Exploring the vascular smooth muscle receptor landscape in vivo: ultrasound Doppler versus near-infrared spectroscopy assessments

1

Exploring the Vascular Smooth Muscle Receptor Landscape In Vivo: 1 Ultrasound Doppler versus Near Infrared Spectroscopy (NIRS) Assessments 2 3 Stephen J. Ives1,2,7, Paul J. Fadel4, R. Matthew Brothers5, Mikael Sander6, and D. Walter 4 Wray1,2,3 5 6 7 1Geriatric Research, Education, and Clinical Center 8 George E. Whalen VA Medical Center, Salt Lake City, Utah 9 10 2Department of Internal Medicine, Division of Geriatrics 11 University of Utah, Salt Lake City, Utah 12 13 3Department of Exercise and Sport Science 14 University of Utah, Salt Lake City, Utah 15 16 4Medical Pharmacology & Physiology 17 University of Missouri, Columbia, Missouri 18 19 5Department of Kinesiology and Health Education 20 University of Texas at Austin, Austin, Texas 21 22 6Department of Cardiology 23 Copenhagen University Hospital, Hvidovre, Denmark 24 25 7Department of Health and Exercise Sciences 26 Skidmore College, Saratoga Springs, NY 27 28 29 Running Title: Ultrasound Doppler vs. NIRS 30 31 CORRESPONDENCE D. Walter Wray, Ph.D. Department of Internal Medicine, Division of Geriatrics, University of Utah VAMC SLC, GRECC 182, Bldg 2 Rm 1C03 500 Foothill Drive, Salt Lake City, UT 84148 Phone: 801.582.1565 ext 4-1556 (office) Fax: 801.584.5656 Email: [email protected] 32 Key Words: Vascular Imaging; Alpha adrenergic; ANG-II; Vasoconstriction; Microcirculation 33 Manuscript Word Count: 4,194 34 Total Number of Figures and Tables: 3 35 Article Type: Rapid Report 36 37

Articles in PresS. Am J Physiol Heart Circ Physiol (January 17, 2014). doi:10.1152/ajpheart.00782.2013

Copyright © 2014 by the American Physiological Society.

2

ABSTRACT 38

Ultrasound Doppler and near infrared spectroscopy (NIRS) are routinely used for non-invasive 39

monitoring of peripheral hemodynamics in both clinical and experimental settings. However, the 40

comparative ability of these methodologies to detect changes in microvascular and whole-limb 41

hemodynamics during pharmacologic manipulation of vascular smooth muscle receptors located 42

at varied locations within the arterial tree is unknown. Thus, in ten healthy subjects (25±2 yrs), 43

changes in resting leg blood flow (Ultrasound Doppler; femoral artery) and muscle oxygenation 44

(HbO2+MbO2; vastus lateralis) were evaluated simultaneously in response to intra-arterial 45

infusions of phenylephrine (PE, 0.025 - 0.8 µg/kg/min), BHT-933 (2.5 - 40 µg/kg/min), and 46

angiotensin II (ANGII, 0.5 - 8 ng/kg/min). All drugs elicited significant dose-dependent 47

reductions in leg blood flow and HbO2+MbO2. Significant relationships were found between 48

ultrasound Doppler and NIRS changes across doses of PE (r2 = 0.37±0.08), BHT-933 (r2 = 49

0.74±0.06), and ANGII (r2 = 0.68±0.13), with the strongest relationships evident with agonists 50

for receptors located preferentially “downstream” in the leg microcirculation (BHT-933 and 51

ANGII). Analyses of drug potency revealed similar EC50 between ultrasound Doppler and NIRS 52

measurements for PE (0.06±0.02 vs. 0.10±0.01), BHT-933 (5.0±0.9 vs. 4.5±1.3), and ANGII 53

(1.4±0.8 vs. 1.3±0.3). These data provide evidence that both ultrasound Doppler and NIRS track 54

pharmacologically-induced changes in peripheral hemodynamics, and are equally capable of 55

determining drug potency. However, considerable disparity was observed between agonist 56

infusions targeting different levels of the arterial tree, suggesting that receptor landscape is an 57

important consideration for proper interpretation of hemodynamic monitoring with these 58

methodologies. 59

60

3

INTRODUCTION 61

A variety of non-invasive methods have been devised to assess peripheral hemodynamics in 62

humans. Ultrasound Doppler has grown in popularity due to its non-invasive nature and relative 63

ease of use. However, the spatial resolution of this methodology limits measurements to larger 64

caliber vessels, and in the case of the arm or leg, provides determination of bulk limb blood flow 65

that includes perfusion of skin, bone, and skeletal muscle. In contrast, near infrared spectroscopy 66

(NIRS) has been developed as a viable method of assessing tissue oxygenation, and under 67

steady-state conditions, microcirculatory blood flow (16). While our group (26) and others (4, 7, 68

18) have used these non-invasive methodologies concomitantly in an effort to comprehensively 69

evaluate peripheral hemodynamics, little work has been done to determine to what degree these 70

methods are related, or to establish whether one method is preferable to another under certain 71

experimental or clinical conditions. 72

Knowledge of the interchangeability between these methodologies may be particularly 73

informative when determining changes in vascular tone elicited by pharmacologic agents that 74

target specific receptor subtypes located at distinct levels of the arterial tree. One of the best 75

described examples of this heterogeneous distribution of vascular smooth muscle receptors in the 76

peripheral vasculature is the alpha adrenergic pathway. Using only ultrasound Doppler, we have 77

identified a unique spatial distribution for alpha adrenergic receptor subtypes in humans, with α1-78

adrenergic receptors preferentially localized proximally and α2-adrenergic receptors located more 79

distally in the leg vasculature (27). However, a clear indication of how bulk limb blood flow 80

relates to microcirculatory blood flow is needed to fully understand the functional consequence 81

and potential therapeutic implications for this heterogeneity in the vascular smooth muscle 82

receptor landscape. 83

4

Therefore, the purpose of the current study was to determine the relationship between 84

ultrasound Doppler and NIRS assessments of skeletal muscle hemodynamics, and to evaluate 85

potential differences in this relationship using pharmacologic agents acting on different portions 86

of the arterial tree. We hypothesized that these methods would be significantly related in terms of 87

drug-induced changes in peripheral hemodynamics in response to local drug delivery, and that 88

both methodologies would detect similar levels of drug potency (EC50). However, we expected a 89

difference in the nature of the relationship between methods depending upon the drug used; 90

specifically, we anticipated the best relationship between ultrasound Doppler and NIRS in 91

response to BHT-933 and ANGII, drugs that primarily target distal portions of the leg 92

microcirculation as opposed to phenylephrine, which preferentially targets the more proximal 93

vasculature of the leg (2, 27). 94

95

96

97

5

MATERIALS & METHODS 98

Subjects and General Procedures 99

Ten young, healthy males participated in the present study (Table 1). All subjects were 100

nonsmokers, normotensive, and free from overt cardiovascular disease, as determined by health 101

history questionnaire and physical examination. Protocol approval and written informed consent 102

were obtained according to the guidelines of the local ethics committee of Copenhagen and 103

Frederiksberg, in accordance with Declaration of Helsinki. All studies were performed in a 104

thermoneutral environment with subject in a semi-recumbent position. Subjects reported to the 105

laboratory in a fasted state and without caffeine or alcohol use for 12 and 24 h, respectively. 106

They also had not performed any exercise within the past 24 h. Arterial and venous catheters 107

were placed under local anesthesia (Lidocaine, 5ml, 20mg/ml) in a retrograde fashion in the right 108

common femoral artery and vein using sterile technique. After catheter placement, subjects 109

recovered for 30 min prior to any drug infusions. A portion of the ultrasound Doppler data were 110

generated from previous published studies by our group (2, 27); additional analyses were applied 111

to address the novel hypothesis of this study making direct comparisons to NIRS derived 112

measurements. 113

114

Drugs 115

Phenylephrine (PE; Danish county pharmaceutical corporation, SAD) was used as a specific α1-116

adrenergic agonist. BHT-933 (BHT; Sigma-Aldrich, Denmark) was used as a specific α2-117

adrenergic agonist. Angiotensin-II (ANGII, Clinalfa, Switzerland) was used as an AT receptor 118

agonist. A range of drug doses were administered (PE: 0.025, 0.05, 0.1, 0.2, 0.4, 0.8 µg/kg/min; 119

BHT-933: 2.5, 5, 10, 20, 40 µg/kg/min; ANGII: 0.5, 1, 2, 4, 8 ng/kg/min). Each dose was infused 120

6

for 2-min to achieve a steady state hemodynamic response. Ultrasound Doppler and NIRS 121

measurements were performed concurrently and continuously during each drug infusion. 122

123

Measurements 124

Femoral Blood Flow. The ultrasound machine (model CFM 800, GE Medical) was equipped 125

with a mechanical sector transducer operating at an imaging frequency of 7.5 MHz. Vessel 126

diameter was determined at a perpendicular angle along the central axis of the scanned area, 127

where the best spatial resolution was achieved. The femoral artery was insonated distal to the 128

inguinal ligament for dynamic recordings of diameter throughout a cardiac cycle. The maximum 129

diameter (systole) was used for calculation of blood flow. The blood velocity profile was 130

obtained using the same transducer with a Doppler frequency of 4.0–6.0 MHz, operated in the 131

high-pulsed repetition frequency mode (4–36 kHz) with a sample volume of 5 mm in depth. All 132

blood velocity measurements were obtained with a 46-50 insonation angle. At all sample points 133

we obtained both diameter of the femoral artery (FAD) and, approximately 20–30 s later, an 134

angle-corrected, time- and space-averaged, and intensity-weighted mean blood velocity (Vmean) 135

(Echopac Software, GE Medical and PowerLab, ADInstruments). Using arterial diameter and 136

Vmean, femoral blood flow was calculated as: FBF = Vmean •π (vessel diameter/2)2 • 60, where 137

blood flow is in milliliters per minute (mL/min). 138

139

Near Infrared Spectroscopy. Near infrared spectroscopy (NIRS) (NIRO300, Hamamatsu, Japan) 140

was used to determine muscle oxygenation of the vastus lateralis muscle. Muscle oxygenation 141

was determined by the oxyhemoglobin signal (6), which cannot differentiate between 142

oxyhemoglobin (HbO2) and oxymyoglobin (MbO2); thus, we express the data as a conglomerate 143

7

signal (HbO2 + MbO2). The site over the vastus lateralis was cleaned, and double sided adhesive 144

tape was used to secure the optodes in place. Optodes were positioned inside a rubber holder 145

with a fixed distance of 4 cm between emitting and receiving optodes, for an effective 146

penetrating depth of 2 cm. The optodes were then covered and further secured with an opaque 147

wrap. The data were acquired at 0.5 Hz, and 30 sec averages were created at baseline and during 148

the last minute of drug infusion for each dose. To normalize the data to individual maximal 149

physiological changes, the total labile signal (TLS) was determined by placing a cuff proximal to 150

the NIRS probes inflated to suprasystolic levels (250 mmHg) for 10 min to elicit complete 151

deoxygenation. The pharmacologically induced changes in the NIRS signal were then expressed 152

as a percent of this maximal change (%TLS). 153

154

Data Analysis 155

The EC50 (half-maximal effective concentration) was calculated on an individual basis using a 156

sigmoidal parameter to estimate the vascular sensitivity to the pharmacological agonists 157

(Biodatafit, v.1.02, Castro, CA). To determine the relationship between methods, the slopes and 158

coefficient of determinations were calculated on an individual basis and compared between drug 159

trials. Repeated measures ANOVA and paired t-tests were used where appropriate. The level of 160

significance was established at p < 0.05. Data are presented as mean ± standard error of the mean 161

(mean ± SE). 162

163

164

8

RESULTS 165

Subject characteristics are presented in Table 1. The dose response curves and drug potency 166

(EC50) for PE, BHT-933, and ANGII are presented in Figure 1. There was a significant 167

relationship between ultrasound Doppler and NIRS changes for all drugs (Figure 2), although 168

the nature of this relationship was significantly different between drugs, with PE having the 169

lowest coefficient of determination. A significant drug-related difference in the femoral arterial 170

diameter response to each pharmacological agonist was also observed, with the highest doses of 171

PE inducing a much greater change in diameter (8.66 ± 0.27 to 5.79 ± 0.51 mm) compared to 172

ANGII (8.75 ± 0.28 to 8.36 ± 0.53) and BHT-933 (8.63 ± 0.22 to 7.79 ± 0.22). Ultrasound 173

Doppler responses presented in Figure 1 (panels D, E, and F) and Figure 2 have been reported 174

previously (2, 27), and are presented here for the purposes of comparison with NIRS assessment. 175

176

177

178

9

DISCUSSION 179

The main finding of the current study was that ultrasound Doppler and Near Infrared 180

Spectroscopy (NIRS) methodologies detect similar changes in limb hemodynamics in response 181

to intra-arterial infusion of three distinct vasoconstrictor drugs (PE, BHT-933, and ANGII), both 182

in terms of drug potency (EC50) and efficacy (dose-dependent changes). Significant relationships 183

were found between the two methodologies for all drugs. However, considerable disparity in 184

correlative analysis was observed between agonist infusions targeting different levels of the 185

arterial tree. The best relationships were evident with agonists preferentially targeting receptor 186

groups located more distal in the leg microcirculation (BHT-933 and ANGII) (2, 27), while a 187

more modest correlation was observed for PE, which likely reflects the greater distribution of 188

alpha-1 receptors in the proximal compared to distal portions of the arterial tree. Together, these 189

data provide evidence that both ultrasound Doppler and NIRS are equally sensitive to detecting 190

pharmacologically-induced changes in peripheral hemodynamics and drug potency, and also 191

emphasize that receptor landscape is an important consideration for proper interpretation of 192

hemodynamic monitoring with these methodologies. 193

194

Assessment of Macro vs. Microcirculatory Hemodynamics. While various methods have been 195

devised for assessment of peripheral hemodynamics in humans, ultrasound Doppler and NIRS 196

have emerged as gold standards of non-invasive testing. When equipped with a duplex linear 197

array probe, ultrasound Doppler is capable of simultaneous, high-resolution measurements of 198

both vessel diameter (12 MHz) and blood velocity (5MHz), enabling beat-to-beat determination 199

of limb blood flow. However, ultrasound Doppler measurements are limited to large conduit 200

vessels, and thus are most often used to determine bulk limb blood flow. In contrast, NIRS 201

10

exploits the principle that near-infrared light easily penetrates tissues and is maximally absorbed 202

by large vessels to provide measurements of oxygenated and deoxygenated hemoglobin and 203

myoglobin in the microcirculation (20). Since changes in the absorption of NIR light are 204

proportional to changes in the relative concentrations of these molecules under steady-state 205

conditions when oxygen demand is constant, NIR absorption is thought to reflect changes in 206

oxygen supply, and thus provide an index of microcirculatory blood flow under resting 207

conditions (9). 208

Though these non-invasive methodologies are often used in an effort to comprehensively 209

evaluate peripheral hemodynamics, the degree to which the two methods are able to track 210

hemodynamic changes, and in particular the sensitivity to detect pharmacologically-induced 211

vasoconstriction, is not well understood. In one of the only studies directly comparing these 212

methodologies, Fadel et al. (10) investigated the potential link between ultrasound Doppler and 213

NIRS in both humans and anaesthetized rats. In this study, reflex sympathetic vasoconstriction 214

measured in the forearm with ultrasound Doppler and NIRS were significantly related. These 215

results were confirmed in a rodent model, revealing a significant relationship between the 216

methodologies elicited by direct sympathetic nerve stimulation (10). 217

Findings from the present study extend these earlier findings in several important ways. 218

First, we have identified that ultrasound Doppler and NIRS are equally efficacious in detecting 219

changes in blood flow in the leg (Figure 1), an ambulatory limb with distinct differences in both 220

vascular function (21) and vascular smooth muscle receptor sensitivity (19) compared to the arm. 221

We have also utilized an array of discrete pharmacologic agents to elicit robust vasoconstriction 222

via various vascular smooth muscle receptor types with differing distribution across the arterial 223

tree. This pharmacologic approach also afforded the opportunity to examine the ability of these 224

11

two methodologies to determine drug potency, as quantified by half-maximal effective 225

concentration (EC50). To our knowledge, this is the first study utilizing both ultrasound Doppler 226

and NIRS to assess EC50, and to report comparable values between the two methods (Figure 1). 227

228

Receptor-Specific Hemodynamic Responses. Though a clear relationship was present between 229

ultrasound Doppler and NIRS for all drugs, a clear disparity in the strength of the relationship 230

between the two methods was identified. The best relationships were seen with BHT-933 (an α2-231

adrenergic receptor agonist) and ANGII (an AT receptor agonist), where coefficients of 232

determination exceeded 0.7 for all subjects (Figure 2). In contrast, the relationship between 233

methodologies was substantially lower for PE (an α1-adrenergic receptor agonist). This 234

difference is, at least in part, mediated by differential receptor landscapes across the leg arterial 235

tree. Indeed, we have previously identified functional α1-adrenergic receptors in the upstream 236

portions of the femoral artery capable of reducing arterial diameter by nearly 50% in response to 237

PE, whereas post-junctional α2-adrenergic receptors are preferentially expressed in the more 238

distal portions of the femoral artery and produce minimal changes in conduit artery diameter 239

(27). These previous findings in humans are supported by earlier work in animals indicating a 240

similar pattern of alpha adrenergic receptor distribution (1), and together indicate a hierarchy of 241

receptor subtypes that may be relevant to the regulation of blood flow and arterial blood 242

pressure. 243

In the present study, we observed a 30-40% reduction in femoral artery diameter during 244

the highest doses of PE, while no significant reductions in femoral artery diameter were observed 245

during any dose of ANGII or BHT-933, as reported previously (2, 27). These findings providing 246

evidence for a paucity of α2-adrenergic and ANGII receptors at the level of the conduit vessel, 247

12

providing evidence for a differential “receptor landscape” may partially explain why the 248

relationship between ultrasound Doppler and NIRS is lowest during PE infusion (Figure 2). 249

Indeed, an assessment of microvascular hemodynamics (i.e. NIRS) may not track perfectly with 250

conduit artery limb blood flow measurements due to the ability of PE to bind at multiple sites 251

along the arterial tree, whereas better agreement would be expected when infusing drugs acting 252

predominantly in the skeletal muscle microcirculation (i.e. ANGII and BHT-933). 253

254

Clinical Implications. There is now considerable evidence supporting the concept that 255

peripheral artery disease is characterized by formation of atherosclerotic lesions at the level of 256

the conduit vessels in the lower limbs (15, 22, 24). However, these medium and large caliber 257

vessels are inexorably linked to the downstream skeletal muscle microcirculation, where the 258

large majority of vasomotor regulation occurs. This obvious but often overlooked association 259

between different levels of the arterial tree is increasingly recognized as an important 260

consideration in the etiology and progression of cardiovascular disease, particularly with respect 261

to therapies targeting the vascular endothelium and the sympathetic nervous system (17). In this 262

context, the present data concerning the simultaneous determination of macro and 263

microcirculatory hemodynamics in the peripheral circulation may be particularly important as we 264

seek to better define the integrative relationship between conduit and resistance vessel beds. 265

Findings from the present study also support the utility of concurrent ultrasound Doppler and 266

NIRS measurements for exploring regional patterns of adaptation in patients with peripheral 267

artery disease. 268

269

13

Experimental Considerations. We recognize the known effect of adipose tissue thickness on 270

NIR light absorption and scatter (13, 25), and therefore cannot exclude the possibility that 271

differences in adipose thickness may have affected our NIRS measurements. This concern is 272

somewhat mitigated by use of a large separation between source and detector optodes in the 273

present study, which provides a maximum measurement depth of approximately 2 cm. This 274

depth is more than sufficient to reach skeletal muscle tissue in young healthy adults, where 275

normal adipose tissue thickness of the vastus lateralis is <10 mm (3, 23). We also recognize the 276

potential of high-irradiance NIR light to provoke nitric oxide (NO) release (5, 12). While a very 277

lower power (<2mW/m2) NIR device was employed in the present study, there are currently no 278

data addressing the potential impact of this device on NO bioavailability. Further work is 279

necessary to elucidate the interaction between low-power NIR and NO release in humans. By 280

design, the present study compared the capabilities of ultrasound Doppler and NIRS derived 281

measures to detect hemodynamics changes at varying portions of the arterial tree. Although these 282

are the two most commonly used methods for hemodynamic monitoring, we acknowledge that 283

the addition of magnetic resonance imaging (MRI) or contrast perfusion ultrasound measures 284

could provide a more comprehensive examination of skeletal muscle microvascular function. 285

Finally, it is noteworthy that the technology of both ultrasound Doppler (8) and NIRS devices 286

(11) is continually advancing, including the development of multi-channel, spatially resolved 287

NIR devices (14). Thus, caution is warranted when extrapolating results from the present study 288

to other measurement devices that may differ in spatial or temporal resolution. 289

290

Conclusions. These data provide evidence that both ultrasound Doppler and NIRS track 291

pharmacologically-induced changes in peripheral hemodynamics to a similar degree, and are 292

14

equally capable of determining drug potency. However, disparity in responses with different 293

drugs suggests that receptor location in the arterial tree is an important consideration for proper 294

interpretation of hemodynamic monitoring with these methodologies. 295

296

ACKNOWLEDGEMENTS 297

Funded in part by AHA 0835209N (D.W.W). 298

299

300

15

Table 1: Subject Characteristics 301 302

Variable Age, yrs 26 ± 2 Height, cm 187 ± 2 Weight, kg 81 ± 4 BMI, kg/m2 23 ± 1 Heart rate, beats/min 60 ± 3 Mean Arterial Blood Pressure, mmHg 88 ± 2 Leg Blood Flow, ml/min 403 ± 60 Leg Vascular Conductance, ml/min/mmHg 4.6 ± 0.7

16

FIGURE 1: Dose response curves for the α1-agonist Phenylephrine (PE), the α2-agonist BHT-303 933, and the AT receptor agonist Angiotensin II (ANGII) assessed via Near Infrared 304 Spectroscopy (NIRS; panels A-C) and femoral blood flow (FBF, ultrasound Doppler, panels D-305 F). Ultrasound Doppler responses illustrated in panels D, E, and F have been reported previously 306 (2, 27), and are presented here for the purposes of comparison with NIRS assessment. 307 308 309 310 311 312 FIGURE 2: Relationship between femoral blood flow (FBF, ultrasound Doppler) and muscle 313 oxygenation (NIRS) during pharmacologic vasoconstriction induced by the α1-agonist 314 Phenylephrine (PE, panel A), the α2-agonist BHT-933 (panel B), and the AT receptor agonist 315 angiotensin II (ANGII, panel C). Ultrasound Doppler responses have been reported previously 316 (2, 27), and are presented here for the purposes of comparison with NIRS assessment. 317 318 319

320

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[Ang II] (ng/kg/min)0 2 4 6 8 10

-40

-30

-20

-10

0

[PE] (�g/kg/min)

0.0 0.2 0.4 0.6 0.8 1.0-50

-40

-30

-20

-10

0

[BHT-933] (�g/kg/min)

0 10 20 30 40 50-40

-30

-20

-10

0

[PE] (�g/kg/min)

0.0 0.2 0.4 0.6 0.8 1.0

FBF

( � m

l/min

)

-250

-200

-150

-100

-50

0

[BHT-933] (�g/kg/min)

0 10 20 30 40 50

FBF

( � m

l/min

)

-250

-200

-150

-100

-50

0

[Ang II] (ng/kg/min)

0 2 4 6 8 10

FBF

( � m

l/min

)

-250

-200

-150

-100

-50

0

A

B

C

D

E

F

EC50 = 0.10 ± 0.01 EC50 = 0.06 ± 0.02

EC50 = 4.5 ± 1.3 EC50 = 5.0 ± 0.9

EC50 = 1.3 ± 0.3 EC50 = 1.4 ± 0.8

� H

bO2+

MbO

2(%

TLS)

� H

bO2+

MbO

2(%

TLS)

� H

bO2+

MbO

2(%

TLS)

(Panels D, E, and F represented for comparison from 2,27)

PE

FBF (� mL/min)

-200-150-100-500

-50

-40

-30

-20

-10

0

BHT-933

FBF (� mL/min)-200-150-100-500

-40

-30

-20

-10

0

ANGII

FBF (� mL/min)-200-150-100-500

-40

-30

-20

-10

0

r2 = 0.68 +/- 0.13

r2 = 0.74 +/- 0.06

r2 = 0.37 +/- 0.08

A

B

C

� H

bO2+

MbO

2(%

TLS)

� H

bO2+

MbO

2(%

TLS)

� H

bO2+

MbO

2(%

TLS)

(FBF values represented for comparison from 2,27)