Adaptation to 5 weeks of intermittent local vascular pressure increments; mechanisms to be considered in the development of primary hypertension?

RESEARCH ARTICLE

Integrative Cardiovascular Physiology and Pathophysiology

Adaptation to 5 weeks of intermittent local vascular pressure increments;

mechanisms to be considered in the development of primary hypertension?

O. Eiken, A. Elia, H. Sk€oldefors, P. Sundblad, M. E. Keramidas, and R. K€olegård

Division of Environmental Physiology, Swedish Aerospace Physiology Center, KTH Royal Institute of Technology, Stockholm, Sweden

Abstract

The aims were to study effects of iterative exposures to moderate elevations of local intravascular pressure on arterial/arteriolar stiffness and plasma levels of vasoactive substances. Pressures in the vasculature of an arm were increased by 150 mmHg in healthy men (n = 11) before and after a 5-wk regimen, during which the vasculature in one arm was exposed to fifteen 40-min sessions of moderately increased transmural pressure ( þ 65 to þ 105 mmHg). This vascular pressure training and the pressure- distension determinations were conducted by exposing the subjects ’ arm versus remaining part of the body to differential ambi- ent pressure. During the pressure-distension determinations, venous samples were simultaneously obtained from pressurized and unpressurized vessels. Pressure training reduced arterial pressure distension by 40 ± 23% and pressure-induced flow by 33 ± 30% (P < 0.01), but only in the pressure-trained arm, suggesting local adaptive mechanisms. The distending pressure-diame- ter and distending pressure- flow curves, with training-induced increments in pressure thresholds and reductions in response gains, suggest that the increased precapillary stiffness was attributable to increased contractility and structural remodeling of the walls. Acute vascular pressure provocation induced local release of angiotensin-II (ANG II) and endothelin-1 (ET-1) (P < 0.05), sug- gesting that these vasoconstrictors limited the pressure distension. Pressure training increased basal levels of ET-1 and induced local pressure release of matrix metalloproteinase 7 (P < 0.05), suggesting involvement of these substances in vascular remod- eling. The findings are compatible with the notion that local intravascular pressure load acts as a prime mover in the develop- ment of primary hypertension.

NEW & NOTEWORTHY Adaptive responses to arterial/arteriolar pressure elevation have typically been investigated in cross-sec- tional studies in hypertensive patients or in longitudinal studies in experimental animals. The present investigation shows that in healthy individuals, fifteen 40-min, carefully controlled, moderate transmural pressure elevations markedly increase in vivo stiff- ness (i.e. reduce pressure distension) in arteries and arterioles. The response is mediated via local mechanisms, and it appears that endothelin-1, angiotensin-II, and matrix metalloproteinase 7 may have key roles.

essential hypertension; precapillary remodeling; precapillary stiffness; vascular distensibility; vascular pressure adaptation

INTRODUCTION

It has since long, and repeatedly, been postulated that in humans, the wall stiffness of precapillary resistance vessels increases in response to sustained or periodically elevated arterial pressure, and hence that the two-way stimulation pathway between increased arterial pressure and increased total peripheral blood- flow resistance, may constitute a

“vicious circuit” in the development of primary hyperten- sion (for reviews, see 1 – 4). Thus, already in the nineteenth century, Ewald observed that patients suffering from Brights disease exhibited arteriolar media hypertrophy and sug- gested that this was both caused by and underpinning their hypertension (5). The notion of a causal relationship

between arterial pressure and precapillary wall stiffness has since been con firmed, predominantly in longitudinal experimental studies in animals (for reviews, see 2 – 4) and in cross-sectional studies in humans, suggesting that individuals suffering from chronic hypertension exhibit increased wall thickness-to-lumen ratio in small arteries and arterioles (6 – 8).

A few longitudinal experimental studies have also been performed in healthy humans, after the development of a technique that makes it possible to, in a controlled manner, manipulate the transmural pressure in the vasculature of a human limb suf ficiently to investigate the entire pressure- distension relationship in arteries and arterioles (9). Thus, a 5-wk period of intermittent intravascular pressure elevations

Correspondence: O. Eiken (eiken@kth.se).

Submitted 15 September 2020 / Revised 24 December 2020 / Accepted 20 January 2021

in one arm, substantially decreased the in vivo pressure dis- tension and pressure-induced flow in local arteries (i.e., increased arterial/arteriolar stiffness) (10). Based on the find- ing that arterial/arteriolar stiffness is higher in the legs than in the arms, it can be assumed that this capacity, to adapt the local vascular pressure resistance, constitutes a salient physiological mechanism to cope with hydrostatic pressure gradients in the circulatory system (11). Moreover, precapil- lary wall stiffness appears to comprise a plastic function, since the increased stiffness observed after a 5-wk pressure- training regimen gradually abates during a 4-wk recovery pe- riod (10), and because, even after lifelong exposure to erect posture, the stiffness of leg arteries/arterioles decreases rap- idly upon removal of the intravascular hydrostatic pressure gradients during a 5-wk exposure to sustained recumbency (12, 13). Mechanisms governing the pressure-induced stiff- ness changes in human precapillary blood vessels are to a large extent unknown. It appears, though, that experimen- tally induced precapillary pressure habituation constitutes a local response since it develops only in the vessels of the pressure-habituated limb, without any “spill over” effects to the vessels of the contra-lateral limb (1, 10).

Acute intravascular pressure elevations induce local release of the vasoconstrictive peptides angiotensin-II (ANG II) and endothelin-1 (ET-1), which may serve to limit vascular pressure distention (14 – 16). Longitudinal studies in experi- mental animals suggest that sustained stimulation of hyper- tensive agonists (i.e., ANG II and ET-1) are also capable of stimulating in flammatory cascades, signified by increases in C-reactive protein (CRP) levels (17), and inducing structural remodeling of precapillary walls via downstream mediators (18 – 22), of which matrix metalloproteinase 7 (MMP-7) has been implicated a key player (20 – 22). As regard healthy humans it is, however, unknown whether and to what extent iterative moderate increments of arterial pressure affect local release of vasoconstrictive peptides and their down- stream mediators of vascular remodeling and/or in flamma- tory responses. Accordingly, the aims of the present study were to con firm the functional effects on precapillary vessels of a 5-wk local intravascular pressure-training regimen and to investigate whether such regimen affects the pressure- induced release of ET-1, ANG II, MMP-7, and CRP.

METHODS

Eleven nonsmoking men participated as test subjects;

notably, also women were invited to participate, but none volunteered. The subjects ’ average (range) age, body mass, and height were 24 (21 –28) years, 79 (68–95) kg, and 1.83 (1.75 –1.91) m, respectively. To be included, each subject should be healthy based on medical history, physical exami- nation and an electrocardiogram; speci fic exclusion criteria were any medication, signs of hypertension, frequent extra- systolies or other cardiac dysrhythmias and history of claustrophobia or vascular headache (Hortons or migraine).

Before enrolling, each subject gave his written informed con- sent, being aware that he was free to terminate any single experiment or training session, and also to withdraw from the study, at any time. The study protocol and experimental procedures were approved by the Regional Human Ethics

Committee in Stockholm (Dnr: EPN 2014/1801-32) and con- formed to the standards set by the Declaration of Helsinki.

General Study Protocol

Using a technique by which the transmural pressures in the vasculature of an arm can be elevated as desired, a 5-wk vascular pressure-training regimen was conducted, during which the blood vessels in one arm were exposed to moder- ately elevated transmural pressure ( þ 65 to þ 105 mmHg) 3  40 min per wk (see also below). Using a similar tech- nique, the arterial and arteriolar pressure-distension rela- tionships were measured during step-wise increasing local vascular pressures (up to þ 150 mmHg). These arterial/arteri- olar pressure-distension determinations (see also below) were performed in one arm before, and in both arms after, the pressure-training regimen. During the course of each such vascular pressure-distension provocation, venous blood samples were drawn simultaneously from the arm subjected to vascular pressure provocation and the contra-lateral con- trol arm. Subsequently, plasma concentrations of several vasoactive substances were determined. In this manner, ar- terial and arteriolar pressure-distension relationships and pressure-induced release of vasoactive substances were determined during acute exposure to high intravascular pressure, and any adaptation of such responses to the pres- sure training regimen was established.

Pressure-Distension Determinations and Blood Sampling

The experiments were conducted using a technique described in detail elsewhere (9, 11). Brie fly, the subject was seated in a pressure chamber with one arm ( “test arm”) extended through an opening in the chamber door and maintained at the level of the heart by use of a supporting stand positioned outside the chamber. The test arm was her- metically sealed to the opening slightly distally of the axilla, using a short self-sealing rubber sleeve. A trunk harness was used to stabilize the subject and hence to allow him to sit relaxed and keep the test arm still as the pressure in the chamber was elevated. Ambient temperature was main- tained at 25

C (range 24 –27

C) at the site of the test arm. The mechanisms by which this technique act to elevate local vas- cular pressures have been described in detail previously (1, 9). Brie fly, when chamber pressure is raised, pressure increases in all tissues enclosed in the chamber and the elevated pressure is transmitted undistorted to the blood vessels of the test arm outside the chamber. Consequently, transmural pressures in the vasculature of the outside arm increase in direct proportion to the applied chamber pressure.

The diameter of the brachial artery was measured in the

test arm 5 cm proximal to the cubital fossa using ultraso-

nography, measurements being conducted in B-mode image

during end-diastole (determined from the ECG), as wall-to-

wall distance in the sagittal section, using a 4 –9 MHz linear

array multifrequency transducer (CX-50, Philips Healthcare,

Sweden). To ensure that the same vessel segment was inves-

tigated during the different trials, and that a similar segment

was investigated in the vessel of the contra-lateral arm,

care was taken to record intravascular and extravascular

“landmarks” during the sonography. To minimize investiga- tor bias, all ultrasound-Doppler recordings were coded during the data analyses phase. All measurements were per- formed by the same sonographer. With that ultrasonogra- pher, and using similar equipment and performing identical measurements as in the present study, we have previously found the coef ficient of variation for brachial artery lumen determinations to be 1.7% –1.8% ( 11). In the present study, the expected brachial arterial pressure distension as well as the training-induced changes in such pressure distension were several-fold larger than the resolution of the ultrasound lumen diameter measurements ( <0.04 mm, cf 23, 24).

Brachial artery flow was estimated by simultaneous meas- urements of vessel lumen diameter and mean- flow velocity, using the ultrasound-Doppler system. Flow velocities were collected with the sample volume covering 100% of the ar- terial lumen, and with an angle correction of 50

. Assuming that the artery had a circular cross-section, flow was calcu- lated by multiplying vessel cross-sectional area by the time integral of the mean- flow velocity across five consecutive cardiac cycles.

Heart rate (HR) was derived from electrocardiographic recordings using a bipolar precordial lead (Physiocontrol Lifepak 8; Physiocontrol Corp., Redmond, WA). Systolic and diastolic arterial pressures (SAP, DAP) were measured con- tinuously in the mid phalanx of the third finger inside the chamber using a volume-clamp technique (Portapres; TNO, Amsterdam, The Netherlands). The level of the heart was used as the point of reference for the arterial pressure meas- urements. Changes in forearm tissue volume were estimated from impedance plethysmographic recordings using a tetrapolar constant-current impedance system (Minnesota Impedance Cardiograph Model 304 A; Instrumentation For Medicine, Inc., Greenwhich, CT) with four pairs of standard disposable pregelled electrodes placed on the test arm.

During the vascular pressure provocations, the participant rated his perceived arm pain using a ratio scale (25), in which pain can be rated from 0 = no pain to 10 = very, very strong (almost intolerable).

Venous blood samples were drawn from Te flon catheters (Ven flon, Becton Dickinson & Co, New Jersey) positioned bilaterally in antecubital veins. The samples, which were col- lected in EDTA-prepared tubes (K2EDTA, REF 454410, Greiner Bio-One GmbH, Kremsmunster, Austria), were cen- trifuged (2,000 g for 10 min at 4

C), and the plasma superna- tants were frozen to 80

C for subsequent analyses.

Experimental protocol.

Each experiment started with a 10-min baseline period with normal atmospheric pressure in the chamber. Thereafter, chamber pressure was increased step-wise every 3 min. The pressure plateaus were 30, 60, 90, 120, and 150 mmHg above atmospheric pressure. Following the last plateau, chamber pressure was rapidly released to basal levels (atmospheric pressure). All recordings were obtained during the last 2 min at each pressure plateau. Blood samples (8 mL) were drawn simultaneously from both arms after the 10-min baseline pe- riod and during the last min at the 90- and 150-mmHg pres- sure plateaus. Recordings of arterial diameter and flow velocities for subsequent determination of flow were

obtained in the test arm immediately before the blood sampling.

Prior to the pressure-training regimen (see below), such vascular pressure-distension provocation was performed in one arm (dominant arm). Between 4 and 6 days following the last session of the pressure-training regimen, identical vascular pressure-distension provocations were performed in both arms, provocations being separated by a minimum of 2 hr; the order in which these two provocations were per- formed (pressure-trained arm first or untrained arm first) was alternated among subjects in a balanced fashion. The ra- tionale of performing pressure-distension tests in one arm before and two arms after the pressure training was to avoid any carry-over effects of the pressure provocation on the pressure-training response, assuming that, for the individual subject, the initial pressure-distension responses did not dif- fer between arms (9, 11).

Plasma analyses.

An enzyme-linked immunosorbent assay (ELISA) technique was utilized to assess systemic concentrations of ET-1 (R&D systems, Minneapolis, MN, human endothelin-1, QET00B, sensitivity 0.102 pg/mL; intra-assay variability 4.9%), CRP (R&D systems, Minneapolis, MN, human C-reactive protein, DCRP00, sensitivity 0.022 ng/mL; intra-assay variability

5.3%), and MMP-7 (Biomatik Inc., Wilmington, DE, human matrix metalloproteinase 7, EKE60873, sensitivity 0.1 ng/mL; intra-assay variability 5.3%). ANG II was quanti fied by enzyme immunoassay (RayBiotech Inc., Norcross, GA, human angiotensin II, EIA-ANGII-1, sensi- tivity 0.3 pg/mL; intra-assay variability 2.2%).

Vascular Pressure Training

For each participant, vascular pressure training was per- formed in the arm that was not subjected to the initial (pre- training) pressure-distension provocation (i.e., in the nondominant arm), whereas transmural pressure in the arm vessels was increased by exposing the arm to subatmo- spheric pressures rather than exposing the rest of the body to supraatmospheric pressures (as in the pressure-distension determinations). Thus, during each training session, the sub- ject was sitting with one arm inside an airtight Plexiglas cylinder (length: 66 cm, diameter: 25 cm), and the arm posi- tioned at the level of the heart. The arm was hermetically sealed to the opening of the cylinder slightly distally of the axilla, by use of a short self-sealing rubber sleeve. Pressure in the cylinder was reduced to the desired level by use of a vac- uum pump and the cylinder was equipped with a pressure- release safety valve set to open at a pressure differential of 130 mmHg.

The training regimen comprised 15 sessions conducted

over 5 wks (3 sessions/wk), each session consisting of four

10-min pressure exposures, intervened by 5-min pauses with

normal atmospheric pressure in the cylinder; during each

10-min exposure, local transmural pressures were increased

over a 1-min period and then maintained at the targeted pla-

teau. The aim was to gradually elevate the transmural pres-

sure plateau during the course of the training regimen from

þ 65 mmHg during the first week to þ 75 mmHg during the

second week, and so on, to finally reach þ 105 mmHg during

the last week ’s sessions. The plateau pressure was, however, also gauged so that discomfort/pain in the test arm never exceeded three (moderate) on the 10-graded scale.

Calculations and Statistical Analyses

Brachial artery diameter was determined during end-dias- tole, and hence, arterial distending pressure (DP) was calcu- lated by adding chamber pressure to DAP. As blood flow in the forearm and hand is mainly controlled by local vascular resistance, it can be argued that flow should be treated as a function of arteriolar rather than arterial DP. However, because the average arteriolar DP is not readily determined, the flow pattern in the brachial artery was examined by treating it as a function of peak arteriolar DP (i.e., DP at the upstream end of the arterioles, corresponding to arterial DP;

9). To determine whether a pressure training-induced change in pressure-distension relationship was attributable to a change in the threshold or gain of the distension, linear functions were fitted to the initial four and final three data points, respectively, of every, individual DP-diameter and DP- flow curve. The DP corresponding to the crossing point of the two regression lines was de fined as the threshold DP, and the slope of the regression line of the last three points was de fined as the gain of the pressure distension (cf 1).

Analysis of the normal distribution of the data was per- formed with the Kolmogorov –Smirnov test. Thereafter, a two-way [condition (pretrained vs. posttrained arm vs. post- untrained arm)  distending pressure or applied chamber pressure] linear model repeated measures ANOVA was used to examine the differences between and within conditions.

Mauchly ’s test was conducted to assess for sphericity, and the Greenhouse-Geisser ɛ correction was used to adjust the degrees of freedom, when the assumption of sphericity was not satis fied. When ANOVA revealed significant effects, pair- wise comparisons with Bonferroni post hoc tests were used to assess differences. Differences in pain ratings were eval- uated with Friedman ’s test, followed by a Wilcoxon test. The alpha level of signi ficance was set a priori at 0.05. Unless oth- erwise stated, data are reported as means ± SD. All statistical analyses were performed using Statistica 8.0 (Statsoft, Tulsa, OK) software.

RESULTS

No adverse reactions or events occurred during the course of the study. All subjects tolerated the pressure-training regi- men well and were in general able to adhere to the predeter- mined time-pressure protocol. On a few occasions during the training sessions, transmural pressure in the trained vascula- ture was temporarily reduced below the target plateau (by 5 – 20 mmHg), to reduce pain and/or discomfort in terms of local numbness or paresthesia.

During the vascular pressure-distension determinations, all subjects experienced local pain in the test arm. The pain increased with increasing chamber pressure, and before the pressure-training regimen it was rated 5.0 (3 –8) [=median (range)] at peak chamber pressure (150 mmHg). The pres- sure-training regimen reduced pressure-induced pain in the trained arm [median (range): 2.0 (1 –5); P = 0.005], but not in the untrained arm [4.5 (1 –8)].

Arterial Pressure Responses

In all conditions, arterial pressures increased ( P < 0.001) during the course of the arm blood-vessel pressure provoca- tion, with no signi ficant intercondition differences in the SAP or DAP responses. The SAP/DAP values at basal versus peak (0 vs. 150 mmHg) chamber pressure were as follows:

pretraining 128 ± 12/70 ± 10 versus 144 ± 16/80 ± 15 mmHg;

posttraining untrained arm 119 ± 15/67 ± 13 versus 156 ± 26/

87 ± 16 mmHg; posttraining trained arm 121 ± 18/65 ± 10 ver- sus 150 ± 22/81 ± 12 mmHg.

Vascular Pressure Distension

Before training, brachial artery diameter remained virtu- ally unchanged up to a DP of 181 ± 19 mmHg and then increased ( P < 0.001) by 20.5 ± 9.4% at the highest DP ( Fig.

1). Pressure training reduced brachial artery pressure disten- sion to 12.2 ± 7.7% in the pressure-trained arm ( P < 0.001) but did not affect it in the untrained arm ( D diameter at peak DP = 19.6 ± 9.2%; P = 0.61; Fig. 1).

Likewise, before training, brachial artery flow remained unaltered up to a DP of 178 ± 22 mmHg and then increased ( P < 0.001) 6-fold from a baseline value of 48 mL/min to

0 5 10 15 20 25

Diameter change (%)

Distending pressure (mmHg) Pre-untrained

Post-untrained Post-trained

***

0 50 100 150 200 250 300

Flow (ml/min)

Pre-untrained Post-untrained Post-trained

***

Distending pressure (mmHg)

Figure 1. Relative change in brachial artery diameter (top) and brachial artery

flow (bottom) as functions of distending pressure, before and after the 5-wk

pressure-training regimen in untrained and trained vessels. Statistical sig-

ni ficance between before training and after training, in the trained vessel

(P < 0.001). Values are means (SD), n = 11.

279 mL/min at the highest DP (Fig. 1). Pressure training reduced the pressure- flow response by 33% to 186 mL/min in the pressure-trained artery (P = 0.016) but did not affect it in the untrained artery ( flow at peak DP = 265 mL; P = 0.75; Fig. 1).

Table 1 shows the DP thresholds for the onsets as well as the gains (slopes) of pressure-diameter and pressure- flow relationships. It can be seen that the pressure-training regi- men increased the DP threshold and blunted the gain, both for the pressure-diameter and pressure- flow relations (P <

0.05); the training regimen did not in fluence DP thresholds or gains in the untrained vessel.

Tissue Impedance

During the course of each pressure-distension provocation, forearm tissue impedance in the test arm exhibited a progres- sive decrease with increasing chamber pressure (Fig. 2).

Pressure training reduced the impedance drop in the trained arm (P = 0.001) but not in the untrained arm (P = 0.94).

Endothelin-1 and Angiotensin-II

Pretraining elevation of DP increased ET-1 ( P < 0.001) and ANG II (P = 0.017) concentrations only in the pressurized arm vessels (Table 2). ET-1 was higher at 150 mmHg in the pressurized than the control arm vessels, whereas no between-arm differences were recorded in ANG II (Table 2).

Following training, basal ET-1 concentrations were signi fi- cantly greater compared with pretraining in the pressure- trained arm ( P = 0.001), and there was a trend toward signifi- cance denoted in the untrained arm (P = 0.066; Table 2).

After the training, pressure provocation of the untrained arm signi ficantly increased ET-1 and ANG II concentrations from baseline at 90 mmHg, but not at 150 mmHg. Contrarily, no differences in ET-1 and ANG II concentrations were recorded in the pressure-trained arm vessels during the pres- sure provocation (Table 2; Fig. 3).

C-Reactive Protein

There were no differences in CRP concentrations between arms or pressurized versus nonpressurized vasculature ei- ther pre- or posttraining (Table 2).

Matrix Metalloproteinase 7

MMP-7 concentrations were similar at baseline across arms pre- and posttraining ( P > 0.05). Pretraining elevations in DP signi ficantly reduced MMP-7 concentrations at 150 mmHg in the pressure-provoked arm vessels (P = 0.011), whereas a tendency toward signi ficance was observed in the contralateral arm (P = 0.081; Table 2). Following training, MMP-7 was markedly increased from baseline only during the pressure provocation of the pressure-trained arm vessels.

In addition, no differences were recorded in the untrained arm vessels during pressure provocation or control (Table 2;

Fig. 3).

DISCUSSION

The results demonstrated that before the 5-wk vascular pressure-training regimen, arterial diameter and flow remained virtually unaltered in response to discrete and mod- erate DP elevations but increased markedly once DP exceeded

175 mmHg. The training substantially increased the DP thresholds for diameter and flow increments and reduced the gains of the DP-diameter and DP- flow responses.

Furthermore, before the pressure training, DP elevations increased the concentrations of ET-1 and ANG II in the pres- surized vasculature but not in the unpressurized vessels of the control arm. Pressure training increased the basal con- centrations of ET-1 and attenuated the local plasma levels of both ET-1 and ANG II in response to DP elevations in the pres- sure-trained vasculature, whereas DP-induced releases of ET- 1 and ANG II remained unaltered in the vasculature of the untrained arm. By contrast, before training, DP elevations reduced the MMP-7 concentration in both the pressurized and unpressurized vasculature, whereas after training, the DP elevation increased the MMP-7 concentration, but only in the vasculature of the pressure-trained arm.

Table 1. Distending pressure-diameter and DP- flow rela- tionships in the brachial artery, in terms of the DP thresh- olds for the diameter and flow increases as well as the gains for the DP-induced increases in these variables

Pretraining Untrained Arm

Posttraining Trained Arm

Posttraining Untrained Arm

Brachial diameter

Threshold, mmHg 175 ± 18 188 ± 18, † ^{177 ± 14}

Gain, D%/mmHg 0.97 ± 0.45 0.59 ± 0.41, †† 0.98 ± 0.42 Brachial flow

Threshold, mmHg 175 ± 7 192 ± 13, † ^{182 ± 12}

Gain, mL/mmHg 3.1 ± 1.5 1.8 ± 1.7, † ^{2.9 ± 1.9}

Values are means ± SD (n = 11) from before and after the 5-wk pe- riod during which the vessels in one arm were pressure trained (trained vessels), whereas those of the contralateral arm were not (untrained vessels). DP, distending pressure. Statistically signi fi- cant difference between before training and after training, in the trained arm. † Difference after training in trained vs. untrained arm.  and † ^: P < 0.05;  and †† ^: P < 0.01.

Figure 2. Relative change in forearm tissue impedance as a function of applied chamber pressure before and after the 5-wk pressure-training regimen, in the untrained and pressure-trained arm. Statistical signi fi- cance between before training and after training, in the trained arm (P <

0.001). Values are means (SD), n = 11.

In Vivo Vascular Sti ffness

The intermittent moderate elevations of intravascular pressures during the training period blunted the pressure-di- ameter and pressure- flow responses in the brachial artery by 40% and 33%, respectively. This is in good agreement with the results of a previous study (10) and implies that the stiff- ness of peripheral arteries and arterioles are plastic qualities that readily adapt to changes in the prevailing local trans- mural pressures. Also, the finding that the pressure training reduced the pressure-induced change in forearm impedance supports the notion that the training regimen increased arte- riolar pressure resistance. Thus, such change in forearm im- pedance in response to a stepwise increase in intravascular pressure is attributable to a combination of venous filling/

distension, predominating at discrete DP elevations, and tis- sue edema formation, predominating at high DPs, whence the precapillary resistance vessels are forced open and the capillary filtration pressure is raised ( 9, 11).

Considering that the cumulated time of pressure incre- ments in the arm vessels comprised but a mere fraction (  1%) of the 5-wk training period plus the 4 –6 days elapsing from pressure training to pressure-distension testing, it appears that the precapillary wall stiffness is governed by the local pressure peaks rather than the average arterial pres- sure. Our notion that, in humans, periodic elevations of local transmural pressure act as initiators in the development of stiff precapillary vessels, is in agreement with numerous lon- gitudinal studies in experimental animals (for review, see 2 – 4). The present pressure-induced increase in precapillary wall stiffness is also in keeping with findings in cross-sec- tional studies on humans suffering from chronic hyperten- sion. Collectively, these studies suggest that the elevated arterial pressure leads to structural changes in small arteries and resistance vessels (6 – 8) that eventually will increase the blood- flow resistance ( 3, 7). Even though the magnitude and duration of the pressure stimuli needed to evoke such struc- tural changes in human blood vessels are largely unknown, it must be assumed that the structural changes are preceded

by pressure-induced increments in the myogenic tone of the small arteries and arterioles (for review, see 2).

Thus, in vivo arterial/arteriolar stiffness is determined by both myogenic tone and passive elastic recoil of the vessel wall. The shapes of the present DP-diameter and DP- flow curves are very similar to those observed in arteries with pre- served myogenic tone from experimental animals (26 – 29). It has been concluded that the horizontal portion of the curve at low and moderate DPs re flects the pressure range during which arterial and arteriolar lumen diameters are preserved by adequate autoregulation of the myogenic tone, whereas, at high DPs, vasoconstriction is no longer suf ficient to pre- vent arterial/arteriolar distension and hence the gain of the pressure distension in this DP range is predominantly dic- tated by the passive elastic recoil properties of the vessel (cf 2, 26 – 29). Since the present pressure training not only increased the DP thresholds for arterial and arteriolar pressure distension but also reduced the gains of these responses, it can be assumed that the pressure training increased the myogenic tone of the arteries/arterioles and also increased their elastic recoil properties, presumably by inducing structural changes in the vascular walls. Even though the time course for the development of pressure load-induced structural changes is unknown for humans, it has, based on extrapolation from findings in rodents, been suggested that such changes would be apparent in human vessels after several weeks, and fully developed after a few months of pressure loading (30). Notably, assessments of in vivo distensibility of human conduit artery stiffness are com- monly performed using pulse-pressure wave analyses (e.g., 31, 32). Such techniques are, however, not capable of reveal- ing the full pressure-distension curves of peripheral arteries and arterioles during marked static increases of local trans- mural pressure, which was the purpose of the present study.

Regardless of whether, or to what extent, the present pres- sure training-induced increments in arterial and arteriolar stiffness re flected structural remodeling, it is clear that the stiffness changes were governed by local mechanisms, since there were no pressure-distension changes in the Table 2. Plasma concentrations of ET-1, ANG II, and MMP-7 and relative change in CRP during pressure provocations in the pressurized and nonpressurized vasculature, before and after the 5-wk pressure training regimen in the pres- sure-trained and untrained arm

Pretraining

ET-1, pg/mL ANG II, pg/mL CRP,D% change MMP-7, ng/mL

Pressure Nonpressurized Pressure Nonpressurized Pressure Nonpressurized Pressure Nonpressurized

Timepoints

Baseline 1.29 ± 0.2 1.30 ± 0.3 478 ± 353 491 ± 321 — — 2.13 ± 1.1 2.18 ± 1.3

90 1.75 ± 0.3* 1.47 ± 0.3 541 ± 371* 520 ± 351 14.01 ± 39.5 13.5 ± 18.3 2.16 ± 1.4 2.13 ± 1.5 150 1.68 ± 0.4*

** 1.35 ± 0.3 555 ± 350* 516 ± 377 10.45 ± 36.2 0.13 ± 15.3 1.85 ± 1.0* 1.79 ± 0.9

Posttraining untrained arm

Baseline 1.62 ± 0.5 † ^{1.73 ± 0.4 546 ± 346 541 ± 390 — — 2.06 ± 1.2 1.91 ± 1.1}

90 2.02 ± 0.4*

** 1.65 ± 0.4 693 ± 428* 509 ± 295 11.4 ± 28.3 0.10 ± 19.2 2.23 ± 1.4 1.84 ± 1.2 150 1.89 ± 0.4 1.70 ± 0.5 595 ± 320 531 ± 312 26.0 ± 48.3 4.84 ± 26.1 2.25 ± 1.4 2.01 ± 1.3 Posttraining trained arm

Baseline 1.68 ± 0.2 † ^{1.64 ± 0.3 543 ± 382 568 ± 413 — — 1.79 ± 1.0 2.01 ± 1.1}

90 1.92 ± 0.8 1.66 ± 0.3 575 ± 405 570 ± 405 4.91 ± 30.8 1.29 ± 19.2 2.18 ± 1.4* 1.99 ± 1.1 150 1.80 ± 0.8 1.71 ± 0.3 601 ± 407 552 ± 382 7.44 ± 26.9 21.03 ± 44.6 2.44 ± 1.8* 2.11 ± 1.2 Values are means ± SD for applied pressure at 0 (baseline), 90, and 150 mmHg; n = 10. ANG II, angiotensin-II; CRP, C-reactive protein;

ET-1, endothelin-1; MMP-7, matrix metalloprotein 7. Statistical significance (P < 0.05) from baseline. Statistical significance between

arms. † Statistically signi ficant baseline differences pre- vs. posttraining.

vasculature of the untrained arm. The question arises then as to what mechanism might be involved in the development of increased precapillary wall stiffness.

Acute Pressure E ffects on Vasoactive Substances Present results support the notion that, in healthy humans, acute, marked intravascular pressure elevations stimulate

release of the vasoconstrictive peptides ET-1 and ANG II (14).

The congruent increases of their plasma levels recorded dur- ing pressure provocation likely served a common function in limiting the pressure distention of arteries/arterioles (cf 15, 16). Since these plasma increases were only observed in the pressure-provoked vessels, our findings attest to local intra- vascular release, and as for ANG II, local conversion of angio- tensin I to ANG II (33 – 35). Notably, both with respect to ANG II (34, 35) and ET-1 (36), it is likely that the leakage to the bloodstream re flected but a fraction of the pressure-induced release, with the main share of released substances being directed toward the smooth muscle layer of the wall.

Therefore, and because of the dilution effect of the pressure- induced surge in local blood flow, the present pressure- induced plasma increases of ANG II and ET-1 markedly underestimated the true responses. Several mechanisms evoked by the present pressure provocation have been pro- posed to stimulate intravascular production and release of ANG II and ET-1, including endothelial shear and vessel wall stretch (37 – 39). In addition, there is evidence suggesting that ANG II is a potent stimulator for ET-1 production (40).

Present analyses do, however, not permit us to discern whether such interaction contributed to the ET-1 response.

Before pressure training, the intravascular pressure provo- cation resulted in a systemic reduction ( 13% at 150 mmHg) in plasma MMP-7 concentration (Fig. 3; Table 2). Conceivably, this might suggest a pressure-induced uptake of MMP-7, although its underlying mechanisms and functional conse- quences remains to be investigated; we have found no accounts in the literature of such pressure-induced MMP-7 uptake. Regardless, increasing evidence implicate MMP-7 in the regulation of vascular tone, tissue remodeling, and the ini- tiation of fibrotic and hypertrophic cascades associated with hypertension (17, 20 – 22, 41 – 44). In spontaneous hypertensive rats, simultaneous knockdown of MMP-7 and tumor necrosis factor- a-converting enzyme attenuated ANG II-induced hypertension as well as the development of cardiac hypertro- phy and fibrosis ( 17, 20 – 22). However, somewhat counterin- tuitively, under sustained agonist-stimulation (i.e., ANG II), MMP-7 mRNA levels were reported to be reduced (21).

Long-Term Pressure E ffects on Vasoactive Substances There appears to be no previous information in the litera- ture concerning longitudinal effects on vasoactive substances in healthy humans, of repetitive, experimentally controlled, local intravascular pressure elevations. Notwithstanding, our finding that the 5-wk vascular pressure training induced a sys- temic increase in the basal ET-1 plasma level, is not com- pletely surprising, since several studies have noted higher plasma ET-1 concentrations in hypertensive than normoten- sive individuals (45, 46). Such overexpression of ET-1 has been linked to the pathogenesis of hypertension and the ET-1 role in provoking low-grade vascular in flammation ( 47) and oxidative stress in the vascular wall (48, 49). Considering the short half time of ET-1 in plasma ( < 5 min), it seems likely that our finding of an increased basal ET-1 level reflected long-term in flammatory and/or remodeling processes in the repeatedly provoked vasculature rather than remains from a pressure release (as discussed above), induced by the last training session 4 days before the blood sampling. If, or Figure 3. Relative change in endothelin-1, angiotensin-II, and matrix metallo-

proteinase 7 (MMP-7) as functions of applied chamber pressure in trained and untrained vessels before and after the 5-wk pressure training regimen.

Statistical significance from baseline and § between before training and af-

ter training, in the trained arm (P < 0.05). Values are means (SD), n = 10.

to what extent, such increased basal ET-1 level has any sys- temic effects remains unclear. It appears, however, that it did not affect the mechanical properties of the blood ves- sels, since after the training regimen, both the DP-diame- ter and DP- flow responses of the vasculature in the untrained arm were comparable with those recorded in the nontrained arm before training (Fig. 1). Notably, ANG II appeared to exhibit a similar pattern, with a tendency, albeit not statistically signi ficant (post hoc, pre- vs. post- trained, P = 0.07), for increased basal levels in both arms following pressure training.

Pressure training did not potentiate the pressure release of ET-1 and ANG II. If anything, it appeared that the iterative pressure exposures rather induced a habituation response, with no signi ficant increases in plasma levels of ET-1 and ANG II in response to pressure provocation of the pressure- trained vasculature. However, since pressure training also reduced arterial/arteriolar pressure distension, the release of these substances as functions of wall stretch may have remained relatively unaffected by the pressure training.

Thus, either other vasoconstrictive substances/mechanisms, e.g., prostanoids and/or uridine adenosine tetraphospate (50) must have been responsible for the training-dependent increments in precapillary myogenic tone, or the pressure training sensitized the effector sites of the exposed vascula- ture to ET-1 and/or ANG II. That cyclic stretch of cultivated aortic smooth muscle cells from rats results in downregula- tion of ETA receptors, associated with ET-1-induced vaso- constriction, and in upregulation of ETB receptors, associated with ET-1 vasodilatation as well as with apoptosis and in flammatory processes ( 51), does not favor increased density of ET receptors as a mechanism of the increased vas- cular contractility posttraining. There is, however, evidence to suggest that the increased arteriolar contractility follow- ing long-term exposure to high pressure is brought about by polymerization of actin in vascular smooth muscle cells (52).

Moreover, the decreased gains of the DP-diameter and DP- flow responses ( Table 1) suggest that to some extent the increased arterial/arteriolar stiffness may not merely re flect increased contractility, but also increased elastic recoil of the vessel walls, and hence that the 15 training sessions of increased transmural pressure constituted a suf ficient stim- ulus and time frame to initiate structural remodeling of the walls. Hypertension-associated structural changes in preca- pillary vessels include media hypertrophy and, more com- monly, eutrophic remodeling, characterized by increased wall thickness-to-lumen ratio with reduced lumen area, but unaltered wall thickness (2, 53). In addition, pressure- induced changes in precapillary wall stiffness may comprise rearrangement of the extracellular protein structures, with increased collagen-to-elastin ratio and attachment of fibril- lary components to the smooth muscle cells (4). Following the present pressure-training regimen, we recorded a signi fi- cant pressure-induced increase in plasma MMP-7 concentra- tions in the pressure-trained vasculature. Among the MMPs, MMP-7 possesses the highest degradative activity in the extracellular matrix against a variety of collagen (i.e., type IV and X collagen, gelatin etc.) and noncollagen (i.e., elastin, enactin, and fibronectin etc.) substrates ( 54), with studies denoting MMP-7 as a key player in modulating eutrophic and hypertrophic remodeling of arteries/arterioles (17, 20 –

22, 55). It thus appears reasonable to assume that the increased activity of MMP-7 may have contributed to the increased arterial/arteriolar stiffness in the pressure-trained vasculature. Notably, the local CRP level was unaffected by PT, which may suggest that the PT-induced vascular remod- eling was not associated with any severe in flammatory processes.

In conclusion, repeated pressure provocations markedly increase stiffness in precapillary vessels, an adaptation that is governed by local mechanisms and appears to include increases in both myogenic tone and elastic recoil of the vessel walls, the latter suggesting a degree of struc- tural remodeling. The results suggest that local, acute release of ET-1 and ANG II serves to limit vascular pressure distension, and that both ET-1 and MMP-7 appear to be involved in the training-induced remodeling of precapil- lary wall structure. The findings are compatible with the notion that in humans, the local intravascular pressure load acts as a prime mover in the development of primary hypertension. Thus, that increased intra-arteriolar pres- sure leads to increased flow resistance reveals a positive feedback loop that, under physiological conditions, pre- sumably constitutes an important adaptive mechanism to buffer regional pressure perturbations. For instance, dur- ing hydrostatic provocations, it may serve to reduce the pressure load in dependent vascular beds, and to preserve central arterial pressure. Under pathological conditions, during which the pressure elevations are systemic rather than local, it may by contrast, constitute a prominent fea- ture in the pathogenesis of primary hypertension. That iterative arterial pressure elevations induced by physical exercise do not appear to increase precapillary stiffness but rather reduce the risk of developing hypertension (for review, see 56) warrants further investigation. Thus, the interplay during exercise remains unknown, between on the one hand, pressure-induced local vascular release of vasoconstrictors capable of stimulating smooth muscle growth, and on the other hand, local and systemic long- term vasodilatory mechanisms.

ACKNOWLEDGMENTS

We are grateful to all test subjects for their participation.

GRANTS

This study was supported by Swedish Armed Forces Grant 922: 0905.

DISCLOSURES

No con flicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

O.E., H.S., P.S., M.E.K., and R.K. conceived and designed research; O.E., H.S., P.S., M.E.K., and R.K. performed experiments;

O.E., A.E., and R.K. analyzed data; O.E., A.E., H.S., and R.K. inter-

preted results of experiments; O.E., A.E., and R.K. prepared fig-

ures; O.E., A.E., and H.S. drafted manuscript; O.E., A.E., H.S., P.S.,

M.E.K., and R.K. edited and revised manuscript; O.E., A.E., H.S.,

P.S., M.E.K., and R.K. approved final version of manuscript.

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