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European Heart Journal Advance Access originally published online on August 16, 2006
European Heart Journal 2006 27(19):2362-2369; doi:10.1093/eurheartj/ehl165
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© The European Society of Cardiology 2006. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

A novel hydrodynamic approach to the treatment of coronary artery disease{dagger}

John J. Pacella1,*, Marina V. Kameneva2, Melissa Csikari1, Erxiong Lu1 and Flordeliza S. Villanueva1

1 Department of Medicine, Cardiovascular Institute, University of Pittsburgh School of Medicine, S568 Scaife Hall, 200 Lothrop Street, Pittsburgh, PA 15213, USA
2 McGowan Institute for Regenerative Medicine, Pittsburgh, PA, USA

Received 6 April 2006; revised 28 June 2006; accepted 6 July 2006; online publish-ahead-of-print 16 August 2006.

* Corresponding author. Tel: +1 412 647 5840; fax: +1 412 647 4227. E-mail address: villanuevafs{at}upmc.edu

See page 2272 for the editorial comment on this article (doi:10.1093/eurheartj/ehl234)


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Aims During severe coronary stenosis, capillary resistance increases. Drag-reducing polymers (DRPs) are blood-soluble macromolecules that reduce vascular resistance, possibly by altering blood hydrodynamics and rheology. Thus, we hypothesized that DRPs would enhance myocardial perfusion distal to a severe coronary stenosis.

Methods and results A flow-limiting left anterior descending (LAD) coronary artery stenosis was created in 12 open chest dogs. Coronary driving pressure, flow, trans-stenotic gradient, and radiolabelled microsphere myocardial perfusion were measured. Myocardial contrast echocardiography was performed and videointensity vs. pulsing interval data in the LAD and left circumflex beds were used to derive red cell velocity and capillary volume. Relative to baseline, the stenosis decreased LAD bed capillary volume (P=0.019) and red blood cell velocity (P=0.010). Intravenous DRP (polyethylene oxide, 2.5 ppm) decreased LAD microvascular resistance (P=0.003) and increased microsphere flow (P=0.009), capillary volume (P=0.0006), and red cell velocity (P=0.007) despite the presence of a severe stenosis. DRP did not alter blood viscosity.

Conclusions DRPs improve perfusion to myocardium subserved by a flow-limiting coronary stenosis by decreasing microvascular resistance through an increase in capillary volume. Primary modulation of blood hydrodynamics and rheology to reduce microvascular resistance offers a novel approach to the treatment of ischaemic coronary syndromes.

Key Words: Myocardial ischaemia • Drag-reducing polymers • Myocardial contrast echocardiography • Coronary microcirculation • Ischaemic heart disease


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Autoregulatory changes in microvascular resistance regulate myocardial perfusion during coronary stenosis. Most of the total coronary vascular resistance resides within the smallest arteries and arterioles, where the greatest pressure drop occurs.1–3 Vascular resistance results in frictional energy losses due to viscosity and a progressive decrease in vessel diameter.2 In addition, flow separations and pseudoturbulence at areas of microvascular bifurcation likely contribute to the loss of pressure.1 Hence, under normal physiological conditions, there is a progressive decline in pressure as blood traverses the coronary vasculature, such that precapillary driving pressure, which is crucial for maintaining capillary perfusion, decreases.4

During non-critical coronary stenosis, arteriolar vasodilation decreases microvascular resistance to maintain flow.5 A severe stenosis exhausts autoregulatory vasodilation, microvascular resistance cannot further decrease, and myocardial ischaemia ensues.5 In this setting, myocardial contrast echocardiographic (MCE) images acquired at end-systole show a contrast defect, suggesting a decrease in intramyocardial blood volume. Because end-systolic MCE images depict capillary opacification, these findings have led to the hypothesis that capillary derecruitment occurs during severe flow-limiting stenosis, that this results in an increase in capillary resistance, and ultimately serves to preserve capillary hydrostatic pressure, albeit at the expense of flow.6

Based on these considerations, we hypothesized that myocardial perfusion can be improved during flow-limiting stenosis, even when vasodilator reserve is exhausted, using drag-reducing polymers (DRPs) to modulate the rheological and hydrodynamic properties of blood flow and hence reduce vascular resistance. DRPs are soluble linear macromolecules (MW >106 Da). Minute intravascular concentrations of DRPs reduce flow-associated energy losses and vascular resistance, possibly by forming a flow-organizing ‘scaffold’ within blood.7–9 DRPs suppress flow disturbances and flow separations at simulated vascular bifurcations in vitro.8,9 We hypothesized that in vivo, this could lead to a reduction in pressure loss along the arterial vasculature and higher blood flow through the microvessels, which could benefit patients with coronary artery disease (CAD).

In the present study, we demonstrated that DRPs improve perfusion to canine myocardium subserved by an experimental flow-limiting stenosis. We measured the effect of DRPs on microvascular resistance, and because of the prominent role capillaries are thought to play in limiting flow reserve during coronary stenosis,6 we were particularly interested in the effect of DRPs on this microvascular compartment. Accordingly, we interrogated intramyocardial blood volume using MCE, which provides incremental and unique information for the assessment of microvascular perfusion, over and above that of radiolabelled microspheres; unlike radiolabelled microspheres, MCE provides a real-time in vivo assessment of microvascular perfusion and depicts the regional distribution of perfusion with higher spatial resolution.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Animal preparation
The protocol was approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh. Twelve dogs were induced with intravenous sodium pentobarbital (30 mg/kg), intubated, and mechanically ventilated. Catheters were placed in the forearm for administration of ultrasound contrast, in the femoral arteries for pressure monitoring, blood gas analyses, and radiolabelled microsphere reference sample withdrawal, and in the femoral veins for medication and fluids. Anaesthesia was maintained with sodium pentobarbital (7 mg/kg/h).

A lateral thoracotomy was performed and the heart was suspended in a pericardial cradle. The left anterior descending (LAD) and left circumflex (LCX) coronary arteries were encircled with ultrasonic flow probes (Transonics Inc.). A variable balloon occluder (Research Products International Corp.) was placed around the proximal LAD. The distal LAD was cannulated with a 25G fluid-filled catheter to measure post-stenotic LAD coronary driving pressure. Catheters were placed in the left and right atria for radiolabelled microsphere injection and pressure measurement, respectively.

Myocardial contrast echocardiography
The ultrasound contrast agent (Definity, Bristol Myers Squibb) was comprised of air and perfluoropropane gas-filled phospholipid bilayer microspheres (1.1–3.3 µm diameter, concentration 109/mL) with intravascular kinetics comparable with that of red blood cells.10 The microspheres (1.5 mL) were placed in 50 mL of 0.9% saline and intravenously infused (120–160 mL/h).

Open-chest echocardiography of the left ventricular (LV) short axis at the mid papillary muscle level was performed using either an ultraharmonic (send/receive 1.3/3.6 mHz, Sonos 5500, Philips Corp.) or a second-harmonic (send/receive 1.75/3.5 mHz, Sequoia, Siemens Corp.) imaging system. During continuous intravenous contrast infusion, ECG-gated MCE was performed during end-systole at incremental pulsing intervals. After initial optimization, the gain, depth, mechanical index (0.8–1.0), focus, and dynamic range were held constant. Digitally acquired images were analysed offline as previously described,11–18 whereby average pixel intensity was measured in the LAD and LCX territories (defined ante-mortem by MCE performed during LAD occlusion).

Videointensity in end-systolic frames was plotted against pulsing interval and fit to the exponential function, y=Ax(1–e–ßt), where y is the videointensity measured at pulsing interval (t).18 Each ultrasound pulse is assumed to destroy the microbubbles in the beam elevation. At subsequent ultrasound pulses separated by time ‘t’, the microbubbles replenish the imaging sector at a rate proportional to red cell velocity (ß). At longer pulsing intervals, microbubbles fully replenish the sector and videointensity plateaus at ‘A’. This value is determined by peak microbubble tissue concentration at end-systole or end-systolic intramyocardial blood volume. Because there is ‘milking’ of the larger (>200 µm) intramyocardial arterioles and venules during systole,19,20 the end-systolic intramyocardial volume is effectively comprised of the microcirculation <200 µm in size, 90% of which is constituted by capillaries.21 Hence, ‘A’ is an index of capillary cross-sectional area and the product of Axß correlates with myocardial blood flow.16,18

Finally, to depict regional perfusion, background subtracted images were colour-coded using a map whereby videointensity change for each pixel was displayed in gradations of red, orange, yellow, and white, in proportion to increasing contrast enhancement.11–13,17,18

Measurement of myocardial perfusion
Approximately 2–3x106 of 15 µm radiolabelled microspheres (Perkin-Elmer Inc.) were suspended in 3 mL of 0.9% saline/0.01% Tween 80, injected into the left atrium, and flushed with saline during simultaneous 90 s arterial reference sample withdrawal. One of the following isotopes was used for each stage: 113Sn, 102Ru, 141Ce, 95Nb, 51Cr, 45Sc. Post-mortem, the LV slice corresponding to the MCE image was radially sectioned into 16 pieces, weighed, and placed in a gamma counter (Packard Instruments Inc.) and blood flow was calculated (mL/min/g) as previously described.22

Preparation of DRP
DRP solution was comprised of polyethylene oxide (PEO), Polyox WSR-301 (Union Carbide Inc.), dissolved in saline at a concentration of 0.1% (1000 ppm) and then dialyzed against saline for 24 h using a membrane with 50 kDa MW cutoff (Spectrum Laboratories Inc.). Drag-reducing properties were confirmed in an in vitro saline flow system by noting an increase in flow rate at a given pressure. Viscosity of the PEO solution was measured with a rotational viscometer (Contraves Inc.). The average molecular weight of the dissolved polymer was ~4500 kDa. The stock PEO solution of 1000 ppm concentration was diluted with saline to 250 ppm and mixed for 6–8 h prior to use.

Measurement of blood viscosity
Venous blood (6 mL) was collected from the first five dogs in our series and transferred into EDTA vacutainers. The DRP solution (0.015 mL) was added to 1.5 mL of blood to a final concentration of 2.5 ppm. An equivalent volume of saline was added to another 1.5 mL sample to serve as control. Viscosity vs. shear rate data were generated by a Couette rheometer (Contraves Inc.). Haematocrit was determined in a microhaematocrit centrifuge (International Equipment Co.).

Testing of occluder stability
To ensure that a stable stenosis could be created, the ability of the occluder to mechanically sustain a constant inflation was evaluated. The occluder was placed around tubing (3.0 mm polyethylene), inflated with dye-containing water, submerged in saline (37°C), and connected to a fluid-filled pressure transducer. Balloon pressure was stable and no dye extravasation occurred to suggest a leak during a 1 h observation period, demonstrating that the occluder could maintain a stable stenosis for a time period that exceeded experimental requirements.

Experimental protocol
After establishing a stable preparation, radiolabelled microspheres were injected into the left atrium during arterial reference sample withdrawal, and haemodynamic and MCE data were collected (baseline stage). The occluder was inflated to decrease resting LAD probe flow by at least 50%, and no further manipulations of the occluder were performed. After a stable trans-stenotic pressure gradient was observed for 20 min, radiolabelled microsphere, haemodynamic, and MCE data were obtained. PEO solution (in 25 mL saline) was then intravenously infused over 25 min to achieve a blood concentration of 2.5 ppm, followed 15 min later by radiolabelled microsphere, haemodynamic, and MCE data collection. The LAD was then ligated, MCE was performed, and 30 mL India ink were injected into the left atrium to define risk area borders, followed immediately by euthanasia with KCl and pentobarbital overdose. The heart was excised and processed for radiolabelled microsphere analysis. Microvascular resistance was calculated as distal LAD pressure (mmHg) minus right atrial pressure (mmHg) divided by LAD microsphere flow (mL/min/g).

Statistical analysis
Data are expressed as mean±SD. The following variables were tested for differences as a function of experimental stage (baseline, stenosis, stenosis+DRP): transmural microsphere flow, microvascular resistance, the MCE-derived parameters, flow probe flow, and distal LAD pressure. For each variable, a separate repeated measures fixed-effect ANOVA was performed to ascertain within-group differences between experimental stages using a univariate approach with a Huynh-Feldt Epsilon correction for non-sphericity (non-independent residuals). This yielded a P-value of ≤0.0002 for all comparisons, thus confirming the presence of significant differences in the variables of interest for the experimental conditions outlined earlier. Post hoc paired t-testing was performed to determine where the differences resided, using Tukey's test (two-tailed) to adjust for multiple comparisons.23 Significance by Tukey testing (three comparisons) was calculated as a critical t>2.70, or P<0.02. Based on our previous data in rats receiving DRP, we defined a potentially clinically significant change in microsphere flow resulting from DRP as a difference of at least 0.25±0.2 mL/min/g, which can be detected with a power of 80% and an alpha error of 0.05, using a sample size of at least 12 animals. To detect a difference in blood viscosity of 0.5±0.2 cP at a power of 90% with an alpha of 0.05, five blood samples per group (blood+DRP and blood+saline) were required.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Conclusions
 Acknowledgements
 References
 
Effect of coronary stenosis
The stenosis reduced the LAD probe flow from 32±4 to 13±2 mL/min (P<0.0001) and increased the trans-stenotic gradient from 2±1 to 42±5 mmHg (P<0.0001). There was no significant difference between the pre-DRP stenosis gradient (42±5 mmHg), and the gradient during (46±5 mmHg) or after (46±5 mmHg) DRP infusion, confirming that the stenosis was mechanically stable. There was a strong trend towards a decrease in LAD bed microvascular resistance relative to baseline (P=0.042) (Figure 1).


Figure 1651
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  		Figure 1 LAD microvascular resistance at three experimental stages. With stenosis, microvascular resistance tended to decrease (*P=0.042 vs. baseline). DRP further reduced microvascular resistance. R, resistance; {Delta}P, pressure gradient; Q, flow.

 
The effect of coronary stenosis on capillary volume is shown in Figure 2, which demonstrates MCE images at high pulsing interval and videointensity vs. pulsing interval data before and during the stenosis. At baseline (Figure 2A), there was homogeneous myocardial contrast enhancement, with similar videointensity curves for the LAD and LCX beds (Figure 2B). With a severe LAD stenosis, there was an anterior/anteroseptal contrast defect (Figure 2C), and peak plateau videointensity (‘A’) and the rate of videointensity rise (‘ß’) in the LAD bed were less than that in the LCX bed (Figure 2D). These changes corresponded to a 31% decrease in radiolabelled microsphere flow.


Figure 1652
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  		Figure 2 MCE data at baseline (A and B) and with LAD stenosis (C and D) in one dog. Colour-coded MCE images at high pulsing intervals are shown on the left. There is posterior wall attenuation from LV cavity contrast. Region between arrows is the perfusion defect. Right panels show videointensity vs. pulsing interval data from the LAD and LCX beds.

 
Effect of DRPs during coronary stenosis
LAD microvascular resistance during the severe LAD stenosis significantly decreased after DRP (Figure 1). The LAD perfusion defect in the dog depicted in Figure 2A was no longer present after DRP administration (Figure 3A). The LAD and LCX videointensity-time curves were virtually superimposable (Figure 3B) after DRP.


Figure 1653
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  		Figure 3 MCE data from the same dog shown in Figure 2 after DRP infusion.

 
Figure 4 summarizes the MCE and radiolabelled microsphere flow data for all dogs. Relative to baseline, stenosis caused a significant decrease in LAD bed peak plateau intensity, the ‘A’ term (Figure 4A, P<0.02), the ß term (Figure 4B, P≤0.01), and the product of Axß (Figure 4C, P<0.004). There was a strong trend towards a decrease in radiolabelled microsphere-derived flow with stenosis (Figure 4D, P=0.028). After DRP injection, there was a significant increase in LAD peak plateau intensity (P<0.0007), the ß term (P<0.008), and the product of these two parameters (P<0.00005) back to baseline levels, which was paralleled by a significant increase in LAD bed radiolabelled microsphere perfusion (P<0.01).


Figure 1654
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  		Figure 4 Graph summarizing MCE parameters ‘A’ [peak plateau intensity or capillary volume (A)], ß [rate of microbubble replenishment or red cell velocity (B)], Axß [myocardial blood flow (C)], and LAD flow as measured by radiolabelled microspheres (D) at baseline, stenosis, and after DRP. *P=0.028 vs. baseline.

 
After DRP administration, epicardial LAD coronary artery flow increased by 16±7% and LCX epicardial coronary artery flow increased by 20±12%. There was no significant change in heart rate, arterial oxygen saturation, right atrial, or mean arterial pressures following DRP administration. There was a trend towards a decrease in distal LAD pressure (P=0.18) with DRP.

Effect of DRP on blood viscosity
Figure 5 plots viscosity vs. shear rate for both DRP and saline control. At the concentration used in our study (2.5 ppm), DRP had no effect on blood viscosity.


Figure 1655
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  		Figure 5 Graph of blood viscosity vs. shear rate with DRP and saline control.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Conclusions
 Acknowledgements
 References
 
This is the first study to demonstrate a new approach to treating acute coronary syndromes using DRPs as an agent to modulate the rheological properties of blood. The major finding of this investigation is that in the presence of a fixed, severe, flow-limiting coronary artery stenosis, nanomolar concentrations of blood-soluble DRPs restored resting myocardial perfusion to normal. We demonstrated that the DRP-induced decrease in microvascular resistance and the resulting flow restoration were associated with an increase in MCE-derived capillary volume and red cell velocity.

In this study, MCE provided incremental information for the assessment of microvascular perfusion, beyond that of radiolabelled microspheres, particularly as it pertains to the characterization of the intramyocardial microvascular space. Using gas-filled microbubbles as red blood cell tracers, MCE enables the user to quantitate intramyocardial blood volume and red blood cell velocity, features which radiolabelled microsphere cannot provide, and which were particularly suitable for the study of DRPs because of our hypothesis regarding their effect on resistance at the microvascular level.

Biological effects of DRPs
Drag reduction using linear, high-molecular weight polymers is a well-described hydrodynamic phenomenon, known as the Toms effect.24 PEO is a member of a class of water-soluble polymers used extensively both in industry25 and in pharmaceuticals.26 Although drag reduction was initially observed in turbulent flow, it also occurs in laminar flow27–29 and has been used in fire fighting, irrigation, and petroleum pumping30 (e.g. the Alaska Pipeline) to reduce frictional energy losses in large pipes. More recently, these observations have been extended to vascular systems, and a number of biological effects of DRP have been reported.31–39

The precise mechanism of action of DRPs in vivo is unknown because of their complex fluid dynamic behaviour in conjunction with the non-Newtonian physics of blood flow.2 Although turbulent flow does not occur in the microcirculation, DRPs have been shown to enhance microvascular perfusion in vivo.31,39–41 Microfilm measurements of frog mesenteric capillaries showed an acute increase in the number of functional capillaries after DRP infusion with no change in diameter, arterial pressure, or heart rate.39 Similarly, in diabetic rat cremaster muscle DRPs nearly doubled the number of functional capillaries.31 In rat models of haemorrhagic shock, minute amounts of DRP improved survival and tissue perfusion.40,41 This present study, however, is the first to evaluate the effect of DRPs on perfusion to the heart. Unlike other studies, radiolabelled microspheres, the gold standard for measuring tissue perfusion, were used to corroborate the effects of DRP on myocardial blood flow.

Because of multiple bifurcations, vessel curves, arterial pressure fluctuations, and pseudoturbulence, non-Poiseuillean flow occurs in the circulation.42 This includes flow separation, recirculation, and vortices at bifurcations, and post-stenotic flow disturbances, which cumulatively result in hydraulic energy loss.7 It is theorized that by virtue of their large molecular weight and linear configuration, DRPs organize blood flow, diminish random cell motion, and therefore, suppress flow disturbances and pseudoturbulence in smaller vessel networks. In this way, DRPs have been described as ‘corralling’ red blood cells into laminar flow patterns,7 an effect which could reduce hydraulic resistance and energy loss within a vascular system. By transforming disturbed flow in the microcirculation into more energy efficient flow, DRPs may augment perfusion at the tissue level.8,9

In non-cardiac experimental preparations, minute concentrations of DRPs improved haemodynamics.31–41,43 Unthank et al.43 reported that DRPs increased flow through stenotic canine iliac arteries without changing blood viscosity. Polimeni et al.36 found that aortic flow increased and total peripheral resistance decreased during infusion of three different DRPs. Mostardi et al.38 reported a 60% reduction in aortic wall flow disturbance frequency distal to an experimental partial occlusion after DRP infusion in canines.

The effect of coronary stenosis and DRPs on capillary volume
During the flow-limiting stenosis in our study, there was a decrease in total microvascular resistance. This finding is consistent with previous studies showing that with decreasing coronary driving pressure from progressive stenosis, arteriolar vasodilation lowers microvascular resistance, even after flow has decreased.44,45 Despite this decrease in microvascular resistance, there was a reduction in MCE-derived capillary volume, as seen on the colour-coded images which demonstrated a contrast defect that persisted even at the higher pulsing intervals. These findings are consistent with the concept that during severe stenosis, despite significant arteriolar vasodilation, capillary resistance increases. Jayaweera et al.6 have hypothesized that such a phenomenon adaptively results in the maintenance of capillary hydrostatic pressure in the face of decreasing driving pressure, and that this homeostatically preserves conditions for effective solute transfer, even at the expense of flow.

After DRP, microvascular resistance in the LAD bed decreased further and the MCE-derived capillary volume was restored to pre-stenotic baseline, indicated by resolution of the MCE contrast defect. The increase in MCE-derived capillary volume, ‘A’, was associated with an improvement in microvascular perfusion to the LAD bed as indicated by both MCE (Axß) and radiolabelled microspheres. In addition, red cell velocity by MCE, ß, increased with DRP, and is consistent with our previous findings using intravital microscopy.46 Thus, these findings suggest that flow to the stenotic LAD bed increased after DRP due to both an increase in capillary volume and red cell velocity.

Of note, MCE and radiolabelled microspheres measure ‘total perfusion’ to the myocardium, which is comprised of both anterograde flow through the epicardial coronary artery and adjacent bed collateral flow, which is well developed in canines.47 The LAD and LCX artery flow probe data suggest that the source of the increased LAD bed microvascular perfusion after DRP delivery was partly from LAD anterograde flow, but a predominant portion derived from collateral flow from the LCX artery. Importantly, a primary DRP-induced decrease in microvascular resistance in the LAD bed would be expected to enhance perfusion to that bed, regardless of whether the perfusion originates from collateral flow from the contralateral bed or from anterograde flow through the stenotic coronary artery.

Potential mechanism of action
Figure 6 depicts a hypothesized mechanism of action of DRPs. During flow-limiting stenosis, coronary driving pressure decreases (Figure 6, top panel). In this setting, during autoregulatory compensatory arteriolar dilation, there was a decrease in LAD bed microvascular resistance. This was associated with a reduction in MCE-derived capillary volume, which would increase capillary resistance. Jayaweera et al. have speculated that in this scenario the capillaries derecruit, a response which increases capillary hydrostatic pressure6 (top panel), and that this homeostatic mechanism supercedes the requirement for flow.6


Figure 1656
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  		Figure 6 Schematic of the coronary circulation demonstrating a possible mechanism of action of DRP at the capillary level. See text for details.

 
If DRPs diminish hydraulic energy loss along the vascular tree, driving pressure would be preserved and microvascular resistance would decrease. Thus, pre-capillary driving pressure (Figure 6, lower panel) would be higher than that prior to DRP, capillary hydrostatic pressure would be maintained and capillary volume would be restored. The net effect would be an increase in flow to the LAD bed. It should be noted that such a mechanism should enhance both anterograde- and collateral-derived flow. Finally, systemic haemodynamic changes are unlikely to account for our findings, as heart rate, arterial, and right atrial pressures remained constant throughout the study.

It should be noted that DRP did not appear to have a substantial effect on the LAD stenosis resistance itself, but rather, the effect was predominantly on LAD bed microvascular resistance, as evidenced by the radiolabelled microsphere and MCE data. Although there was some increase in epicardial blood flow through the LAD stenosis with DRP, it could not entirely account for the magnitude of increase in microsphere-derived flow to the LAD bed. This suggests that the mechanism of action of the DRPs was not predominantly through primary alteration of the stenosis resistance itself, but perhaps through its effect on the rheology of blood flow with respect to flow disturbances in the microcirculation. The magnitude of this rheologic effect would be magnified by the multitude of bifurcations which contribute to hydraulic energy loss in the microcirculation, and not just an effect at one, single epicardial stenosis.

Study limitations
Our study presumes that during flow-limiting stenosis, the arterioles are maximally or near maximally dilated, and that the DRP-induced decreases in microvascular resistance were not due to vasodilation. To address this possibility, we have previously performed two separate studies. First, we found no measurable DRP-induced vasodilation through direct observation during intravital microscopy in our studies of rat cremaster microvessels.46 Secondly, we have shown in a rabbit hindlimb model of pharmacologically induced maximal vasodilation that DRPs still increased flow despite abolition of vasomotor tone.48

We did not perform placebo. The confounding features that would be addressed by a placebo would be: (1) the volume effects that might explain the observed increase in flow to the LAD bed after the volume of DRP infusion; and (2) the time-related changes that might favour an improvement in flow were the stenosis mechanically unstable. Both of these are unlikely because (1) the volume in which DRP was administered was small (25 cc) relative to the dog's total blood volume and slowly infused; and (2) in separate experiments (detailed earlier), we validated the stability of our mechanical occluder for even longer periods than that required in this study.

DRPs could have changed blood viscosity, which is a major determinant of microvessel resistance.2 However, the DRPs used in our experiments did not alter blood viscosity.

PEO, the DRP used in this study, is sometimes referred to as polyethylene glycol (PEG), which is non-toxic, and has been approved by the Food and Drug Administration for internal consumption.25 Toxicity studies of PEG showed that it can be safely administered intravenously in animals.49 In vitro, DRPs mechanically degrade to lower molecular weight polymers in fluid flow.50 In humans, PEG is excreted by the kidney.25 Our own experience in animals using concentrations five times higher than that used in the current study, revealed that haematuria and haemolysis occurred through unknown mechanisms (unpublished). We did not observe haematuria or haemolysis at the lower DRP doses used in the current experiments.

As canines are known to have substantial myocardial collateral flow, it was not possible, in this model, to study the effects of DRPs on myocardial perfusion independent of collateral contributions. However, during myocardial ischaemia, augmenting total perfusion to the jeopardized bed is the over-riding therapeutic endpoint. Although it might be useful to study the effects of DRPs in a collaterally deficient species, such as pigs, the ability of DRPs to enhance total perfusion to myocardium subserved by a severe stenosis is of obvious clinical import.

Finally, the hypotheses we present here focus primarily on rheological and fluid dynamic explanations. Other unelucidated, non-rheological effects of DRPs, which are not addressed by our experimental design, could play a role in their physiologic effects.

Clinical implications
Current strategies for treating acute coronary syndromes focus on a combination of pharmacologically decreasing myocardial oxygen demand and restoring blood flow. The present study takes a highly novel approach, whereby enhancement of microvascular flow with DRP targets the hydrodynamic and rheological properties of blood. By decreasing microvascular resistance, DRPs may improve myocardial perfusion independently of the status of the epicardial coronary artery, both through anterograde and collateral pathways. This strategy could have major therapeutic benefit in acute coronary syndromes, while patients are awaiting traditional mechanical interventional approaches. That DRPs might enhance collateral flow, as suggested by our study, could have important clinical implications, because many patients with coronary disease have extensive collateral networks,47,51 which are typically underestimated by angiography, as most collaterals are less than 100 µm in size and below the resolution of angiography. As collateral flow is largely pressure-dependent, treatments that reduce collateral resistance and provide higher pressure to the jeopardized microcirculation, such as DRP administration, could have significant therapeutic impact by reducing angina or myocardial infarct size in patients with collateral-dependent myocardium.


   Conclusions
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
Acknowledgements
 References
 
In a canine model of flow-limiting coronary artery stenosis, DRPs reduced microvascular resistance, increased MCE-derived capillary volume, and improved myocardial perfusion. Although the mechanisms are unknown, based on the body of in vitro work described earlier, we speculate that DRPs may diminish energy-dissipating flow patterns occurring in the vascular network proximal to the capillaries, and hence preserve pre-capillary pressure in the setting of coronary stenosis. Primary manipulation of the hydrodynamic properties of blood may be a powerful new approach to the treatment of ischaemic coronary syndromes and other manifestations of ischaemic heart disease characterized by microvascular dysfunction.


   Acknowledgements
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
References
 
We acknowledge Bristol Meyers Squibb for providing the contrast agent (Definity) used in this study, Joie Marhefka for her help in preparing the DRPs and Erxiong Lu, David Fischer, Eli Friedman, Rekhi Varghese, and Vijay Ramanath for their technical assistance. We thank Thomas Kamarck for his assistance with the statistical analysis. Supported in part by a Post-Doctoral Fellowship grant from the American Heart Association, Pennsylvania-Delaware Affiliate (J.P.).

Conflict of interest: None declared.


   Footnotes
 
{dagger} This work was presented at the 75th Scientific Sessions of the American Heart Association. Back


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 

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