The Pennsylvania State University The Graduate School Department of Kinesiology THE MECHANISMS AND FUNCTIONAL IMPLICATIONS OF ALTERED MICROVASCULAR VASOREACTIVITY IN HEALTHY AND ESSENTIAL HYPERTENSIVE MEN AND WOMEN A Dissertation in Kinesiology by Rebecca Sue Bruning  2013 Rebecca Sue Bruning Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2013 The thesis of Rebecca Bruning was reviewed and approved* by the following: W. Larry Kenney Professor of Physiology and Kinesiology Chair of Committee Lacy M. Alexander Assistant Professor of Kinesiology Dissertation Advisor Nilam Ram Associate Professor of Human Development and Psychology Timothy R. McConnell Professor of Exercise Science Chair of the Exercise Science Department Bloomsburg University Nancy I. Williams Professor of Kinesiology and Physiology Head of the Department of Kinesiology *Signatures are on file in the Graduate School iii ABSTRACT Cutaneous vasodilation enables nutrient delivery to the skin and effectively transfers heat away from the body core to the environment for thermoregulatory purposes. Direct local skin heating elicits a hyperemic response that is reflective of global changes in microcirculation and in endothelial function. Local skin heating is commonly used as a microvascular vasoreactivity test because it is largely reliant on nitric oxide (NO)-dependent mechanisms to elicit vasodilation. NO-dependent vasodilation elicited from local skin heating is reduced in populations with cardiovascular risk factors such as aging and essential hypertension, but further studies are needed to characterize the up- and down-stream mechanisms mediating NO-dependent signaling during local skin heating. Further, the effects of systemic cardiovascular therapies on microvascular function can be examined using skin vasoreactivity tests in both healthy and clinical populations. Drug therapies that alter microvascular function could have functional thermoregulatory consequences during heat stress. The purpose of this series of studies was to investigate and characterize the mechanisms mediating NO-dependent vasodilation in middle- aged and essential hypertensive men and women. In addition, we planned to investigate the functional thermoregulatory consequences of systemic antithrombotic therapy during rest and exercise in the heat. In the first study we investigated the specific nitric oxide synthase (NOS) isoforms responsible for mediating NO-dependent vasodilation during local skin heating in healthy young and middle-aged men and women. Human aging is associated with attenuated cutaneous vasodilation but little is known about the middle-aged cohort, which is commonly used for comparison with clinical populations. We tested the hypotheses that endothelial NOS (eNOS) is the primary isoform mediating NO production during local heating and that eNOS-dependent vasodilation would be reduced in middle-aged skin. Vasodilation was induced by local heating (42°C) and during acetylcholine dose-response (Ach-DR: 0.01, 0.1, 1.0, 5.0, 10.0, 50.0, 100.0mmol/L) protocols. Four intradermal microdialysis fibers were placed in the skin of 12 middle-aged (531yrs) and 12 young (231yrs) men and women. Sites served as control, non- selective NOS inhibited (NOS-I; L-NAME), inducible (i)NOS inhibited (1400W), and neuronal (n)NOS inhibited (NPLA). After full expression of the local heating response, L-NAME was perfused at all sites. Laser-Doppler flowmetry (LDF) was used to measure cutaneous vascular conductance (LDF flux/mean arterial pressure) and normalized to maximum (%CVC : sodium max iv nitroprusside). L-NAME reduced %CVC at baseline, all phases of the local heating response, max and at all Ach concentrations compared to all other sites. iNOS inhibition reduced the initial peak (53±2vs. 60±2%CVCmax; p<0.001); however, there were no other differences between control, nNOS, and iNOS inhibited sites during the phases of local heating or Ach-DR. When age cohorts were compared, middle-aged men and women had reduced NO-dependent vasodilation during local heating (526 vs. 684%CVC ; P = 0.013) and Ach perfusion (50mmol/L: 83±3 vs. 93±2; max 100mmol/L: 83±4 vs. 92±3%CVC ; both p=0.03). There were no differences in NOS isoform max expression obtained from skin biopsy samples between groups (all p>0.05). These data suggest that eNOS mediates the production of NO during local heating and that it is attenuated in middle- aged skin. In the second study we used spectral analysis (fast Fourier transformation) to characterize the skin flowmotion in LDF recordings during local heating-induced vasodilation before and after NOS-I (L-NAME) in essential hypertensive (HTN) and age-matched normotensive (NTN) men and women. We hypothesized that HTN reduces total power spectral densities (PSD), specifically in the frequency intervals (FI) associated with intrinsic endothelial and neurogenic control of the vasculature. Furthermore, we hypothesized that NOS-I would attenuate the endothelial FI in both groups. LDF recordings during local heating experiments from 18 HTN (MAP: 108±2mmHg) and 18 NTN (MAP: 88±2mmHg) men and women were analyzed. Local heating-induced vasodilation increased total PSD for all FI (all p<0.001). HTN lowered total PSD (HTN:30±6AU2, NTN:14±3AU2, p=0.03) and neurogenic FI (HTN:6±1AU2, NTN:17±3AU2, p<0.01) versus NTN. As a percentage of total PSD, HTN had reduced neurogenic (HTN:41±3%, NTN:51±1%, p<0.001) and higher myogenic contributions (HTN:29±3%, NTN:24±1%, p=0.04) to the total spectrum. NOS-I decreased total PSD (p<0.001) for both groups, but HTN exhibited lower endothelial (p<0.01), neurogenic (p<0.05), and total PSD (p<0.001) FI compared to NTN. These data suggest that HTN results in altered neurogenic and NO-dependent control of skin flowmotion and support the use of spectral analysis as a non-invasive technique to study vasoreactivity. Antithrombotic therapy with low dose aspirin (ASA) or clopidogrel bisulfate (CLO; Plavix®) is associated with attenuated skin vasodilator response and a greater rate of rise in core temperature in healthy middle-aged individuals during passive heating in a water perfused suit. In the third study we hypothesized that ASA and CLO therapy would have negative v thermoregulatory and cardiovascular consequences during rest and exercise in a hot environment. This was a double-blind, placebo-controlled, crossover study that examined the functional consequences of 7 days of ASA (81mg), CLO (75mg), and placebo (sucrose) in 14 healthy, middle-age (50–65 yr) men and women during passive heating in air (40 min at 30ºC, 40% relative humidity) followed by exercise (60% ̇ ). Oral temperature (T ) was measured in 2peak or the antechamber (23.0ºC±0.1ºC) before entering a warm environmental chamber. After 40 min of rest, subjects cycled on a recumbent cycle ergometer for up to 120 min. Esophageal temperature (T ) and laser- Doppler flux were measured continuously, and assessed as %CVC . Before es max entry into the environmental chamber there were no differences in T among treatments. or However, after 40 min of rest in the heat, T was significantly higher for ASA and CLO versus es placebo (37.2ºC±0.1ºC, 37.3ºC±0.1ºC, vs. 37.0ºC±0.1ºC, both P<0.001), a difference that persisted throughout exercise (P<0.001 vs. placebo). The mean body temperature thresholds for the onset of cutaneous vasodilation were shifted to the right for both ASA and CLO during exercise (P<0.05). These data suggest that ASA and CLO elevate core temperatures during passive heat stress and shift the onset of peripheral thermoeffector mechanisms toward higher body temperatures during exercise in the heat. vi TABLE OF CONTENTS LIST OF FIGURES ................................................................................................................. ...ix LIST OF TABLES ................................................................................................................... ..xii LIST OF ABBREVIATIONS .................................................................................................. .xiii ACKNOWLEDGEMENTS………………………………………………………………….....xv Chapter 1 INTRODUCTION ................................................................................................... 1 Background and Significance .......................................................................................... 1 Local Skin Heating ................................................................................................... 2 Acetylcholine Dose Response .................................................................................. 3 Spectral Analysis ...................................................................................................... 3 Skin Blood Flow and Platelet Inhibition .................................................................. 4 Summary .................................................................................................................. 5 Specific Aims and Hypothesis ......................................................................................... 5 Chapter 2 REVIEW OF THE LITERATURE ......................................................................... 8 Mechanisms Mediating Vasodilation during Local Skin Heating ........................... 9 Primary Aging and Cutaneous Vasodilation during Local Skin Heating ................. 11 Essential Hypertension and Cutaneous Vasodilation during Local Skin Heating .... 13 Essential Hypertension and Skin Flowmotion ......................................................... 13 Platelet Inhibition and Skin Blood Flow .................................................................. 16 Chapter 3 ENDOTHELIAL NITRIC OXIDE SYNTHASE MEDIATES CUTANEOUS VASODILATION DURING LOCAL SKIN HEATING AND IS ATTENUATED IN MIDDLE-AGED HUMAN SKIN .............................................................................. 21 Introduction ...................................................................................................................... 21 Methods ............................................................................................................................ 22 In Vivo Analysis of Vascular Function..................................................................... 23 In Vitro Analysis of Vascular Function .................................................................... 25 Data Acquisition and Statistical Analysis ................................................................ 25 Results .............................................................................................................................. 26 Discussion ........................................................................................................................ 28 Limitations ............................................................................................................... 30 Summary .................................................................................................................. 32 Chapter 4 ALTERED SKIN FLOWMOTION IN HYPERTENSIVE HUMANS .................. 40 Introduction ...................................................................................................................... 40 Materials and Methods ..................................................................................................... 41 Subjects .................................................................................................................... 41 Local Skin Heating and Assessment of Skin Blood Flow ........................................ 42 vii Power Spectral Density (PSD) Analysis .................................................................. 43 Statistical Analysis ................................................................................................... 43 Results .............................................................................................................................. 44 Discussion ........................................................................................................................ 46 Chapter 5 ASPIRIN AND CLOPIDOGREL ALTER CORE TEMPERATURE AND SKIN BLOOD FLOW DURING HEAT STRESS .......................................................... 55 Introduction ...................................................................................................................... 55 Methods ............................................................................................................................ 56 Blinded Drug Treatments ......................................................................................... 57 Subjects .................................................................................................................... 57 Experimental Protocol .............................................................................................. 58 Instrumentation and Measurements.......................................................................... 58 Experimental Timeline ............................................................................................. 59 Data Acquisition and Statistical Analysis ................................................................ 60 Results .............................................................................................................................. 60 Discussion ........................................................................................................................ 62 Aspirin, Clopidogrel and Passive Thermal Stress .................................................... 62 Aspirin, Clopidogrel and Exercise in the Heat ......................................................... 63 Limitations ............................................................................................................... 67 Chapter 6 CONCLUSION AND FUTURE DIRECTIONS .................................................... 74 Nitric Oxide Synthase Isoforms and Local Cutaneous Vasodilation ............................... 74 Implications .............................................................................................................. 75 Future Directions ...................................................................................................... 75 Altered Skin Flowmotion in Essential Hypertension ....................................................... 76 Implications .............................................................................................................. 76 Future Directions ...................................................................................................... 78 Implications .............................................................................................................. 80 Future Directions ...................................................................................................... 81 Appendix A: NOVEL METHODOLOGY FOR ASSESSING AGE-RELATED CHANGES IN CUTANEOUS VASODILATION DURING LOCAL SKIN HEATING ........................................................................................................................ 83 Methods ............................................................................................................................ 83 Subjects .................................................................................................................... 83 In Vivo Vasoreactivity Studies ................................................................................. 84 Local Heating Protocol .................................................................................................... 85 Mathematical Modeling ........................................................................................... 85 Statistical Analysis ................................................................................................... 86 Preliminary Findings ........................................................................................................ 86 Figure Legends ................................................................................................................. 88 Appendix B: EXPANDED METHODS AND THEORY FOR SPECTRAL ANALYSIS .... 95 Rationale .......................................................................................................................... 95 viii Laser-Doppler Flowmetry Pre-Signal Processing ............................................................ 96 Data Collection and Acquisition .............................................................................. 96 Sampling Rate of Laser-Doppler Flowmetry Signal ................................................ 96 Signal Processing ............................................................................................................. 96 Fast Fourier Transformation ..................................................................................... 96 Estimation of Power Spectral Density Using Fast Fourier Transformations ........... 97 Methods for Power Spectral Density Estimation ..................................................... 98 Step-by-Step WinDAQ and Excel File Preparation ......................................................... 99 Converting WinDAQ Data Files into a .CSV File ................................................... 99 MATLAB Program .......................................................................................................... 102 Appendix C: INFORMED CONSENT FORMS ..................................................................... 106 NOS Isoform and Spectral Analysis Studies ................................................................... 106 Mathematical Modeling of Local Cutaneous Vasodilatory Responses with Aging Study ........................................................................................................................ 115 Platelet Inhibition and Exercise Study ............................................................................. 125 Bibliography ............................................................................................................................ 139 ix LIST OF FIGURES Figure 1-1. Schematic depicting ways in which primary human aging and essential hypertension can reduce NO bioavailability and attenuate local cutaneous vasodilation during direct local skin heating. The signaling mechanisms which act to reduce NO bioavailability are in gray. L-orn: L-ornithine; L-cit: L-citrulline; BH : tetrahydrobiopterin; L-arg: L-arginine; ONOO: peroxyntirite; S-NO: S- 4 nitrosating, NOS: nitric oxide synthase. .......................................................................... 7 Figure 2-1. Schematic illustrating the mechanisms mediating the thermal hyperemic response to direct local skin heating. Both primary aging and essential hypertension result in attenuated NO-dependent dilation during local skin heating. Further studies are needed to characterize pathological changes in neural, myogenic, and endothelial control of cutaneous vasodilation during local skin heating in aged and essential hypertensive populations. Adapted from Roustit and Cracowski 2013. ......................... 19 Figure 2-2. Schematic illustrating the putative mechanisms and thermoregulatory consequences of altered microvascular function during systemic platelet inhibition with low dose aspirin (ASA; 81mg) and clopidogrel bisulfate (CLO, 75mg). ................ 20 Figure 3-1. A time course representative tracing of the control site during the local heating protocol. The baseline, initial peak, plateau, and post-L-NAME plateau are identified. ......................................................................................................................... 34 Figure 3-2. Mean±SE as a percentage of maximal cutaneous vascular conductance (%CVC ) during A) baseline, B) initial peak, C) plateau, and D) non-specific NOS max inhibition with L-NAME for the microdialysis drug treatment sites. iNOS inhibition with 1400W decreased the initial peak and non-specific NOS inhibition with L-NAME decreased baseline, initial peak, and the plateau during local heating. *P<0.001 Significant difference from the control site. .................................................... 35 Figure 3-3. Mean±SE as a percentage of maximal cutaneous vascular conductance (%CVC ) during an acetylcholine (Ach) dose response. There was no effect of max selective nNOS or iNOS inhibition. Non-selective NOS inhibition with L-NAME attenuated %CVC at all concentrations of Ach. *P<0.001 Significant difference max from the control site. ........................................................................................................ 36 Figure 3-4. Group mean±SE as a percentage of maximal cutaneous vascular conductance (%CVC ) during the plateau and post-L-NAME plateau of the local heating max response. The post-L-NAME plateau was increased and total NO-dependent vasodilation, illustrated as the difference between the plateau and the post L-NAME plateau was attenuated in the middle aged group. *P=0.013 Significant difference versus young. ................................................................................................................... 37 Figure 3-5. Group mean±SE as a percentage of maximal cutaneous vascular conductance (%CVC ) during an acetylcholine (Ach) dose response in the control sites. Ach- max induced vasodilation was attenuated in the middle-aged at the two highest concentrations of Ach (50 and 100mM). *P=0.03 Significant difference versus young. ............................................................................................................................... 38 x Figure 3-6. iNOS, nNOS, eNOS, VASP, and pVASP. Expression of the three NOS enzymes, and VASP, and pVASP activity were determined by western blotting. GAPDH was used as loading control. Sample blot is shown in first panel. Densitometry analysis was performed using ImageJ software (NIH). ............................. 39 Figure 4-1. Mean power spectral density plots for (A) baseline, (B) local skin heating, (C) non-specific nitric oxide synthase inhibition with L-NAME, and (D) SNP-induced maximum laser Doppler recordings during a local skin heating protocol in normotensive (unfilled circles) and essential hypertensive (black circles) men and women. Spectra were estimated from the fast Fourier transformation of the laser Doppler recordings. The frequency intervals 0.01Hz (0.008-0.02Hz), 0.04Hz (0.02- 0.05Hz), and 0.1Hz (0.05-0.15Hz) are considered to correspond to endothelial, neurogenic, and myogenic activity, respectively. ............................................................ 52 Figure 4-2. Power spectral density (PSD) during (A) baseline, (B) local skin heating, (C) non-specific NOS-I with L-NAME, and (D) SNP-induced maximum laser Doppler responses during local skin heating protocol in normotensive (white bars) and essential hypertensive (gray bars) men and women. The power was calculated from the fast Fourier transformation of the laser Doppler signal around 0.01Hz (0.008- 0.02Hz), 0.04Hz (0.02-0.05Hz), and 0.1Hz (0.05-0.15Hz) frequency intervals (FI), which are considered to correspond to endothelial, neurogenic, and myogenic activity, respectively. The total power was calculated from the of the non- overlapping FI. Baseline spectral power in all FI were lower than all other periods of the local heating response in both groups (P<0.001). L-NAME plateau spectral power was lower for all FI compared to the heating plateau and SNP-induced max (P<0.05). *P<0.05, **P<0.01, ***P<0.001 Significantly different than normotensive men and women. †P<0.05 Significantly different than baseline, local skin heating, and reduction with L-NAME for the endothelial FI in essential hypertensive subjects. Data are represented as box and whisker plots with the whiskers representing the 95th percentile. The solid lines within the box represent the median PSD and the dashed lines represent the average PSD. ..................................................... 53 Figure 4-3. Pie chart representing the mean relative power spectral density (PSD) as a percent contribution of the calculated total power during (A) baseline, (B) local skin heating, (C) non-specific NOS inhibition with L-NAME, and (D) SNP-induced maximum laser Doppler recordings. The relative energy contribution of each frequency interval (FI) was assessed around the 0.01Hz (0.008-0.02Hz), 0.04Hz (0.02-0.05Hz), and 0.1Hz (0.05-0.15Hz) FI, considered to correspond to endothelial, neurogenic, and myogenic activity, respectively. The total power was calculated from the of the non-overlapping FI. The size of the pie chart is proportional to the absolute total power with the exception of the baseline period which was increased in size so the data would be visible (proportional between the normotensive and essential hypertensive group). #P<0.05 Significantly different than the neurogenic FI in the baseline, L-NAME plateau, and SNP max periods. *P<0.05 Significantly different than normotensives. ........................................................................................... 54 Figure 5-1. Individual differences in T between placebo and systemic drug treatments es after 40 minutes of mild passive heat stress (30°C, 40% relative humidity) in an environmental chamber. Mean responses±SE for low-dose ASA (graph A) and