVEC in experimental cultures retain differentiated characteristics.

Nitrite/nitrate determination. NO is a molecule with relatively short half-life in aqueous solution. On release into the culture, it quickly converts to NO and NO, the stable end products of NO (15). Measurement of NO and NO gives the total NO production. NO levels were measured with a quantitative fluorometric assay. The assay was modified from Ref. 30 to improve the sensitivity. This assay is based on the reaction of nitrite with 2,3-diaminonaphthalene (DAN; Aldrich, Milwaukee, WI) under acidic conditions to form the highly fluorescent product 1-(H)-naphthotriazole. A volume of 80 µl of freshly prepared DAN reagent (25 µM in 0.62 M HCl) was added into each 800-µl medium sample and mixed immediately. After a 10-min incubation at 20°C, the reaction was terminated with the addition of 40 µl 2.8N NaOH. Formation of 1-(H)-naphthotriazole was measured with a spectrofluorophotometer (model RF-5000; Shimadzu, Kyoto, Japan) with excitation at 380 nm and emission at 405 nm. Nitrite concentrations were determined relative to a standard curve. Sodium nitrite (Sigma) was used as the standard for calibration of the assay between 10 and 1,000 nM. This assay gives linear fluorescence intensity with regard to nitrite concentration with a sensitivity of 5 nM. Total nitrite production values were normalized to the number of cells in each group. To assess the NO component, in separate tests, nitrate in HUVEC-conditioned medium was converted to nitrite with 50 mU of nitrate reductase from Aspergillus niger (Sigma) and 10 µM -NADPH for 1 h at room temperature. This protocol converted >95% of nitrate to nitrite. After this step, nitrite was then determined by the above-described procedures. We obtained the same result as without nitrate reductase conversion, confirming that the nitrate component could be neglected and that the nitrite level reflects the total NO production.

Northern blot analysis. Northern hybridization analysis was used to assay for gene induction associated with elevated flow or vessel collapse. Following standard procedures (36), total RNA from guanidine isothiocyanate extracts of experimental and control silicone rubber tube cultures was isolated by centrifugation through cesium chloride and then separated by 1.2% formaldehyde-agarose gel electrophoresis, transferred to nylon membranes, and hybridized in the presence of 49% formamide at 42°C to specific ³²P-labeled eNOS cDNA probes. Autoradiographs on a Kodak X-OMAT AR X-ray film were obtained and quantitated with laser scanning densitometry (Molecular Dynamics, Sunnyvale, CA) with Image Quant 3.0 software. The intensity of the glyceraldehyde-3-phosphate dehydrogenase band in each lane was used for normalization to correct for differences in RNA loading.

Statistical analysis. Data are expressed as means ± SE. When data from more than two groups were compared, one-way ANOVA followed by post hoc Scheffé's test was used. Differences were considered statistically significant when P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

As shown in Fig. 3, intermittent pulsatile flow generated by EPC stimulates NO production in HUVEC. Nitrite level increased rapidly within the first hour of exposure to flow. During this time, nitrite levels in the FC and F groups were about twice those in C and Control groups, whereas in the Static Control group, there was essentially no nitrite production. Interestingly, no significant difference in nitrite levels was observed between CF and F groups or between C and Control groups. Two phases of nitrite production rate in cultures exposed to flow could be discerned: phase A (0-1 h), which was characterized by a rapid nitrite release, and phase B (1-6 h), which was characterized by diminished but sustained nitrite release. Average nitrite production rates during phase A were 12.5 nmol · 10⁶ cells¹ · h¹, whereas the corresponding nitrite levels in phase B were 2.2 nmol · 10⁶ cells¹ · h¹. Nitrite production rates in each phase were calculated as the slope of the cumulative nitrite production.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Cumulative nitrite production in conditioned medium collected each hour (n = 3 for each group). Data were normalized to number of cells in each group. ANOVA followed by Scheffé's test was used to compare different groups. Experimental condition is listed in Table 1. F and FC groups are significantly different from C and Control groups (P < 0.01); C and Control groups are significantly different from Static Control group (P < 0.01).

Figure 4 shows the nitrite production under different peak levels of shear stress. Condition F with peak shear stress set at different levels (1, 10, 20, and 40 dyn/cm²) was tested against the Static Control group (0 dyn/cm²). It is interesting to note that intermittent pulsatile flow at peak shear stresses of only 1 dyn/cm² stimulated NO production compared with the Static Control group. However, maximum NO production was reached only when peak shear stress attained 10 dyn/cm². NO production was almost identical when peak shear stress was 10, 20, or 40 dyn/cm².

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Cumulative nitrite production under different peak shear stress. F group with peak shear stress set at different levels (1, 10, 20, 40 dyn/cm2) was tested against Static Control group (0 dyn/cm2). The flow rate at each peak shear stress is 0.3, 3, 6, and 12.5 ml/s, respectively.

To determine whether the observed increase in nitrite level under flow conditions reflected increased NO production, experiments were done with L-NAA, a NOS inhibitor. Treatment with L-NAA for 60 min before and during exposure to flow completely blocked the flow-induced nitrite accumulation (Fig. 5).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Nitrite production after 6-h experimental period with and without 100 µM NG-amino-L-arginine (L-NAA; n = 3). Significant difference between treated and untreated groups: *P < 0.05, **P < 0.01.

HUVEC cultures under flow were incubated with specific inhibitors for NOS to determine the enzyme involved in flow-induced NO production. S-methyl-L-thiocitrulline, which inhibits nNOS, and N-(3-aminomethyl)benzylacetamidine, which inhibits iNOS, had no observable effect on NO production in medium from HUVEC culture under EPC, whereas N-nitro-L-arginine methyl ester, which is a nonspecific inhibitor for NOS, dramatically diminished the production of NO (Fig. 6).

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Cumulative nitrite levels in flow-only group (F) in presence of 1 µM S-methyl-L-thiocitrulline, 0.5 µM N-(3-aminomethyl)benzylacetamidine, and 1 µM N-nitro-L-arginine methyl ester (n = 3). *Significant difference from other groups (P < 0.01).

Northern blot analysis of mRNA expression showed no changes in eNOS gene expression at 1 h (data not shown). Expression of eNOS was, however, increased at the 6-h time point by factors of 2.08 ± 0.25 in F tubes and of 2.11 ± 0.21 in FC tubes relative to control. There was no significant change in the compression only (C) group (Fig. 7). When peak shear stress in F and FC groups was set at a lower level (10 and 20 dyn/cm², respectively), no upregulation of eNOS was observed (Fig. 8).

View larger version (46K): [in this window] [in a new window]   Fig. 7.   Experiments were done at the conditions listed in Table 1. A: Northern blot analysis of mRNA expression of endothelial nitric oxide synthase (eNOS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at 6 h. B: densitometric analysis of eNOS mRNA expression. All data (n = 3) were first normalized to GAPDH and then to Control group. FC and F groups are significantly greater than other groups (P < 0.05).

View larger version (40K): [in this window] [in a new window]   Fig. 8.   Experiments were done at the conditions listed in Table 1 except that the peak flow rate in F and FC groups was 3 ml/s and the peak shear stress in F and FC groups was 10 and 20 dyn/cm2, respectively. A: Northern blot analysis of mRNA expression of eNOS and GAPDH at 6 h. B: densitometric analysis of eNOS mRNA expression. All data (n = 3) were first normalized to GAPDH and then to Control group. No significant difference was found between groups.

Experiments using different modes of EPC indicate that NO production and eNOS mRNA expression respond differently to the time cycle of compression. There is no significant difference in NO production from 0 to 1 h. However, from 1 to 6 h, the production rate with a cycle period of 60 s is 2.2 ± 0.33 nmol · 10⁶ cells¹ · h¹, whereas the rates with 30- and 120-s periods are 1.7 ± 0.2 and 0.77 ± 0.18 nmol · 10⁶ cells¹ · h¹, respectively (Fig. 9). Northern blot analysis showed that the upregulation of eNOS mRNA expression can be influenced by the different cycles of EPC. Of the three modes of EPC tested, the group with pulsed flow every 30 s expressed the highest level of eNOS mRNA, followed by the groups with a period of 60 and 120 s (Fig. 10).

View larger version (17K): [in this window] [in a new window]   Fig. 9.   Nitrite production rate between 0 and 1 h (A) and between 1 and 6 h (B) under different external pneumatic compression (EPC) cycles (n = 3). Peak flow rate and shear stress are the same as those of the F group in Table 1: peak flow rate is 12.5 ml/s for 4 s, and peak shear stress is 40 dyn/cm2. *Significant difference compared with group with 120-s cycle (P < 0.05).

View larger version (53K): [in this window] [in a new window]   Fig. 10.   Northern blot analysis (A) and densitometric analysis (B) of eNOS mRNA expression under different EPC cycles. Peak flow rate and shear stress are the same as the F group in Table 1: peak flow rate is 12.5 ml/s for 4 s, and peak shear stress is 40 dyn/cm2 (n = 3). *P < 0.05, **P < 0.01 compared with Control group.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The VFS system was designed to study the influence of vessel collapse and intermittent pulsed flow, two conditions associated with EPC, on endothelial function. By this means, the cells are simultaneously subjected to the patterns of wall shear stress and vessel wall strain experienced in vivo during EPC. Using this system, we investigated endothelial NO production under the influence of EPC. The important observations of this study are that intermittent pulsed flow generated by EPC stimulates NO release by cultured HUVEC and also upregulates eNOS mRNA expression at 6 h. Both NO production and expression of eNOS mRNA are dependent on the peak shear stress and time cycles of EPC.

EC NO production and eNOS mRNA expression under steady shear stress have been studied by other groups. Ranjan et al. (34) cultured HUVEC and bovine aortic EC (BAEC) under steady laminar shear stress and found that, in both cell types, eNOS mRNA was elevated within 3 h of flow exposure at 25 dyn/cm² and remained elevated at 6 and 12 h of flow exposure. Flow exposure at 4 dyn/cm² caused no enhancement of eNOS levels in either cell type. In a separate study (28), steady shear stress was found to induce a time-dependent increase in eNOS mRNA in BAEC (2.9- and 3.6-fold after 6 h at 4 and 20 dyn/cm², respectively). Some experimental data also demonstrate that EC can sense different patterns of shear stress that trigger both common and different signaling pathways (40). For example, compared with laminar flow, turbulent flow does not upregulate eNOS or increase NO release (33). Oscillatory shear stress does not upregulate eNOS expression in BAEC, whereas unidirectional shear stress does (44, 45).

Consistent with earlier studies (22), we found shear stress to be a potent stimulator of NO release by EC. Compared with controls with a low, steady flow with no collapse, we observed a much higher initial rate of NO release followed by a sustained elevated release. NO release under intermittent pulsatile flow was also found to be dependent on the peak shear stress level. Maximum NO production was reached only when peak shear stress exceeded 10 dyn/cm². However, NO production does not increase further when peak shear stress increases beyond this level. Previous studies in EC (22) showed that NO increases biphasically, with an initial burst that is independent of shear stress (1.8-25 dyn/cm²) followed by a sustained, shear stress-dependent phase. The signaling mechanisms that lead to different shear stress- or flow-induced NO responses are not yet identified. It has been proposed that EC respond to mechanical stimuli, which activate eNOS by at least two separate pathways, the Ca²⁺/calmodulin-dependent and Ca²⁺/calmodulin-independent pathways (2, 7). The Ca²⁺/calmodulin-dependent pathway mediates the rapid, transient response to fluid shear stress involving activation of NOS and ion transport. In contrast, the Ca²⁺/calmodulin-independent pathway mediates a slower response involving the sustained activation of NOS and changes in cell morphology and gene expression (22). It therefore appears that EC NO production rate exhibits two distinct phases after the onset of flow, first a burst of release and then a relatively slower rate of release under sustained flow.

With EPC, the flow is highly pulsatile, with short periods of elevated flow alternating with long rest periods of zero or low flow. It is therefore likely that both of the pathways mentioned above are involved in the activation of eNOS, although the effect of time-varying flow patterns cannot be discerned from the previous work. To consider this effect, we measured NO release over a range of different patterns of high flow and no flow in an attempt to identify conditions of maximum effect. Only those patterns that can be produced by EPC were considered, so each consisted of a short period of elevated flow followed by a much longer period of zero flow, corresponding to the time for the vein to refill via the capillary bed. Our results indicate that the highest level of eNOS mRNA expression was achieved in the group with pulsed flow every 30 s and the group with pulsed flow every 60 s exhibited the highest level of NO production during the 6-h experiment. It is possible that because of the extended resting period, there may be a relatively greater contribution from the Ca²⁺/calmodulin-dependent pathway leading to enhanced activation of eNOS, which has a higher rate of release of NO than the Ca²⁺/calmodulin-independent pathway. In the 30-s group, although EC are exposed to shear stress for a longer total amount of time, the Ca²⁺/calmodulin-independent pathway may predominate, leading to a pattern of behavior more closely aligned to that seen with constant shear stress. Regardless of mechanism, these results showing variable NO production and eNOS expression under various types of EPC suggest that different EPC cycles may influence the pattern and amount of NO release and thereby have impact on the capability of the method to prevent DVT. Of the three patterns of flow we have tested, the 30-s cycle period appears better suited for long-term use because it upregulates eNOS mRNA expression, whereas the 60-s cycle maximizes short-term NO release.

It is interesting to observe that even a very low level of flow (0.3 dyn/cm² in C and Control groups) stimulates HUVEC to produce a much greater level of NO than static, no-flow conditions, indicating that EC production of NO exhibits an exquisite sensitivity to flow. This is consistent with other studies showing that EC produce NO under shear stress as low as 0.2 dyn/cm² (20). How EC sense such low shear is not clear. Experiments on EC have shown that fluid flow alters the boundary layer concentration of ATP, resulting in ATP-mediated increases in intracellular Ca²⁺ (32), which might be responsible for the activation of NOS. Clinically, the results imply that venous blood flow, even at low flow rate, offers better protection than no blood flow because it stimulates moderate levels of NO production. However, it appears from our experiments that this low level of shear is insufficient to upregulate eNOS expression and might therefore be a short-term effect that could be quickly exhausted.

Intermittent pulsatile flow, on the other hand, not only stimulates NO release throughout the experimental period but also upregulates eNOS mRNA expression at 6 h. Because NO production increases immediately after the onset of flow, even before eNOS mRNA upregulation, it must be due to direct activation of eNOS enzyme rather than increased protein. Although the importance of eNOS upregulation compared with NO production is not fully understood, it is believed that eNOS upregulation contributes to the long-term vasomotor regulatory function of EC under flow. For example, eNOS mRNA transcription levels increase in response to chronic exercise (37), and acetylcholine-stimulated NO production is markedly enhanced in large coronary arteries and microvessels after chronic exercise compared with control. On the other hand, decreased eNOS expression has been implicated in endothelial dysfunction in atherosclerosis (12, 13).

Vessel collapse, and the associated wall strain of up to 10%, alone seems to exert little influence on NO production and eNOS expression. This is similar to the study of Ziegler et al. (44), which showed that shear stress represents the major mechanical factor inducing an increase in eNOS expression in BAEC. Also, the shear stress effect appears to saturate at high levels of shear. In the FC group, even though the peak shear (80 dyn/cm²) is considerably higher than in the flow-only group (F; 40 dyn/cm²) because of the reduction in cross-sectional area during compression, NO production follows nearly an identical trend.

L-NAA blocked flow-induced increases in nitrite, indicating that these nitrite measurements reflect increased NO production and that the nitrite measured is not an artifact of the assay system due to the presence of cell debris in conditioned media. Specific inhibitors for NOS isoforms I and II do not inhibit NO production from cultures exposed to EPC, strongly suggesting that EPC-induced NO production in HUVEC was not due to the induction of iNOS or nNOS.

Our cell culture results are consistent with previous studies in animal models (26), which showed that pneumatic compression causes vasodilation in arteries and veins. In those experiments, vasodilation reached maximum levels 30 min after initiation of compression and could be completely blocked by an inhibitor of NOS. In a different study (27), in which intermittent pneumatic compression with different inflation rates but the same peak pressure duration was applied to the legs of rats, the results showed that faster inflation rates cause greater vasodilation. When the duration of peak pressure was varied while the inflation rate was held constant, the degree of vasodilation was unchanged. These findings suggest that inflation rate is a dominant factor in vasodilation induced by EPC. Our results show that to stimulate NO release, a peak shear stress above 10 dyn/cm² is needed. For eNOS mRNA to be upregulated, peak shear stress should rise to the level of 40 dyn/cm². Studies have shown that peak shear stress induced by EPC is directly related to inflation rate. Therefore, to generate a desired level of shear stress, faster inflation rates may be needed.

Choosing the right cell type is important to the interpretation of our results. HUVEC were chosen because they have been proven to be a reliable model for standardized investigation of venous EC function by many investigators. Nevertheless, cells from leg veins are closer to our investigation, anatomically and possibly functionally. Deep vein EC, presumably, would be the best choice for our study. However, these cells are difficult to obtain. The next most appropriate cell type is saphenous vein EC. These cells are routinely available in our lab from discarded saphenous vein segments obtained in the operating room during coronary artery bypass or femoropopliteal bypass procedures. However, these cells show large variability depending on source because they are usually obtained from an aged population. EPC, on the other hand, is used on a wide range of medical and surgical patients and is especially effective in orthopedic surgery and abdominal surgery. Because of the variability in saphenous vein EC obtained from older patients and the fact that they may not be most reflective of the target population, these cells do not necessarily provide the best model system for studying the basic mechanisms of EPC. For these reasons, umbilical vein EC were chosen in our experiments on the assumption that the fundamental mechanisms of EC gene regulation by EPC should be reasonably well modeled by the HUVEC used in the present study. However, because HUVEC may behave differently from venous EC from the legs, it will be essential that we validate these findings in vivo and test new designs in the clinical setting.

The observed characteristics of NO production under EPC in this work may have clinical implications for using the EPC device in different ways in different situations. In the short term, rapid release of NO follows EPC. In the longer term, EPC has the beneficial effect of upregulating several thromboresistance genes (e.g., eNOS, tPA), thus implying that at differing levels of recovery, ambulation, perioperative trauma, etc., the utility and mode of EPC might be optimally and strategically varied. We also studied the influence of different modes of EPC on NO release and eNOS mRNA expression, which is helpful for improving the design of the device. Combined with the in vitro cell culture studies, future clinical studies on healthy volunteers and patients are essential to establish the optimized protocol of EPC prophylaxis.