Articles in PresS. Am J Physiol Heart Circ Physiol (March 13, 2015). doi:10.1152/ajpheart.00017.2015
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
Angiotensin II Type I Receptor Blockade Attenuates Reflex Cutaneous Vasoconstriction in Aged but not Young Skin James A. Lang1 and Kelsey E. Kolb1
Des Moines University Department of Physical Therapy1 Des Moines, IA 50312 USA
American Journal of Physiology: Heart and Circulatory Physiology Running Title: Angiotensin II and Reflex Vasoconstriction with Age Key Words: microdialysis, skin blood flow, aging, temperature regulation, rho kinase Total Word Count: 3,380
Correspondence: James A. Lang, Ph.D. Assistant Professor Department of Physical Therapy Des Moines University 3200 Grand Avenue Des Moines, IA 50312 USA Phone: 515-271-1733 Email: [email protected]
Copyright © 2015 by the American Physiological Society.
44
ABSTRACT
45
Stimulation of angiotensin II type I receptors (AT1R) elicits vasoconstriction (VC) that may be
46
occurring through the activation of a pathogenic vascular pathway such as Rho kinase (ROCK).
47
We hypothesize that reflex cutaneous VC to whole-body cooling (Tsk = 30.5oC) in older humans
48
relies in part on AT1R activation, which may explain greater ROCK activity attendant with aging.
49
Two microdialysis (MD) fibers were placed in the forearm skin of 10 young (Y) (24 ± 1 years) and
50
10 older (O) (70 ± 2 years) individuals for infusion of 1) lactated Ringer’s solution (switched to
51
fasudil, a ROCK antagonist, after cooling) and 2) AT1R blockade with losartan. Laser Doppler flux
52
(LDF) was measured over each MD site and cutaneous vascular conductance (CVC) was
53
calculated (CVC = LDF/mean arterial pressure) and expressed as percent change from baseline
54
(%ΔCVCBASELINE). In older individuals the VC response to whole-body cooling was blunted (Y = -34
55
± 2, O = -17 ± 3 %ΔCVC) and was further attenuated at the losartan site (Y = -34 ± 3, O = -9 ± 3
56
%ΔCVC; p < 0.05). The VC response to an exogenous 10 µM dose of angiotensin II (Y = -27 ± 3, O
57
= -42 ± 5 %ΔCVC) was completely blocked in sites pretreated with losartan or with fasudil.
58
These data suggest that AT1R activation contributes to the reflex VC response in aged but not
59
young skin. Furthermore, the angiotensin II component of the VC response appears to occur
60
primarily through a ROCK-mediated mechanism.
1
61
INTRODUCTION
62
Reflex cutaneous vasoconstriction (VC) is an immediate and sustained response to whole-body
63
cold exposure that minimizes convective heat loss to the environment. This response is
64
mediated by multiple parallel signaling pathways that ultimately increase intracellular calcium
65
in vascular smooth muscle. Loss of redundancy and impaired function in older men and women
66
results in a blunted reflex VC response to cold exposure and increased susceptibility to excess
67
heat loss and hypothermia even in mildly cool ambient conditions (11, 19, 42).
68 69
The RhoA/Rho-kinase (ROCK) pathway is a proconstrictor vascular mechanism that may remain
70
functionally intact or potentially upregulated in aged skin (40). It was demonstrated that
71
approximately 50% of the reflex cutaneous VC response in aged skin was mediated by ROCK
72
(23). Despite activation of this pathway, the VC response in older adults remains blunted.
73
Although upregulated ROCK may strengthen the effectiveness of cold-induced VC from a
74
thermoregulatory standpoint, this may be occurring at the expense of microvascular function.
75
Increased ROCK activity precedes several vascular pathologies including atherosclerosis, stroke,
76
diabetes, endothelial dysfunction, and hypertension (5, 12, 24, 29-32, 34, 43). Thus, increased
77
ROCK activation may confer some thermoregulatory compensation benefit in a cold
78
environment but this may also indicate a shift toward preclinical signaling changes in the
79
vasculature.
80 81
Although the source for ROCK activation is unclear, animal and in vitro studies have identified
82
that ROCK activation increases in the presence of angiotensin II (AngII) (6, 16, 31). The classic
83
view is that AngII is produced from an endocrine pathway involving multiple organ systems that
84
originates with renin secretion from the kidney. However, it is increasingly evident that human
85
skin alone expresses a complete renin angiotensin system (RAS) for the production and
86
activation of AngII (35). Thus, AngII can be synthesized locally and have paracrine effects
87
independently of systemic RAS and may have a more central role in cutaneous vasomotor
88
function. This is supported by evidence indicating that elevated AngII is in part responsible for
89
blunted NO-mediated cutaneous vasodilation (VD) in low-flow postural tachycardia syndrome 2
90
(POTS) patients (37). However, the importance of AngII in the VC response or as a trigger for
91
increased ROCK activation in the cutaneous microvasculature has yet to be elucidated.
92 93
Increased AngII signaling and activation of downstream targets such as ROCK may provide a
94
causative link between primary aging and microvascular dysfunction. In light of that, the
95
purpose of this study was to determine the functional role of AngII in the reflex cutaneous VC
96
response in young and aged skin. We hypothesize that intradermal administration of an
97
angiotensin type I receptor (AT1R) antagonist, losartan, will have a greater effect in reducing the
98
reflex VC response to whole-body cooling (Tsk = 30.5 oC) in aged skin. We further hypothesize
99
that AngII may be eliciting cutaneous VC in part through downstream activation of ROCK.
3
100
METHODS
101
Subjects
102
All experimental procedures were approved by the Des Moines University Institutional Review
103
Board and conformed to the standards set by the Declaration of Helsinki. After providing
104
voluntary verbal and written informed consent, 10 younger (24 ± 1 years; 4 men, 6 women) and
105
10 older (70 ± 2 years; 3 men, 7 women) subjects participated in the study (Table 1). All
106
subjects were normotensive, not obese, nondiabetic, normal lipid profile, non-smokers, and not
107
taking any prescription medications that would otherwise alter thermoregulatory or vascular
108
function (e.g., statin or blood pressure lowering drug). Young women were normally
109
menstruating and tested during the low hormone phase (days 2-7) of the menstrual cycle or
110
during the placebo week of oral contraceptives (n=3) (7). All older women were
111
postmenopausal and not taking hormone replacement therapy. On the day of the
112
experimental visit, subjects reported to the laboratory after abstention from alcohol and
113
caffeine (>12 h) as well as strenuous physical activity or vitamin supplement (> 24 h).
114 115
Instrumentation
116
Subjects arrived to the laboratory (room temperature = 23 oC) in the morning (0800-1300) and
117
were positioned comfortably in a semirecumbent position for instrumentation. Two
118
microdialysis (MD) fibers (10 mm, 30 kDa molecular weight cutoff membrane, MD 2000
119
Bioanalytical Systems, West Lafayette, IN) were aseptically placed in the dermis of the ventral
120
forearm. Prior to placement, the entry and exit site for each fiber were marked ~2.5 cm apart
121
in an area of skin free of larger superficial veins. Ice was applied to the forearm for 5 min to
122
temporarily anesthetize the skin (17). Immediately thereafter, two 25-gauge needles were
123
inserted, one for each fiber site, in approximation to the marked areas of skin. Then, a MD fiber
124
was threaded through the beveled end of each of the needles and then withdrawn leaving the
125
membrane component (10 mm) of the MD fiber submerged in the dermis. After which, MD
126
fibers were secured with tape and then perfused for 60-90 minutes at 2.0 μL/min (Baby Bee
127
syringe drive and Hive Controlled; Bioanalytical Systems) to allow resolution of localized
128
hyperemia from needle insertion trauma. 4
129 130
To control skin temperature, subjects donned a water-perfusion suit that covered the entire
131
body with the exception of the face, feet, hands, and instrumented forearm. The unweighted
132
mean skin temperature (Tsk) was collected from six copper-constantan thermocouples taped on
133
the skin of the thigh, calf, abdomen, chest, back, and arm. Local skin temperature was
134
controlled by placing a heater (moorVMS-HEAT; Moor Instruments, Axminster, UK) directly over
135
each MD fiber site. The local temperature was set at 33 °C for the duration of the experimental
136
protocol. This was important to ensure that any vasomotor change detected is reflex in origin
137
and not confounded by locally derived stimuli.
138 139
Red blood cell flux (LDF), an index for skin blood flow, was continuously measured by multifiber
140
integrated laser Doppler probes (moorVMS-LDF; Moor Instruments) fitted into the local heaters
141
such that the probe was positioned perpendicular to the skin surface. Arterial blood pressure
142
was measured every 5 min on the contralateral arm using an automated cuff (Suntech Tango
143
M2) that was verified with brachial auscultation. The mean arterial pressure (MAP) was
144
calculated as the diastolic blood pressure plus one-third the pulse pressure. Heart rate was
145
monitored using a lead II electrocardiogram (CT-1000 cardiotachometer, CWE inc). Cutaneous
146
vascular conductance (CVC) was calculated as the ratio of laser Doppler flux (LDF) to MAP and
147
expressed as percent change from baseline (%ΔCVCBASELINE).
148 149
Protocol
150
After instrumentation, MD fiber sites were randomly assigned for continuous perfusion of
151
either 1) lactated Ringer’s solution serving as the control site or 2) 2 μg/L losartan, an
152
angiotensin II receptor type I (AT1R) antagonist. All drugs were weighed, dissolved in lactated
153
Ringer’s solution, and filtered with syringe microfilters (Acrodisc, Pall, Ann Arbor, MI)
154
immediately prior to use. Drug dosages were based from previous studies and verified with
155
pilot experiments (23, 38). The optimal AngII dose was determined as the dose that maximally
156
affected the VC response, and the losartan dose ensured complete blockade of AngII-mediated
5
157
VC without altering baseline CVC. After 60 min of drug perfusion, baseline values were
158
collected while Tsk was maintained at 34°C.
159 160
Following baseline, cold water was circulated through the suit to induce a reflex cutaneous
161
vasoconstriction response. Skin temperature was gradually reduced from 34°C to 30.5°C over a
162
30 min period and maintained at 30.5°C for an additional 10 min. The peak cooling stimulus
163
was not severe enough to elicit a shivering response and this was verified by asking participants
164
to describe their thermal discomfort or propensity to shiver. Rewarming for approximately 30
165
min followed to return Tsk to 34°C. Then, 10 μM angiotensin II was perfused through both MD
166
fibers for 10 minutes to verify the blockade established by losartan. After a 20 min washout
167
period, MD fibers were reassigned such that the losartan site was switched to perfuse lactated
168
Ringer’s solution and the control site was switched to receive 3 mM fasudil to inhibit ROCK.
169
Following 60-90 min of drug perfusion, the 10min AngII perfusion was repeated. After a 20 min
170
washout period, vascular responsivity was tested by briefly (~3 min) increasing the local heaters
171
to 42°C at each site until an increase in laser Doppler flux was observed.
172 173
Data Acquisition and Analysis
174
Data was collected at 1000 Hz and stored for offline analysis (LabChart v7.0 and PowerLab
175
16/35; ADInstruments, Bella Vista, NSW, Australia). Specifically, LDF data was processed into 1
176
sec averages every 4th sec throughout the protocol. CVC was calculated toward the end of a
177
baseline period, for each 0.5°C in Tsk during cooling, and during AngII perfusion. Normal
178
distribution was evaluated using Shapiro-Wilk tests. A three-way mixed model repeated
179
measures ANOVA followed by Bonferroni post hoc tests were conducted (SPSS version 22,
180
Chicago, IL) to detect age and treatment effects during both whole-body cooling and AngII
181
administration. Physical characteristics between age groups were analyzed using student’s
182
unpaired t-tests. All tests were two-sided and statistical significance was set at α = 0.05. An a
183
priori sample size analysis was performed, which indicated that 10 participants were needed to
184
detect significant differences at p < 0.05 and 80% power. Values are represented as mean ± SE.
185 6
186
RESULTS
187
The physical characteristics of the age groups are presented in Table 1. Groups were well
188
matched with respect to anthropometric parameters, resting MAP, and lipid profile. Although
189
total and LDL cholesterol was higher in older adults (p < 0.05), there was no difference with
190
respect to HDL cholesterol or cholesterol ratio.
191 192
The absolute CVC values (calculated as laser Doppler flux • mmHg-1) for each MD fiber site is
193
represented in Table 2. At the losartan site, CVC values during AngII perfusion differed from the
194
control site in older adults (p < 0.05). After MD fiber sites were reassigned so that fasudil was
195
perfusing skin, CVC was elevated both before (i.e., baseline) and during AngII perfusion (p
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
Angiotensin II Type I Receptor Blockade Attenuates Reflex Cutaneous Vasoconstriction in Aged but not Young Skin James A. Lang1 and Kelsey E. Kolb1
Des Moines University Department of Physical Therapy1 Des Moines, IA 50312 USA
American Journal of Physiology: Heart and Circulatory Physiology Running Title: Angiotensin II and Reflex Vasoconstriction with Age Key Words: microdialysis, skin blood flow, aging, temperature regulation, rho kinase Total Word Count: 3,380
Correspondence: James A. Lang, Ph.D. Assistant Professor Department of Physical Therapy Des Moines University 3200 Grand Avenue Des Moines, IA 50312 USA Phone: 515-271-1733 Email: [email protected]
Copyright © 2015 by the American Physiological Society.
44
ABSTRACT
45
Stimulation of angiotensin II type I receptors (AT1R) elicits vasoconstriction (VC) that may be
46
occurring through the activation of a pathogenic vascular pathway such as Rho kinase (ROCK).
47
We hypothesize that reflex cutaneous VC to whole-body cooling (Tsk = 30.5oC) in older humans
48
relies in part on AT1R activation, which may explain greater ROCK activity attendant with aging.
49
Two microdialysis (MD) fibers were placed in the forearm skin of 10 young (Y) (24 ± 1 years) and
50
10 older (O) (70 ± 2 years) individuals for infusion of 1) lactated Ringer’s solution (switched to
51
fasudil, a ROCK antagonist, after cooling) and 2) AT1R blockade with losartan. Laser Doppler flux
52
(LDF) was measured over each MD site and cutaneous vascular conductance (CVC) was
53
calculated (CVC = LDF/mean arterial pressure) and expressed as percent change from baseline
54
(%ΔCVCBASELINE). In older individuals the VC response to whole-body cooling was blunted (Y = -34
55
± 2, O = -17 ± 3 %ΔCVC) and was further attenuated at the losartan site (Y = -34 ± 3, O = -9 ± 3
56
%ΔCVC; p < 0.05). The VC response to an exogenous 10 µM dose of angiotensin II (Y = -27 ± 3, O
57
= -42 ± 5 %ΔCVC) was completely blocked in sites pretreated with losartan or with fasudil.
58
These data suggest that AT1R activation contributes to the reflex VC response in aged but not
59
young skin. Furthermore, the angiotensin II component of the VC response appears to occur
60
primarily through a ROCK-mediated mechanism.
1
61
INTRODUCTION
62
Reflex cutaneous vasoconstriction (VC) is an immediate and sustained response to whole-body
63
cold exposure that minimizes convective heat loss to the environment. This response is
64
mediated by multiple parallel signaling pathways that ultimately increase intracellular calcium
65
in vascular smooth muscle. Loss of redundancy and impaired function in older men and women
66
results in a blunted reflex VC response to cold exposure and increased susceptibility to excess
67
heat loss and hypothermia even in mildly cool ambient conditions (11, 19, 42).
68 69
The RhoA/Rho-kinase (ROCK) pathway is a proconstrictor vascular mechanism that may remain
70
functionally intact or potentially upregulated in aged skin (40). It was demonstrated that
71
approximately 50% of the reflex cutaneous VC response in aged skin was mediated by ROCK
72
(23). Despite activation of this pathway, the VC response in older adults remains blunted.
73
Although upregulated ROCK may strengthen the effectiveness of cold-induced VC from a
74
thermoregulatory standpoint, this may be occurring at the expense of microvascular function.
75
Increased ROCK activity precedes several vascular pathologies including atherosclerosis, stroke,
76
diabetes, endothelial dysfunction, and hypertension (5, 12, 24, 29-32, 34, 43). Thus, increased
77
ROCK activation may confer some thermoregulatory compensation benefit in a cold
78
environment but this may also indicate a shift toward preclinical signaling changes in the
79
vasculature.
80 81
Although the source for ROCK activation is unclear, animal and in vitro studies have identified
82
that ROCK activation increases in the presence of angiotensin II (AngII) (6, 16, 31). The classic
83
view is that AngII is produced from an endocrine pathway involving multiple organ systems that
84
originates with renin secretion from the kidney. However, it is increasingly evident that human
85
skin alone expresses a complete renin angiotensin system (RAS) for the production and
86
activation of AngII (35). Thus, AngII can be synthesized locally and have paracrine effects
87
independently of systemic RAS and may have a more central role in cutaneous vasomotor
88
function. This is supported by evidence indicating that elevated AngII is in part responsible for
89
blunted NO-mediated cutaneous vasodilation (VD) in low-flow postural tachycardia syndrome 2
90
(POTS) patients (37). However, the importance of AngII in the VC response or as a trigger for
91
increased ROCK activation in the cutaneous microvasculature has yet to be elucidated.
92 93
Increased AngII signaling and activation of downstream targets such as ROCK may provide a
94
causative link between primary aging and microvascular dysfunction. In light of that, the
95
purpose of this study was to determine the functional role of AngII in the reflex cutaneous VC
96
response in young and aged skin. We hypothesize that intradermal administration of an
97
angiotensin type I receptor (AT1R) antagonist, losartan, will have a greater effect in reducing the
98
reflex VC response to whole-body cooling (Tsk = 30.5 oC) in aged skin. We further hypothesize
99
that AngII may be eliciting cutaneous VC in part through downstream activation of ROCK.
3
100
METHODS
101
Subjects
102
All experimental procedures were approved by the Des Moines University Institutional Review
103
Board and conformed to the standards set by the Declaration of Helsinki. After providing
104
voluntary verbal and written informed consent, 10 younger (24 ± 1 years; 4 men, 6 women) and
105
10 older (70 ± 2 years; 3 men, 7 women) subjects participated in the study (Table 1). All
106
subjects were normotensive, not obese, nondiabetic, normal lipid profile, non-smokers, and not
107
taking any prescription medications that would otherwise alter thermoregulatory or vascular
108
function (e.g., statin or blood pressure lowering drug). Young women were normally
109
menstruating and tested during the low hormone phase (days 2-7) of the menstrual cycle or
110
during the placebo week of oral contraceptives (n=3) (7). All older women were
111
postmenopausal and not taking hormone replacement therapy. On the day of the
112
experimental visit, subjects reported to the laboratory after abstention from alcohol and
113
caffeine (>12 h) as well as strenuous physical activity or vitamin supplement (> 24 h).
114 115
Instrumentation
116
Subjects arrived to the laboratory (room temperature = 23 oC) in the morning (0800-1300) and
117
were positioned comfortably in a semirecumbent position for instrumentation. Two
118
microdialysis (MD) fibers (10 mm, 30 kDa molecular weight cutoff membrane, MD 2000
119
Bioanalytical Systems, West Lafayette, IN) were aseptically placed in the dermis of the ventral
120
forearm. Prior to placement, the entry and exit site for each fiber were marked ~2.5 cm apart
121
in an area of skin free of larger superficial veins. Ice was applied to the forearm for 5 min to
122
temporarily anesthetize the skin (17). Immediately thereafter, two 25-gauge needles were
123
inserted, one for each fiber site, in approximation to the marked areas of skin. Then, a MD fiber
124
was threaded through the beveled end of each of the needles and then withdrawn leaving the
125
membrane component (10 mm) of the MD fiber submerged in the dermis. After which, MD
126
fibers were secured with tape and then perfused for 60-90 minutes at 2.0 μL/min (Baby Bee
127
syringe drive and Hive Controlled; Bioanalytical Systems) to allow resolution of localized
128
hyperemia from needle insertion trauma. 4
129 130
To control skin temperature, subjects donned a water-perfusion suit that covered the entire
131
body with the exception of the face, feet, hands, and instrumented forearm. The unweighted
132
mean skin temperature (Tsk) was collected from six copper-constantan thermocouples taped on
133
the skin of the thigh, calf, abdomen, chest, back, and arm. Local skin temperature was
134
controlled by placing a heater (moorVMS-HEAT; Moor Instruments, Axminster, UK) directly over
135
each MD fiber site. The local temperature was set at 33 °C for the duration of the experimental
136
protocol. This was important to ensure that any vasomotor change detected is reflex in origin
137
and not confounded by locally derived stimuli.
138 139
Red blood cell flux (LDF), an index for skin blood flow, was continuously measured by multifiber
140
integrated laser Doppler probes (moorVMS-LDF; Moor Instruments) fitted into the local heaters
141
such that the probe was positioned perpendicular to the skin surface. Arterial blood pressure
142
was measured every 5 min on the contralateral arm using an automated cuff (Suntech Tango
143
M2) that was verified with brachial auscultation. The mean arterial pressure (MAP) was
144
calculated as the diastolic blood pressure plus one-third the pulse pressure. Heart rate was
145
monitored using a lead II electrocardiogram (CT-1000 cardiotachometer, CWE inc). Cutaneous
146
vascular conductance (CVC) was calculated as the ratio of laser Doppler flux (LDF) to MAP and
147
expressed as percent change from baseline (%ΔCVCBASELINE).
148 149
Protocol
150
After instrumentation, MD fiber sites were randomly assigned for continuous perfusion of
151
either 1) lactated Ringer’s solution serving as the control site or 2) 2 μg/L losartan, an
152
angiotensin II receptor type I (AT1R) antagonist. All drugs were weighed, dissolved in lactated
153
Ringer’s solution, and filtered with syringe microfilters (Acrodisc, Pall, Ann Arbor, MI)
154
immediately prior to use. Drug dosages were based from previous studies and verified with
155
pilot experiments (23, 38). The optimal AngII dose was determined as the dose that maximally
156
affected the VC response, and the losartan dose ensured complete blockade of AngII-mediated
5
157
VC without altering baseline CVC. After 60 min of drug perfusion, baseline values were
158
collected while Tsk was maintained at 34°C.
159 160
Following baseline, cold water was circulated through the suit to induce a reflex cutaneous
161
vasoconstriction response. Skin temperature was gradually reduced from 34°C to 30.5°C over a
162
30 min period and maintained at 30.5°C for an additional 10 min. The peak cooling stimulus
163
was not severe enough to elicit a shivering response and this was verified by asking participants
164
to describe their thermal discomfort or propensity to shiver. Rewarming for approximately 30
165
min followed to return Tsk to 34°C. Then, 10 μM angiotensin II was perfused through both MD
166
fibers for 10 minutes to verify the blockade established by losartan. After a 20 min washout
167
period, MD fibers were reassigned such that the losartan site was switched to perfuse lactated
168
Ringer’s solution and the control site was switched to receive 3 mM fasudil to inhibit ROCK.
169
Following 60-90 min of drug perfusion, the 10min AngII perfusion was repeated. After a 20 min
170
washout period, vascular responsivity was tested by briefly (~3 min) increasing the local heaters
171
to 42°C at each site until an increase in laser Doppler flux was observed.
172 173
Data Acquisition and Analysis
174
Data was collected at 1000 Hz and stored for offline analysis (LabChart v7.0 and PowerLab
175
16/35; ADInstruments, Bella Vista, NSW, Australia). Specifically, LDF data was processed into 1
176
sec averages every 4th sec throughout the protocol. CVC was calculated toward the end of a
177
baseline period, for each 0.5°C in Tsk during cooling, and during AngII perfusion. Normal
178
distribution was evaluated using Shapiro-Wilk tests. A three-way mixed model repeated
179
measures ANOVA followed by Bonferroni post hoc tests were conducted (SPSS version 22,
180
Chicago, IL) to detect age and treatment effects during both whole-body cooling and AngII
181
administration. Physical characteristics between age groups were analyzed using student’s
182
unpaired t-tests. All tests were two-sided and statistical significance was set at α = 0.05. An a
183
priori sample size analysis was performed, which indicated that 10 participants were needed to
184
detect significant differences at p < 0.05 and 80% power. Values are represented as mean ± SE.
185 6
186
RESULTS
187
The physical characteristics of the age groups are presented in Table 1. Groups were well
188
matched with respect to anthropometric parameters, resting MAP, and lipid profile. Although
189
total and LDL cholesterol was higher in older adults (p < 0.05), there was no difference with
190
respect to HDL cholesterol or cholesterol ratio.
191 192
The absolute CVC values (calculated as laser Doppler flux • mmHg-1) for each MD fiber site is
193
represented in Table 2. At the losartan site, CVC values during AngII perfusion differed from the
194
control site in older adults (p < 0.05). After MD fiber sites were reassigned so that fasudil was
195
perfusing skin, CVC was elevated both before (i.e., baseline) and during AngII perfusion (p
