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J Appl Physiol 100: 1842-1850, 2006; doi:10.1152/japplphysiol.00618.2005
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Chronic hypoxia augments uterine artery distensibility and alters the circumferential wall stress-strain relationship during pregnancy

Stephanie N. Mateev,1,2 Rhonda Mouser,1 David A. Young,3 Robert P. Mecham,4 and Lorna G. Moore1,5

1Center for Women's Health Research and Cardiovascular Pulmonary Research Laboratory, 3Department of Preventive Medicine and Biometrics, and 5Department of Health and Behavioral Sciences, University of Colorado at Denver and Health Sciences Center, Denver, Colorado; 2Department of Pediatrics, University of California, Davis Medical Center, Sacramento, California; and 4Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri

Submitted 24 May 2005 ; accepted in final form 12 January 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Pregnancy-associated increases in uterine artery (UA) blood flow are due, in part, to vasoactive and growth-related changes that enlarge UA diameter. Although active and passive mechanical factors can contribute to this enlargement, their role is less well understood. We hypothesized that pregnancy increased UA distensibility and/or decreased myogenic tone. Given the fetal growth restriction and lower UA flow seen under chronic hypoxia, we further hypothesized that chronic hypoxia opposed these normal active and passive mechanical changes. UA were isolated from 12 nonpregnant and 12 pregnant (0.7 gestation) guinea pigs housed under normoxia or chronic hypoxia (3,960 m) and studied by pressure myography. Pregnancy increased UA diameter similarly under normoxia and hypoxia. Although chronic hypoxia raised resting tone in UA from nonpregnant guinea pigs to ~20% and tone was greater in preconstricted pregnant chronically hypoxic vs. normoxic UA (both P < 0.01), there was an absence of myogenic response (i.e., an increase in tone with rising pressure) in all groups. Pregnancy increased UA distensibility 1.5-fold but did not change stiffness or the stress-strain relationship. Compared with vessels from normoxic pregnant animals, hypoxic pregnancy raised UA distensibility fourfold, decreased stiffness (rate constant b = 3.80 ± 1.06 vs. 8.92 ± 1.25, respectively, P < 0.01), lowered elastin by 50%, and shifted the stress-strain relationship upward such that four times as much strain was present at a given stress. We concluded that increased distensibility and low myogenic tone contribute to enlarging UA diameter and raising UA blood flow during pregnancy. Chronic hypoxia exaggerates the rise in distensibility and alters the stress-strain relationship in ways that may provoke vascular injury.

myogenic tone; preeclampsia; elastin; collagen; intrauterine growth restriction

MARKED MATERNAL HEMODYNAMIC changes occur during pregnancy. Systemic blood pressure and vascular resistance fall, enabling a 40% rise in cardiac output. Much of the rise is directed to the uteroplacental circulation where uterine artery (UA) blood flow increases 40- to 60-fold near term (35). Such remarkable flow alterations occur as the result of an especially marked fall in uteroplacental vascular resistance owing, in turn, to the establishment of the placental circulation and the enlargement of the UA, mesometrial, and other uteroplacental arteries (4, 20).

Several mechanisms help to enlarge the UA during pregnancy. Hypertrophic eccentric growth augments luminal diameter because of cellular proliferation in each layer of the vessel wall (5, 20). Dynamically, UA vasodilator response is enhanced and vasoconstrictor response reduced (26, 29, 45, 47, 48). Distensibility and myogenic tone (passive and active mechanical factors, respectively) are two less well-studied but also important factors influencing vessel diameter. Previous studies have shown that UA distensibility increases with pregnancy in sheep (14), but the effects of pregnancy on myogenic tone in uterine vessels are more variable, with both increases in mesometrial vessels and decreases in the UA being reported (13, 33, 44).

We hypothesized that pregnancy decreases UA myogenic tone and increases distensibility, thus serving to enlarge UA diameter and help to maintain the high UA blood flow characteristic of normal pregnancy. Although the UA is not normally thought of as a resistance vessel, it makes a substantial contribution to uteroplacental vascular resistance during pregnancy in species with deeply invasive hemochorial placenta such as higher primates, guinea pigs, and rodents. Unlike species with epitheliochorial placenta in whom terminal uteroplacental channels constitute the major site of vascular resistance, two-thirds of the uteroplacental vascular resistance reside in the myometrial, arcuate, mesometrial (synonomous radial) and UA in hemochorial species (28, 38). Thus we considered that pregnancy-associated changes in UA distensibility and myogenic tone could be important for facilitating the normal increase in UA blood flow seen during pregnancy in guinea pigs, the experimental animal used in this report.

We also hypothesized that the absence of such changes might contribute to the pregnancy complications of fetal growth restriction and preeclampsia. We (and others) have reported that the chronic hypoxia of residence at high altitude increases the frequency of both these complications (18, 34). Supporting the likelihood that chronic hypoxia plays an etiologic role, we have also shown that chronic hypoxia opposes the normal, pregnancy-associated increase in UA growth, flow vasodilation, and near-term UA blood flow (26, 36, 47). However, whether distensibility and/or myogenic tone are also affected by chronic hypoxia is unknown.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals for these experiments were 24 guinea pigs (12 nonpregnant and 12 pregnant), half of whom were kept at the laboratory altitude of 1,600 m (normoxia, inspired PO2 = 125 Torr) and the other half housed at a simulated altitude of 3,960 m in a hypobaric chamber (chronic hypoxia, inspired PO2 = 90 Torr). There were six animals in each of four groups: normoxic nonpregnant, normoxic pregnant, hypoxic nonpregnant, and hypoxic pregnant. Animals were placed in the hypobaric chamber within 3 days of conception or for an equivalent period in the nonpregnant state and studied after 45 days (0.7 gestation, term = 63 days). We chose this time because peak UA DNA synthesis had been achieved (20) and hence structural changes in the UA likely complete. The University of Colorado Health Sciences Center Animal Care and Use committee approved all animal handling and study procedures.

Isolated vessel preparation.   Animals were euthanized with pentobarbital sodium (6.5%, 200 mg/kg) and one or both of the main UA removed. Several ~4-mm-long segments without collaterals were carefully dissected to avoid mechanical damage. These were from the middle segment of the UA, approximately equidistant between the cervical and ovarian ends. The changes in inner (ID) and outer diameter (OD) in response to increasing intraluminal pressure were measured using pressure myography. As previously described (37), both ends of the vessel were cannulated by using a dual pipette system in which an inner pipette is inserted into each end of the vessel and then advanced into an outer holding pipette connected to the microscope stage with the use of four-axis micromanipulators (Spiderwort Design, Colorado Springs, CO). The vessels were visualized by using an inverted binocular light microscope (model CK, Olympus; Tokyo, Japan), and signals were displayed on a monochrome monitor (model BWM 12A, Javelin Electronics). Approximately 2 mm of the vessel were visible under the microscope. The mounted vessel assembly was submerged in a 2-ml perfusion bath maintained at 37°C by a thermal control circuit (model 16060, Love Controls; Wheeling, IL). The vessels were bathed externally with a modified physiological salt solution containing (in mM) 145 NaCl, 5.0 glucose, 4.7 KCl, 2.75 NaOH, 2.0 MOPS, 2.0 CaCl2, 2.0 pyruvate, 1.17 MgSO4, 1.12 NaH2PO4, and 0.02 EDTA and was maintained at pH 7.40 ± 0.04 as previously described (9). The vessel lumen was perfused with the same solution with the addition of 1% bovine albumin (A-2934, Sigma Chemical, St. Louis, MO) and 5% dextran 40 (S-9631, Braun; Irvine, CA). The system was equipped with upstream and downstream pressure monitors and a servo-controlled pump (Living Systems Instrumentation; Burlington, VT). All measurements were made under conditions of no flow through the vessel lumen.

Experimental protocol.   The mounted vessels were pressurized to 20 mmHg, which we had previously determined to be the optimal pressure or that at which the greatest contractile responses to KCl and phenylephrine (PE) occurred. Changes in ID in response to chemicals added to the bath or increasing flow were considered to represent the contractile or dilator responses. After a 30-min equilibration period, the vessels were contracted with 40 mM KCl, rinsed, and allowed to return to resting diameter. Vessel dimensions were then measured at a constant (20 mmHg) intraluminal pressure before any further manipulation of the vessel under what we termed "resting conditions." Then test doses of PE (10–6 M) and ACh (3 x 10–5 M) were administered, followed by a complete contractile dose response to PE (10–8 to 10–5 M). Vessels were considered viable if they contracted at least 50% to PE across the full dose response range and dilated at least 50% in response to ACh. The nonlinear regression curve was fit to the percent maximum contraction values for each vessel at each PE dose. The resultant curve used to determine the negative log of the half-maximal contractile response as a measure of contractile sensitivity.

To evaluate myogenic tone, vessel ID and OD were measured after step increases in intraluminal pressures from 5 to 70 mmHg, with an initial increase of 5 mmHg (to 10 mmHg), then in 10-mmHg increments thereafter. This range was chosen to encompass sub- and supraphysiological pressures in the guinea pig [systemic mean arterial pressure = 55–60 mmHg and 30–40 mmHg in the UA (16, 28)]. In a preliminary study, intraluminal pressure was increased to 120 mmHg and no change in vessel diameter was observed above 70 mmHg. Vessel diameters stabilized quickly with pressure increases. Measurements were performed 5 min after each pressure step. Vessel ID and OD were recorded in the absence of PE preconstriction under resting conditions and then repeated in UA that had been preconstricted to 50% of the previously observed maximum. The dosage of PE required to produce 50% maximal constriction did not differ between the pregnant and nonpregnant groups [1.76 x 10–6 vs. 1.59 x 10–6 M, P = not significant (NS)] or between any groups (Table 1). To evaluate the effect of nitric oxide (NO) on myogenic tone, these measurements were repeated after addition of the NO synthase (NOS) inhibitor NG-nitro-L-arginine (L-NNA, 200 µM) to the vessel bath.


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  		Table 1. Body weight and UA dimensions in normoxic or chronically hypoxic, nonpregnant and pregnant groups

 
To evaluate distensibility, a final set of ID and OD determinations was made after treatment with the smooth muscle relaxant papaverine (100 µM). In preliminary studies, we and others (12, 25) found that administration of papaverine had similar effects as those produced by removal of calcium from the perfusate and vessel bath.

Collagen and elastin contents in fresh UA segments were determined by measuring hydroxyproline and desmosine. Fresh vessel segments (2–3 mm) were hydrolyzed in 6 N HCl in sealed tubes at 110°C for 24 h. The amino acid composition of the dried hydrolysates was then determined with a Beckman model 6300 analyzer, first for total protein and desmosine by using a modified gradient for resolving cross-linking amino acids (3), and subsequently with a second program for hydroxyproline analysis. Results for the hydroxyproline and desmosine analyses were normalized to the amount of total protein present to allow direct comparisons between vessels.

Calculations.   ID and OD were recorded at each pressure. ID measured in the presence of papaverine was used to assess distensibility, defined as the mechanical stretch a vessel underwent in response to increasing intraluminal pressure. Values are reported as percent change from the initial diameter at 5 mmHg. To further describe the vessel's passive mechanical properties, wall strain or the response of an artery to an increase in intraluminal pressure was calculated as (ID2 – ID1)/ID1 where ID1 is the initial ID measured at 5 mmHg and ID2 is the ID at each new pressure. Circumferential stress or the force exerted on the vessel wall per unit tissue was calculated as (P x ID)/(2 x WT), where P is the transmural pressure (in mN/mm2; 1 mmHg = 0.133 mN/mm2), ID is the inner diameter (in µm) at that pressure, and WT is the wall thickness measured as (OD – ID)/2 (in µm). The rate constant b was determined as an index of vessel stiffness for each stress-strain relationship by using the equation y = aebx, where y is stress, a is the stress at the initial diameter of 5 mmHg, and x is the strain. Lower rate constant values are indicative of a less stiff vessel.

Pressure-induced tone, or the ability of the vessel to constrict in response to increasing intraluminal pressure, was calculated as [(IDPAP – ID)/IDPAP] x 100, where IDPAP is the intraluminal diameter with papaverine and ID is the intraluminal diameter at the same pressure in the untreated or PE-preconstricted vessel.

Statistics.   Data are expressed as means ± SE. Comparisons of body weight, PE contractile response, vessel dimensions, and vessel composition were performed using one-way ANOVA with Student-Newman-Keuls multiple comparisons. The effects of pregnancy and/or chronic hypoxia on UA myogenic tone and distensibility were assessed by the SAS procedures MIXED and IML (SAS, Chapel Hill, NC), which employ a linear mixed-effects modeling approach to account for variability between animals and variability between multiple measurements on the same animal (22, 24). The process consisted of fitting polynomials of increasing order to the data for all groups and then selecting and validating the best-fitting lines with likelihood tests. Comparisons of differences between groups were made by using Scheffé-type simultaneous contrasts that controlled for multiple comparisons (50). Comparisons were considered significant when P < 0.05 and reported as trends when 0.05 > P < 0.10. Two-tailed probability values were employed unless the direction of the comparison was specified in advance, in which case one-tailed values were used.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Resting vessel dimensions and composition.   Table 1 shows the body weights and characteristics of the arteries for each group of animals used in the experiment. As expected, total body weight was greater in pregnant than nonpregnant animals.

In normoxic animals, pregnancy increased UA ID by 50% and OD by 38%. Chronic hypoxia alone decreased UA diameter by 36%. Pregnancy increased ID and OD to an even greater extent than seen under normoxia (148 and 90%, respectively) such that diameters were similar in vessels from the pregnant hypoxic and normoxic groups (Table 1). Wall thickness (WT) was similar in all four groups, although there was a trend toward greater WT in the nonpregnant hypoxic vessels. Comparisons of vessel diameters among groups were similar whether or not they were controlled for maternal body weight.

Pregnancy tended to lower collagen content in both the normoxic and hypoxic groups. Elastin content decreased with pregnancy, with a greater decrease occurring in the hypoxic than normoxic vessels (51 vs. 38%). The collagen-elastin ratios were similar in all four groups.

Pressure-induced (myogenic) tone.   Resting tone (measured in the absence of PE preconstriction) was low in UA from normoxic animals and unchanged by pregnancy (Fig. 1A, dashed lines, P = NS). Chronic hypoxia raised resting tone in the UA from nonpregnant animals to ~20% (Fig. 1B, dashed lines, P < 0.01). Pregnancy lowered resting tone in the vessels from chronically hypoxic animals to achieve levels similar to those seen in the pregnant normoxic group. Resting tone changed little with rising pressure, indicating an absence myogenic response.


Figure 1
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  		Fig. 1. Myogenic tone in uterine artery (UA) from normoxic (A) and chronically hypoxic (B) guinea pigs. Resting (not preconstricted) UA from normoxic nonpregnant (open symbols, dashed lines) or pregnant (solid symbols, dashed lines) had essentially no myogenic tone. Chronic hypoxia raised resting tone in the vessels from nonpregnant animals; pregnancy lowered resting tone to similar levels as seen in the vessels from the normoxic pregnant animals. After partial phenylephrine (PE) preconstriction (solid lines), tone increased but did not differ in UA from nonpregnant vs. pregnant animals under normoxic or chronically hypoxic conditions. Vessels did not exhibit myogenic response (i.e., an increase in tone with rising pressure) in any group. NS, not significant.

 
PE preconstriction increased overall tone in each group, but, again, there was little myogenic response; rather, tone fell with increasing intraluminal pressure. Concerning the effect of pregnancy, although tone at the lowest pressure was greater in the normoxic nonpregnant than pregnant vessels (70 vs. 40%), there was no effect on tone across the full range of pressures (Fig. 1A, solid lines). Similarly, there was no effect of pregnancy nor any myogenic response in the UA from hypoxic nonpregnant and pregnant animals (Fig. 1B, solid lines). Chronic hypoxia had no effect on tone in nonpregnant UA (Fig. 2A) but raised tone in the preconstricted vessels from pregnant animals (Fig. 2B). Finally, inhibition of NOS with the administration of L-NNA to the vessel bath did not alter tone in any of the four groups (Fig. 3).


Figure 2
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  		Fig. 2. Myogenic tone in UA from nonpregnant (A) and pregnant (B) guinea pigs that had been housed under conditions of normoxia (open symbols) or chronic hypoxia (solid symbols). In preconstricted UA, chronic hypoxia did not alter tone in vessels from nonpregnant animals but increased tone in pregnant animals.

 

Figure 3
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  		Fig. 3. Nitric oxide (NO) synthase inhibition by the administration of NG-nitro L-arginine (L-NNA) to the vessel bath did not alter tone or affect the myogenic response in the UA from any group. A: normoxic, nonpregnant group. B: normoxic, pregnant group. C: hypoxic, nonpregnant group. D: hypoxic, pregnant group.

 
UA distensibility and stress-strain relationships.   UA from nonpregnant normoxic and hypoxic animals were moderately distensible, with ID increasing 46 and 40%, respectively, in response to rising pressure (Fig. 4). Pregnancy increased distensibility within the UA from normoxic animals, demonstrating a 1.5-fold greater ID enlargement (68 vs. 46%, P < 0.05). Chronic hypoxia potentiated this effect, with ID increasing fourfold (170 vs. 40%, P < 0.0001) in UA from chronically hypoxic pregnant vs. nonpregnant animals. The net effect of the greater increase in distensibility was for ID in the chronically hypoxic UA to be 2.5-fold greater at the highest pressures than in the UA from normoxic pregnant animals.


Figure 4
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  		Fig. 4. Pregnancy increased UA distensibility under both normoxic (A) and chronically hypoxic (B) conditions. The overall ANOVAs are shown for comparisons of the nonpregnant (open symbols) and pregnant (solid symbols) responses, with pairwise differences significant at P < 0.05 being shown by asterisks.

 
Assessing the stress-strain relationships permits evaluating the amount of change in ID (strain) in response to a pressured-induced increase in force per unit tissue (stress) (Fig. 5). In the normoxic groups, pregnancy increased the amount of stress 1.7-fold at the highest intraluminal pressure (119 ± 23 mN/mm2 vs. 71 ± 9 mN/mm2, P < 0.01) but did not change the stress-strain relationship (Fig. 5A). Greater stress occurred as a result of the pregnancy-associated increase in ID and the somewhat, although not statistically significant, greater fall in WT with rising pressure (Fig. 6, leftmost two sets of columns). There was no change in the rate constant b, indicating that UA stiffness was the same in the pregnant and nonpregnant normoxic groups (Fig. 5A, 6.91 ± 1.53 vs. 5.51 ± 0.77, respectively, P = NS).


Figure 5
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  		Fig. 5. Under normoxia (A), pregnancy raised the level of stress present at the highest intraluminal pressure but did not change the UA stress-strain relationship. Chronic hypoxia (B) alone did not alter the stress-strain relationship. Pregnancy combined with chronic hypoxia raised the level of maximal stress and strain, shifting the stress-strain relationship both rightward and upward (all P < 0.01). Overall ANOVAs are shown for comparisons of the nonpregnant (open symbols) and pregnant (solid symbols) responses.

 

Figure 6
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  		Fig. 6. UA wall thickness decreased with increasing pressure in all but the chronically hypoxic nonpregnant groups. Across all pressures, wall thickness in the chronically hypoxic pregnant group was greater than in the other 3 groups (2-way ANOVA, P < 0.01).

 
Chronic hypoxia altered the stress-strain relationship in the UA from pregnant animals, displacing the curve rightward such that strain was fourfold greater at maximal stress (0.39 ± 0.07 vs. 1.64 ± 0.41, P < 0.01) and shifting it rightward with the result that maximal stress was increased 2.1-fold (104 ± 12 mN/mm2 vs. 49 ± 8 mN/mm2, P < 0.01) (Fig. 5B). These alterations in the stress-strain relationship were due to the pregnant vessels' greater distensibility (Fig. 4B) and greater WT at initial pressures (Fig. 6, rightmost columns). Correspondingly, the UA from chronically hypoxic pregnant animals were approximately half as stiff than those from the other groups, as demonstrated by lower values for the constant b (hypoxic pregnant = 3.80 ± 1.06 vs. hypoxic nonpregnant = 8.92 ± 1.25, normoxic pregnant = 6.91 ± 1.53 or normoxic nonpregnant = 5.51 ± 0.77, P < 0.01 for all comparisons).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
There were three major observations in this study. First, as hypothesized, chronic hypoxia increased UA resting tone in vessels from nonpregnant animals and tone in preconstricted vessels from pregnant animals. Second, pregnancy increased UA distensibility, but this increase was greater in the chronically hypoxic UA, not smaller as hypothesized. Third and unexpectedly, chronic hypoxia altered the stress-strain relationship such that there was much greater strain was present for a given level of stress in the UA from pregnant animals.

Consistent with previous studies, our results showed that pregnancy increased UA diameter. This occurred as the result of hyperplasia in each layer of the vessel wall, resulting in eccentric growth and proportional increases in ID and OD without a change in WT. There may be some species variability in the time course of these growth-related changes. In the guinea pig, the species chosen for the present study, the rise in DNA synthesis is maximal at midgestation whereas it occurs closer to term in the rat (5, 20). We studied vessels from 0.7 gestation because our prior work showed that the rise in DNA synthesis was complete by this time and hence we reasoned that structural changes in the vessel would have also occurred (20).

Because we previously observed that guinea pigs exposed to chronic hypoxia had approximately half the pregnancy-associated rise in DNA synthesis (36), we expected UA diameters to be smaller than in their normoxic counterparts. This was the case in the nonpregnant animals, whose UA diameter was about one-third smaller than in the normoxic nonpregnant animals. However, the chronically hypoxic and normoxic pregnant animals had similar UA diameters. It is important to recall that vessel diameter is not a fixed entity but depends on intraluminal pressure as well as other measurement conditions. Although we measured all vessels in the same way and at the same intraluminal pressure, the pressure used had been previously determined as optimal for assessing contractile response (20 mmHg), but this was below physiological values (30–40 mmHg). Thus it is unknown whether UA diameters differed in the normoxic vs. chronically hypoxic animals at physiological pressures or in vivo. If the UA diameters are not different, it is possible that increased cell size (hypertrophy) in the pregnant animals may have compensated for their reduced hyperplasia. Future studies are required for measuring cell size and cell numbers in vessels from chronically hypoxic as well as normoxic animals.

Previous studies have shown variable effects of pregnancy on myogenic tone. Pregnancy reduced tone in the rat renal artery but not in human subcutaneous or mesenteric vessels (7, 12, 23, 27, 42). Posterior cerebral arteries had a biphasic response, with pregnant vessels having greater tone in the physiological range but less tone than nonpregnant controls at high pressures (6). In the mouse uteroplacental circulation, pregnancy lowered UA tone (44) but increased tone in the next downstream vessel, the mesometrial or radial artery (13). Interestingly, these changes in tone were site-specific such that pregnancy increased tone in the proximal portion but reduced it in the more distal portion of the mesometrial arteries supplying placental (as opposed to myometrial) tissue (13). In our study, the low levels of resting tone seen in nonpreconstricted vessels from normoxic nonpregnant animals made it unlikely that pregnancy could reduce tone further. In the chronically hypoxic nonpregnant animals, resting tone was increased relative to their normoxic counterparts and, although still low (~20%), it was within the range seen in coronary arteries (46). But even in this setting of elevated UA tone, pregnancy did not raise tone in the UA from chronically hypoxic animals but rather reduced it to levels that were equally as low as those seen in their normoxic pregnant counterparts. Perhaps because pregnancy is known to prompt dedifferentiation of UA smooth muscle cells from a contractile to a synthetic phenotype (41), differences in cellular differentiation may also have contributed to the observed lack of myogenic response.

Because vessels in vivo are exposed to circulating as well as locally released vasoconstrictors, we preconstricted UA to 50% of their maximal values and repeated the tone studies. Although the preconstricted vessels had greater pressure-induced tone than did the resting vessels, pregnancy still did not have any effect on tone in the either the normoxic or chronically hypoxic animals. However, the level of tone present in the preconstricted vessels was slightly (and significantly) greater in the preconstricted vessels from chronically hypoxic compared with normoxic pregnant animals. An unexpected finding was the pattern of diminishing tone with increasing pressure seen in the resting as well as the preconstricted vessels. Because myogenic response is defined as constriction in response to rising pressure, it appears that although the UA had some, albeit low levels of tone, they did not have a myogenic response. Species and/or gestational age variability is again evident insofar as a myogenic response has been described in the main UA of the near-term pregnant mouse and the next downstream vessel, the mesometrial artery, of the rat (13, 44). A 50% diet restriction has recently been shown to increase tone in the rat mesometrial arteries from pregnant animals (43). In this study, the mesometrial arteries from the nonpregnant rats had little myogenic response, similar to our observations in nonpregnant as well as pregnant animals. However, we are unaware of any studies in which myogenic response actually fell with increasing pressure. Perhaps such a pattern of falling tone helps maintain blood flow at the relatively low pressures present in the uteroplacental circulation in the guinea pig, a species with an especially invasive, hemochorial placenta.

We expected NOS inhibition to increase myogenic tone on the basis of the previous studies in mesometrial vessels (13, 43). Thus it appeared that altered NO synthesis or metabolism was not the cause of the greater tone in the preconstricted UA from chronically hypoxic pregnant vs. nonpregnant animals, nor the explanation for lack of an effect of pregnancy in the normoxic animals. Previous reports, however, also show that the effects of NOS inhibition on tone in uteroplacental vessels is variable. NOS inhibition has no effect on tone in distal mesometrial arteries or vessels from diet-restricted or nonpregnant animals as well as in other vessels (13, 21, 30, 43). Such variability may relate to the time during pregnancy when studies were performed. Our studies were performed at the beginning of the third trimester, earlier than the previous reports, which were all near term.

The effects of pregnancy on distensibility have previously been shown to vary among vascular beds. Posterior cerebral arteries show no change in distensibility (6) whereas a reduction occurs in mesenteric vessels (25). Our observation of increased UA distensibility agrees with earlier studies in sheep UA and in rat mesometrial arteries (14). Preeclampsia does not appear to alter distensibility in myometrial, omental, or subcutaneous arteries relative to values seen in normal pregnant women (31, 42). However, vessels from nonpregnant women were not studied and hence the effects of pregnancy remain unclear. To the best of our knowledge, the effect of chronic hypoxia on exaggerating the pregnancy-associated rise in UA distensibility has not previously been described.

Changes in distensibility may or may not be accompanied by alterations in stiffness (the rate constant b) or the stress-strain relationship, because these are a function of the relative alterations in diameter and WT as well. Thus, despite greater distensibility in the normoxic pregnant compared with nonpregnant UA, there were no changes in stiffness or the stress-strain relationship. This was due to the proportionate nature of changes in UA diameter and WT in the normoxic groups. Nevertheless, even though the stress-strain relationship was unchanged, the absolute level of stress was nearly twofold greater at the highest intraluminal pressure in the pregnant compared with the nonpregnant UA.

In chronically hypoxic animals, the pregnant UA were only half as stiff as the nonpregnant vessels. In addition, the stress-strain relationship was shifted upward and rightward such that at the highest intraluminal pressures there was twice as much stress on the vessel wall and four times as much strain. Chronic hypoxia alone had no effect on vessel stiffness or the stress-strain relationship. However, chronic hypoxia modified the effects of pregnancy such that there was a greater distensibility and greater circumferential wall strain. In addition to the differences in stiffness, there were differences in WT behavior. At low pressures, WT was greater in the chronically hypoxic pregnant group that, in turn, narrowed UA diameter and enabled a greater decline in WT with increasing pressure (Fig. 6). These results are somewhat similar to those recently reported in myometrial arteries from pregnancies complicated by preeclampsia or intrauterine growth restriction. Although the differences in stress-strain relationship were not significant, preeclamptic and intrauterine growth restriction curves were right-shifted at high intraluminal pressures and WT was greater than in normal pregnancies (32).

It is unknown whether greater circumferential wall strain was present in vivo in the chronically hypoxic animals, but, if it was, an intriguing possibility is that increased strain could induce oxidative stress in endothelial cells as the result of mechanical deformation and elaboration of reactive oxygen species and lipid peroxidation products (17). Thus we speculate that the greater strain present for a given level of stress in the UA from pregnant animals may cause oxidative stress and endothelial injury and, in turn, contribute to the impaired UA flow vasodilation and lower UA blood flow reported previously in chronically hypoxic vs. normoxic pregnancy (26, 51).

Several sources of evidence suggest that the pregnancy-associated alterations in distensibility likely involve changes in collagen and/or elastin content. Increased elastin levels have been reported in UA from near-term pigs (15). In rat cerebral or mesenteric arteries, changes in distensibility paralleled those occurring in collagen or elastin content (2, 8, 25). Our study did not identify any changes in collagen or elastin at midgestation or by chronic hypoxia alone, but the combination of pregnancy and chronic hypoxia lowered UA elastin and tended to reduce collagen as well. The lack of clear alterations in collagen levels may have been due to the differential effects of pregnancy-associated changes on endothelial and vascular smooth muscle cell types, decreasing collagen synthesis in the former but increasing it in the latter (39, 40). Although it seems counterintuitive that lower elastin levels would increase distensibility, lower elastin levels in combination with increased distensibility have been seen in vessels from heterozygous elastin-knockout mice (11). Reduced elastin levels and increased WT have also been reported in umbilical arteries of babies born to preeclamptic women (19). Because hypoxia is known to downregulate elastin expression as the result of decreased mRNA stability (1, 10), our observations together with those of previous studies suggest that a hypoxia-induced reduction in elastin and increase in oxidative stress may be responsible for raising WT, distensibility, and thereby strain at a given level of stress in the UA from chronically hypoxic vs. normoxic pregnant animals.

In summary, these studies indicated that normal (normoxic) pregnancy had no effect on UA myogenic tone but increased UA distensibility in the guinea pig. We used the guinea pig for evaluating these passive and mechanical changes because it has a deeply invasive hemochorial placenta and is thus probably the most suitable model in which to study uteroplacental vascular adjustments to human pregnancy, next to primates. We found that the UA has essentially no tone without preconstriction, and even when preconstricted it has no myogenic response (rise in tone with increasing pressure). Such low myogenic tone and greater distensibility would both act to increase UA diameter and thus may be important passive and active mechanical factors that contribute to the rise in UA blood flow during normal pregnancy. Moreover, the eccentric nature of UA growth during pregnancy and greater distensibility do not alter the relationship between change in diameter (strain) and the force exerted per unit tissue (stress) in the UA.

Chronic hypoxia altered these normal effects of pregnancy by raising tone in preconstricted vessels, but chiefly by exaggerating the increase in distensibility, reducing stiffness, and altering the stress-strain relationship. Together these changes produced up to fourfold greater strain at a given level of stress. We speculate that this increased strain could predispose the vessel to endothelial injury, which could in turn, contribute to the reduction in NO production, absence of flow-induced vasodilation, and UA blood flow seen during chronically hypoxic vs. normoxic pregnancy (26, 47, 49).


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National Heart, Lung, and Blood Institute Grants HL-60131, HL-14985, and HL-07607 supported this study.


   ACKNOWLEDGMENTS
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REFERENCES
 
We thank Wendy MacCannell and Colleen Julian for help with preparation of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. N. Mateev, Univ. of California, Davis Medical Center, 2516 Stockton Blvd., 2nd Floor, Sacramento, CA 95817 (e-mail: stephanie.mateev{at}ucdmc.ucdavis.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
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 RESULTS
 DISCUSSION
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 ACKNOWLEDGMENTS
 REFERENCES
 

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