Time Course of Arterial Wall Changes with DOCA Plus Salt Hypertension in the Rat ROBERT H.

Transcription

1 Time Course of Arterial Wall Changes with Plus Salt Hypertension in the Rat ROBERT H. COX SUMMARY Segments of carotid and tall artery, and thoracic aorta from control and hypertensive animals ( + salt) were used for the study of mechanics and/or chemical composition. Pressure-diameter measurements were made on intact segments under conditions of actire (145 mm-k+) and passive (O-Ca ++ and 2 mm-egta) smooth muscle. Segments were used for chemical analyses of connective tissue content, water spaces, and electrolyte content. The passive stiffness of carotid and tail arteries increased monotonically with time. The carotids showed significant changes after two weeks of hypertension while the tail arteries only after 12 weeks. The collagen and total connective tissue content of the hypertensive arteries was decreased while collagen/elastln was unchanged. Smooth muscle activation produced larger changes in diameter of hypertensive arteries especially at higher values of transmural pressure. Maximum active force development was increased in carotid arteries at each time period from weeks on while it was Increased for the tall arteries only at and weeks. Relative cellular volume of these arteries was monotonically increased with hypertension. Maximum active force normalized to cellular content was not significantly different for carotid arteries from control and rats. For hypertensive tall arteries normalized on this basis force development remained elevated at weeks but was significantly reduced at weeks. Not all of the responses to smooth muscle activation are monotonic with duration of hypertension, nor can all of these changes be explained on the basis of changes in cellular volume. (Hypertension 4: 27-38, 1982) KEY WORDS smooth muscle mechanics connective tissue cell volume extracellular water electrolyte content IT is generally accepted that changes in arterial wall properties occur with the development and maintenance of hypertension. 1 " 7 These include changes in mechanics, composition, and biochemistry, among others. In general, however, there is some lack of unanimity with regard to the exact nature of some of the arterial wall changes in hypertension. For example, recent studies of changes in the passive stiffness of arteries (i.e., in the absence of smooth muscle tone), during the development of hypertension, have indicated in some cases an increase 8 ' * and in others a decrease in stiffness. 7-1 ' " In two previous studies from this laboratory it was found in one that the passive stiffness of the rat carotid From the Bockus Research Institute, The Graduate Hospital, and Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania. Supported by Research Grant HL from the Department of Health, Education, and Welfare. Address for reprints: Dr. Robert H. Cox, Bockiu Research Institute, Graduate Hospital, One Graduate Plaza, Philadelphia, Pennsylvania Received January 8, 1981; revision accepted May 22, artery decreased" and in the other increased* with + salt and renal (Goldblatt) hypertension. The reason for the difference in findings was not clear but could have been due to differences in the duration of hypertension or to the initial age of the animals when hypertension was produced in the two studies. Most previous studies of force development by vascular smooth muscle, particularly in experimental hypertension, have generally indicated a decrease in maximum force development by hypertensive arteries. 1 - ia Recently, studies from this as well as other laboratories have indicated that maximum active force development is increased in arteries from animals with both experimental and genetic forms of hypertension.* 114 ~ 17 At least a portion of this increased force development was due to an increase in wall thickness of the hypertensive arteries.* 114 ~ 17 There are a number of possible explanations for the differences in the results of the various studies quoted above. The most obvious relate to differences in the animals and blood vessels employed as well as in the methodology employed in the various studies. Another very important factor is the duration of hypertension. It may be that the time course of 27

2 28 HYPERTENSION VOL 4, No 1, JANUARY-FEBRUARY 1982 changes in arterial wall properties associated with the production of experimental hypertension are not monotonic in nature. As a result, the duration of hypertension may have a very important impact on the results of experiments presented by various investigators. Accordingly, it was the principal objective of the experiments described in this study, to determine the time course of changes in arterial wall properties associated with experimental hypertension in the young rat. A second objective was to perform studies at two arterial sites (i.e., the carotid and the tail artery) to determine the uniformity of changes associated with hypertension. Finally, detailed chemical studies were performed in order to obtain a measure of relative cell content of the arteries studied for the purpose of normalizing active force development on the basis of relative cell volume of the various arteries studied. Methods Animals These experiments were performed using male Wistar rats obtained at an initial age of 7 days (Charles River Breeding Laboratories). The animals were maintained in standard laboratory cages with two rats per cage. Initially, they were given food and water ad libitum. The animals were maintained for 2 weeks prior to the surgical procedures. During this period of time, body weight, blood pressure, and heart rate were periodically recorded. Systolic blood pressure was determined by the indirect tail cuff method (Programmed Electro-Sphygmomanometer, Narco Biosystems). At an age of 12 weeks the animals were subjected to the experimental surgery. They were anesthetized with pentobarbital (4 mg/kg i.p.). The left kidney was exposed by a retroperitoneal approach, and removed. The incision was closed in layers. A desoxycorticosterone acetate () impregnated silastic strip (Dow Chemical Silastic, 382 Medical Grade Elastomer) was implanted subcutaneously in each animal. Each strip contained approximately 8 mg of. The animals were allowed to recover and were given saline supplemented with potassium (.1%) as drinking fluid. animals were treated in a similar manner except the mobilized kidney was returned to its original position. These animals received tap water to drink. The animals were monitored weekly for body weight, blood pressure and heart rate. and hypertensive animals were removed in pairs from the colony and studied 2, 4, 8 and 12 weeks post surgery. A control animal was studied at the same time as a hypertensive one. At the time of study, the animals were anesthetized with pentobarbital (5 mg/kg i.p.). The heart, the carotid arteries, the tail artery and the thoracic aorta were quickly exposed and removed from the animals. The thoracic aorta from the left subclavian branch to the diaphragm was trimmed of fat and loose connective tissue as were the other arteries. The atria and large vessels were removed from the heart. The trimming procedures were performed with the tissues in an oxygenated physiological salt solution (PSS) maintained at 37 C. One carotid artery and a proximal 2 cm segment of the tail artery were used for the mechanical studies. The other carotid artery, the remaining segment of tail artery, and the entire thoracic aorta were placed in PSS in a metabolic incubator for use in chemical analyses. The ventricles were immediately weighed using an analytical balance. The PSS was aerated with a 95% O, - 5% CO, mixture and had the following composition in millimoles/ liter: NaCl, 22.5 NaHCO,, 1.2 NaH,PO 4, 2.4 Na,SO 4, 4.5 KC1, 1.2 M B SO 4, 2.5 CaCl, and 5.6 dextrose. Mechanics For mechanical studies, arteries from the control and hypertensive animals were mounted in random order in the experimental apparatus as has been previously described in detail.* 1 " l8 The segments were mounted horizontally on hypodermic needles in a temperature controlled bath. One end of the segment was coupled to a force transducer for the measurement of axial wall force. The other end of the vessel was coupled to a manifold on a movable slide assembly. Upon mounting, the length of the segments was stretched to a point equivalent to their in vivo value. This was accomplished by noting the retraction of the arteries upon excision and using this value to determine the amount of in vitro stretch. The external diameter of the vessel was continuously measured using a semiconductor cantilever transducer pivoted from above the segment. 1 ' The segments were inflated with air introduced through the manifold. The inflation pressure was generated using an electropneumatic transducer (Conoflow, Model T25) which was driven from a compressed air supply at 15 psi. A current controlled valve in the transducer was driven by a function generator (Hewlett-Packard, Model 331OB) to obtain the desired transmural pressure waveform within the vessel segment. Following mounting, the segments were allowed to equilibrate for 6 min at zero transmural pressure. The bath was then drained and refilled with a high K + - PSS where K + was substituted for Na + on an equimolar basis. After ten minutes in the solution, the pressure-diameter response to slow continuous inflation at a rate of.1 mm Hg/sec was obtained and recorded on an X-Y plotter (Hewlett-Packard, Model 746). Only the response to inflation was recorded under activated conditions. At the end of the response pressure was returned to zero, the bath was drained, rinsed and refilled with a Ca ++ -free solution containing 2 mm EGTA. Pressure was then continuously cycled between and about 25 mm Hg for 3 minutes. At this time, the effects of activation had been reversed, and the response to one complete infla-

3 ARTERIAL WALL RESPONSE TO HYPERTENSION/Cox 29 tion-deflation cycle was recorded on the plotter. Addition of potassium or norepinephrine to the bath at this point did not produce any effect on the pressurediameter curve indicating the absence of smooth muscle contractile responsiveness, i.e., passive smooth muscle. When data acquisition was complete for a vessel, it was removed from the bath, its unstressed length measured, and its wet weight determined using an analytical balance. The records from the X-Y plotter were used for subsequent data analysis. Pressure-diameter curves under conditions of active and passive smooth muscle were digitized using a Talos digitizer. Only data from the ascending limb of the passive curve were used." Digital data were transferred to a computer for subsequent numerical computations (PDP 11/34). Values of average wall stress (<r) were computed from transmural pressure (P) using the following equation: 18 a =: (1) where a and b are the internal and external radii, respectively. Values of b were obtained directly from values of external diameter while values of a were determined using values of b, stressed length, and segment wet weight. Values of incremental elastic modulus (E lnc ) were obtained from pressure diameter data assuming the arterial wall to be isotropic using the following equation: 1 ' ldc _ 2a»b AP b»-a» Ab where AP/Ab represents the slope of the pressureradius curve at a specific point. The slope was determined using a polynominal regression method based upon a least squares fit of data points above and below the one of interest. Treatment of arterial wall mechanics in this manner is clearly an approximation of its true properties. However, previous studies have shown that values of elastic moduli determined in this manner are reasonable approximations of values of tangential elastic moduli (E e ) computed from an anisotropic analysis. 11 This analysis was applied to data recorded under conditions of active and passive smooth muscle. The mechanical performance of vascular smooth muscle in these preparations was quantitated in two ways as previously described. 1 ' In the first, values of active stress response were computed from the increase in tangential wall stress at a given value of external diameter using pressure-diameter data under active and passive conditions and equation 1. This analysis was performed for a variety of values of diameter and produced data that are equivalent to isometric force development divided by wall crosssectional area." In the second method, values of active diameter response were computed using the difference in values of diameter at a specific transmural pressure for active and passive conditions. This diameter (2) difference was normalized by dividing by the value of passive diameter at each pressure level. These responses are equivalent to isobaric constriction responses for arterial smooth muscle. 18 The validity and applicability of these methods for the study of arterial smooth muscle mechanics have been documented in previous publications. 8 ' " " Values of various mechanical parameters were averaged from the experiments on the two vessel sites for control and hypertensive animals at the various age levels in pressure increments of 5 mm Hg. Mechanical data presented in the figures are expressed in terms of mean ± 1 SE. Statistical comparisons were made using the double ended Student's t test with a p value of less than.5 taken as a measure of a significant difference. Chemical Analysis The arterial segments used for mechanical studies were subsequently used for the determination of connective tissue content as previously described." The collagen and elastin fractions of these arteries were separated by heat and pressure. The hydroxyproline content of the soluble and insoluble fractions was then determined, and used to compute collagen and elastin contents, respectively. The remaining segments were used for the determination of water and electrolyte content. The extracellular water space was measured using * Co chelated to EDTA as an extracellular marker." After 2 hours of incubation in normal PSS at 37 C, the arterial segments were transferred to an identical solution containing the isotopic marker for a period of 2 minutes. They were then removed, lightly blotted to remove adventitial and intimal surface water and placed in polyethylene vials. Tissue weights were determined before and after oven drying at 9 C for 2 hours. The segment vials along with standard solutions were counted in a well-type gamma counter (Nuclear Chicago, Model 185). From the radioactivity taken up by the samples, the * Co-EDTA space was determined and assumed to be representative of the extracellular water space in these tissues. This marker has a distribution space equivalent to that of sucrose." The difference between "Co-space and total water space was assumed to represent cellular water content of the tissues. The total cell volume of the specimens was determined from the sum of the cell water content and the cell solid content. Cell solid content was estimated as the difference between total solid content and total connective tissue content of these specimens. Samples used for determination of water spaces were subsequently used for the analysis of electrolyte content. 94 Samples were ashed in 3% peroxide at 9 C for 2 hours. The ash was then dissolved in.1 N HNO, containing 1 mm LiNO,. Electrolyte content of these samples was determined by atomic absorption spectrophotometry using appropriate standards (Perkin Elmer, Model 33). Electrolyte content was expressed on the basis of tissue dry weight.

4 3 HYPERTENSION VOL 4, No 1, JANUARY-FEBRUARY 1982 Results The time course of changes of systolic pressure and heart rate in control and hypertensive animals is summarized in figure 1. Values of heart rate in the hypertensive animals tended to be somewhat lower than in their control counterparts with the differences at 2 and 12 weeks post surgery being statistically significant. The hypertensive procedure was followed by a rapid increase in systolic pressure which was significantly elevated two weeks post surgery and reached a plateau from 8 to 12 weeks post surgery. Systolic pressures at this plateau region averaged about 13 mm Hg in the control animals and about 2 mm Hg in the animals. The heart and body weight responses to the increase in arterial pressure are shown in figure 2. The surgical procedure produced an interruption of normal weight gain in these animals. The difference was statistically significant at two weeks post surgery but then became normalized with increasing age. There was a trend for a decline in the ratio of heart weight to body weight in control animals with age over the time course studied herein. Hypertension was associated with an increase in this ratio which was significant after four weeks post surgery. At 8 weeks post surgery, heart weight/body weight ratio reached a maximum value then declined over the next four weeks at the same rate as the decline in the control animals. These curves suggest that the first 8 weeks represent a period of development of hypertension whereas the last 4 weeks represent a period of maintenance of hypertension. The remaining data will be discussed in accordance with this definition of time frame. In the case of the carotid artery under passive conditions, changes in pressure-diameter relations oc- E 2 E Ixl OC z> CO CO UJ QL a. o _l o CO CO 15 1 ~ 4 c "E LLJ < 35 cr LJJ X 3 h**' AGE (wks) E Q O CO < UJ -> 5 E ui Q Om r AGE (wks) AGE (wks) AGE (wks) 24 FIGURE 1. Time course of systolic pressure and heart rate changes in control (o) and hypertensive animals (*). Symbols are means and vertical lines ± 1 SEM. Arrow shows time of surgical procedure (+ weeks). FIGURE 2. Time course of changes in heart weight body weight ratio and body weight from the two groups. Symbols are as given in figure I.

5 ARTERIAL WALL RESPONSE TO HYPERTENSION/Cox 31 curred primarily in the low pressure range. Values of external diameter at 1 mm Hg were not significantly altered during the time course of development of hypertension (table 1). Values of lumen radius for the carotid artery from hypertensive animals were significantly smaller than those of their control counterparts after 4 weeks of hypertension. On the other hand, the changes in the tail artery passive pressure-diameter data with the development of hypertension were somewhat different. Values of diameter became progressively smaller for the animals at higher values of pressure with increasing duration of hypertension (table 1) compared to controls. Values of incremental elastic moduli under passive conditions at specific values of transmural pressure were lower in the hypertensive animals compared to their control counterparts in the case of the carotid artery. They were not significantly different in the case of the tail artery (table 1). Values of passive elastic moduli at prevailing values of systolic pressure for both sites were always higher in the compared to the control animals at both sites. As shown in figure 3, there was a progressive shift to the left of passive stress-strain curves for arteries from hypertensive animals relative to that of the controls. The magnitude of this leftward shift increased with duration of hypertension. This leftward shift of passive stress-strain curve indicates an increase in passive stiffness of these blood vessels. In the case of the carotid artery, changes were statistically significant from two weeks post surgery onward. In the case of the tail artery, however, the changes were only statistically significant at 12 weeks post surgery. Table 2 summarizes values of connective tissue content of arteries from control and hypertensive animals at the various ages. There was a monotonic decline in CAROTID ARTERY wks CM c ( O in LJ cr I - id LJ CD -z. < o TAIL ARTERY wks L5r NORMALIZED EXTERNAL DIAMETER (D/D o ) FIGURE 3. Summary of average values of tangential wall stress versus normalized external diameter under passive conditions for arteries from control (o) and animals ( ). Data for carotid arteries are shown in the upper row and for tail arteries in the lower row. Symbols are means and bars ± 1 SEM. Panels show data at various times after the surgical procedure. External diameter was normalized by dividing by the value at zero pressure. The number of arteries used in each group is the same as those given in table 1.

6 32 HYPERTENSION VOL 4, No 1, JANUARY-FEBRUARY 1982 TABLE 1. Summary of Some Mechanical Data at 1 mm Hg N L/Lo Group Carotid artery Tail artery ± ± ± ± ± ± ± ± ± ± ± ± ±.3* 1.64 ±.1* 1.44 ± ± ± ± ± ± ± ± ±.2.97 ±.3.97 ±.3.99 ± ± ±.2 D(mm) 1.24 ± ± ± ±.4.96 ±.3.97 ±.2 1. ±.3.97 ±.3* p <.5. fp<.1. Legend: N = number of animals/group; L/L» = axial extension ratio; L = in vivo stretched vessel length; L» = unstretched vessel length; D = external diameter, R/h = mid-wall radius wall thickness ratio; a = internal radius; Eh* = passive incremental modulus (1* dyn/cm 2 ). collagen content of the hypertensive arteries with the duration of the hypertension. While there were changes in the elastin content of arteries from these animals (decreased), they were not uniform with age or arterial site. The total connective tissue content was significantly reduced in arteries from the hypertensive compared to the control animals at all arterial sites and ages. The decrease in total connective tissue content increased monotonically with the duration of hypertension. No significant changes in the ratio of Q Q j^ LJ CO -z. o Q_ CO UJ Q: QC LJ I - LJ < Q LJ CAROTID ARTERY 2OO 4 4 collagen to elastin content were found in these studies. As shown in figure 4, active midwall diameter responses were significantly larger in the case of hypertensive carotid arteries compared to control ones at pressure levels above 1 mm Hg. The difference between control and hypertensive animals increased with the duration of hypertension. At 8 and 12 weeks post surgery, the differences in active midwall diameter response at pressures below 1 mm Hg were also significantly higher in the case of carotids.3 O O.8r 2 < TRANSMURAL PRESSURE (mmhg) FIGURE 4. Summary of average values of active midwall diameter response to high-k + activation. Panels and symbols are as defined in figure 3.

7 ARTERIAL WALL RESPONSE TO HYPERTENSION/Cox 33 TABLE 1. (Continued) R/h a (mm) E 14.7 ± ± ± ± ± ± ±.9t 9.1 ± ±.6 59 ±.1.57 ±.1.6 ±.1.62 ±.1.61 ±.1 57 ±.2 55 ±.1* 57 ±.1* 53 ±.3* 12.9 ± ± ± ±.7 1. ± ± ±.9f 7.7 ±.5f 85 ± ± ± ± ± ± ± ± ± ±.4f.41 ±.2.41 ±.2.43 ±.2.46 ±.2.46 ±.1.41 ±.1.42 ±.2.44 ±.2.4 ± O.Olf 11.1 ± ± ± ± ± ± ± ± ±1.7 from hypertensive animals. In the case of the tail arteries no significant differences existed at values of transmural pressure below 2 mm Hg. At higher values of transmural pressure, however, tail arteries from the hypertensive animals exhibited a larger capacity for decreasing blood vessel diameter. Figure 5 shows a summary of values of active diameter response computed from changes in lumen diameter under active and passive conditions. For the CAROTID ARTERY- 1. r carotids the difference between control and hypertensive animals was exaggerated compared to midwall diameter responses. Significant differences extend over nearly the entire pressure range from four weeks post surgery to 12 weeks. Only values of active internal diameter response from 1 to 16 mm were statistically significant 2 weeks post surgery, however. The reason for the differences between results in figures 4 and 5 are related to the increase in carotid 2 O \ 2 O 2 4 O 4 TRANSMURAL PRESSURE (mmhg) FIGURE 5. Summary of average values of active internal diameter response to high-k + activation Panels and symbols are as defined in figure 3.

8 34 HYPERTENSION VOL 4, No 1, JANUARY-FEBRUARY 1982 TABLE 2. Group C Thoracic aorta 7 15, 13 16, 13 12, 15 16, 19 Carotid artery 1 13, 1 12, 9 1, 9 12, 12 Tail artery Summary of Connective Tissue Data N Collagen Elastin D 8 11, 9 12, 9 11, 1 11, ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±.8f 18.5 ± ±.6T 27.3 ±.6* 26.9 ± 1.* 272 ± 1.3* 21.2 ±.9f 342 ± ± ±12* 26.3±l.lj 38.1 ± ± ± ± ±.9* 38.1 ± ± l.ot 37.5 ± ±.8t 34.6 ± ± ± ± ± Lit 34.6 ± ±.6* 33.2 ± ± ± ± ± ± ± ± ± ± ±1.4* *p <.5; tp <.1. Connective tissue content is given as percent of dry weight. Collagen + elaetin 57.5 ± ± ± ± ± Lit 58.8 ± ± 1.3t 58.2 ± ± l.oj 61.3 ± ± ± ± ± l.lf 63. ± ± 1.4* 65.3 ± ±.6t 45.9 ± ± ± ± ± ± ± 1.5t 46.2 ± ± Lit Collagen/elastin 51 ±.7 53 ±.9.56 ± ±.7.5 ± ±.7.65 ± ±.8.66 ± ± ±.1.75 ± ±.8.78 ± ± ± ±.5.74 ± ± ± ± ± ± ± ± ± ±.26 TABLE 3. Summary of Arterial Wall Electrolyte Content (mmoles/kg dry weight) Group Ca Mg K+ Na+ Thoracic aorta 28.5 ± ±.7 16 ±4 368 ± ± ± 2.5* 19.7 ± ± ±3 134 ±6t 338 ±22 37 ± ± ± 1.7t 12 9 ± ± 1.7* 92 ±5 118 ±9* 314 ± ± ± ± Lit 15.6 ± ±.6 11 ±7 147±lit 384 ±29 39 ± ± ± ± ±.8 97 ±3 136 ±6t 237 ±21 37 ± 23* 1 artery Carotid Tail artery p <.5. tp < ± ± ± ± ± ± ± ± 2.9* 27.5 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.3* 28.2 ± ± ± 2.t 19.8 ± ± ±3 112 ±2 119 ±6 11 ±2 229 ± ±9 215 ± ± ± ± 7t 13 ±8* 146 ±6t 144 ± 12t 197 ±7 192 ± ± ±8 41 ±57 47 ±35 45 ± ± ± ±2 42 ± ± ± ± 51 4 ±24 45 ± ± ± ± ± ± 25* 429 ±66 ci- 47 ± ±29 399± ± ±2 412 ± ± ± ± ±41 62 ± ±2 598 ± ± ± ± ±5 395 ± ± ± 23* 416 ± ± ±23 53 ±33 484±33t 586 ±2 44 ±93

9 ARTERIAL WALL RESPONSE TO HYPERTENSION/Cox 35 wall thickness that occurred with the development and maintenance of hypertension (table 1). In the case of the tail artery, the conclusions for internal diameter changes are the same as those for midwall diameter changes, that is, significant differences only existed at high values of transmural pressure, i.e., from 25 to 4 mm Hg. The reason for this is due to the fact that tail arteries from control and hypertensive animals under conditions of maximal activation constricted luminal diameters to an essentially zero value which was maintained at pressures up to 2 mm Hg, and no differences in wall thickness occurred. Values of active stress response for arteries from the two animal groups are summarized in figure 6. Vessel diameter was normalized to obtain these data in the following manner: the diameter at which maximum active stress response occurred was set equal to a normalized value of one. The value of external diameter at which active stress response was zero was set to a value of zero. All other diameter values were normalized according to this scheme. Values of active stress were then determined at specific values of normalized diameter and averaged for the various animal groups. This normalization procedure was used to minimize the dispersion produced by averaging results from different animals. In the case of the carotid artery, there was a significant increase in the maximum active stress developed in response to high K + in the hypertensive animals. The increase in active stress development was found to increase with the duration of hypertension for the carotids. In the case of the tail arteries, values of maximum active stress development were higher at two and four weeks post surgery. At 8 weeks post surgery, no significant differences existed in values of maximum active stress response at any value of muscle length. At 12 weeks post surgery, the maximum value of active stress development for tail arteries from the animals were lower than that of the control animals, but the differences were not statistically significant. A summary of chemical data (water and electrolyte composition) is given in table 3. The only consistent change was an increase in K + content of aorta and carotid arteries from the hypertensive animals. No consistent trends existed in Ca ++, Na + and Cl~ contents. However, in the case of the thoracic aorta Ca ++ and water contents were increased in hypertensive animals at most time periods. While there was a tendency for water content of arteries from the hypertensive animals to be higher, this result was variable. Significant differences did exist, however in the distribution of water (Table 4). Values of M Co-EDTA space were smaller in the case of arteries from hypertensive animals. Computed values of cell water content were elevated in carotid arteries and thoracic aortae from hypertensive animals. The differences tended to be larger with increasing duration of hypertension. As shown in table 2, total connective tissue content was lower in arteries from hypertensive animals. This suggests that cellular solids increased in arteries from hypertensive animals. The fractional cellular content of arteries from hypertensive animals was larger than CAROTID ARTERY TAIL ARTERY NORMALIZED DIAMETER FIGURE 6. Summary of average values of active stress response to high-k + activation for arteries from control (o) and hypertensive { ) animals. Data for carotid arteries are shown in the row of panels on the left and those of the tail arteries on the right. that of the control counterparts. The differences tended to increase with the duration of hypertension. Maximum values of active stress response were normalized on the basis of the relative cell content of control and hypertensive arteries (table 5). No significant difference was found between values of active cellular stress development for carotid arteries from control and hypertensive animals. Values of active cell stress development at 4 weeks post surgery for tail arteries from the animals were significantly larger than those of the control tail arteries. However, at 12 weeks post surgery, values of active cellular stress development were significantly lower in the case of tail arteries from the compared to those of the control animals.

10 36 HYPERTENSION VOL 4, No 1, JANUARY-FEBRUARY 1982 TABLE 4. Water Content (% wet weight) Group Thoracic artery 67. ± ± ± ± ±.5 Carotid artery 69.3 ± ± ± ± ±.8 Tail artery 78.3 ± ± ± ± ± 1.4 *p <.5; fp<.1. Total 67.3 ±.9f 68.4 ± ±.6f 68.7 ± l.lj 71.1 ±1.3* 72.9 ± ± ± ± ± ± ± 1.1 "CO-space 46.9 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±.6* 42.1 ± ± ± ± 7J ± ± 25f 51.7 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.9 Cell 24.9 ± 1.3f 28.9 ±.9f 29.9 ± 1.2* 3.2 ±3.1* 25.7 ± 1.4* 32.7 ± 3.5* 3.1 ± ± 4.6* 24.4 ± ± ± ± 2.9 TABLE 5 Summary of Computed Maximum Active Cell Stress Response Cell solids, % Cell fraction (%) Groupi Carotid artery 727 ± ± ± 62* 1.3 ±.3* 36. ± ± ± ± ± 2.8* 854 ± ± ± 46 Tail artery 1284 ± ± ± ± ± ±48* 126 ± ±53f 1666 ± ±112* 17% ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±.2t 12.6 ±.3* 11.1 ± ±^t 16.1 ±.3t 33.2 ± ±3. 39 ± ± ± ± ± ±2 * 44. ± 2.9f 37. ± ± ± ± 2.6* A< (Ted 219 ± ± ± ± ± ± ± ± ± ± 359 p < 5; jp<.1. Aa wa ]i, maximum value of active stress computed by the basis of wall cross-sectional area (1 3 dyn/cm 2 ), value of active stress computed on the basis of cell cross-sectional area (1 3 dyn/cm 2 ) ± ± ± ± ± ± 274* 559 ± ± 243* n, maximum Discussion The passive mechanics of carotid and tail arteries showed monotonic increases in stiffness with the duration of hypertension in these experiments. The onset of the changes was faster and of a greater magnitude in the carotid compared to the tail artery. At 12 weeks post surgery, the passive mechanics of both carotid and tail arteries from hypertensive animals were significantly stiffer compared to arteries from similar sites in control animals. The results of previous studies on the relationship between arterial wall mechanics and connective tissue content would have predicted from the changes in collagen and elastin found in this study that the passive stiffness of the arterial wall should have

11 ARTERIAL WALL RESPONSE TO HYPERTENSION/Cox 37 decreased with hypertension, 2 * As this did not occur, it must be concluded that other more subtle changes in the connective tissue matrix of the arterial wall at the secondary and tertiary levels may have occurred and were responsible for the increased stiffness found in this study. For example, the manner in which collagen fibers are recruited to support wall forces with increasing wall strain may differ in the hypertensive compared to the normotensive animal. With hypertension it may be that as new elements of the connective tissue matrix are synthesized, they may be organized in a different manner. Differences in the organization of connective tissue elements and perhaps in the interconnection between the elastin and collagen could potentially explain the differences in arterial wall mechanics found herein. In addition, more subtle differences in the connective tissue matrix may exist in hypertension such as differences in intermolecular and intramolecular crosslinking in the collagen matrix" 1 " and/or differences in the type of alpha chains synthesized by the smooth muscle cells.**-" Also, differences in amino acid content of the individual chains of collagen could possibly exist. 2 "'" While numerous possible alterations of the connective tissue matrix exist along these lines, any further discussion would be overly speculative at this time. The results of these studies document an increase in "contractility" of vascular smooth muscle in arteries from hypertensive animals. These changes, in the case of the carotid artery, were monotonic in nature over the 12-week course of this study. On the other hand, the changes found in the case of the tail artery were more complexly related to the duration of hypertension. There was an increased maximal constriction capacity of carotid arteries from hypertensive animals. In addition, arteries from hypertensive animals were better able to maintain constrictions at higher values of transmural pressure. These results suggest that the functional changes of the arterial wall in hypertension may act to maintain the control by smooth muscle over arterial wall properties at prevailing values of blood pressure which, of course, is elevated in the -treated animals. This "adaptive response" would be expected to maintain neurohumoral control of blood pressure and vascular resistance. The results from the luminal constriction responses of the carotid arteries support the thesis of Folkow and coworkers" concerning the role of wall thickness increases in the pathogenesis of hemodynamic changes in hypertensive subjects. In the analysis of the data from these experiments, values of active force development were normalized on the basis of cellular cross-sectional area. The latter was estimated using connective tissue and extracellular water contents. While this analysis is obviously fraught with potential problems and pitfalls, it allows the estimation of cell volume. The results of this analysis demonstrated an increase in the proportion of the wall cross-section composed of cells. When values of active wall stress development were normalized on the basis of cellular cross-sectional area, no significant differences in values of maximum active cell stress response were found for the carotid arteries from normotensive and hypertensive animals at any age. This analysis suggests that the increase in active wall stress response found in the carotid arteries of -hypertensive rats was the result of an increase in the relative cell volume of the arterial wall. This conclusion is similar to the one presented by Mulvany and his colleagues 1 *' 17 for active force development of small mesenteric arteries. In the case of the tail artery, however, after 4 weeks of age, values of active cell stress response were found to be significantly larger for tail arteries from the hypertensive animals. On the other hand, after 12 weeks of hypertension, active cell stress development of hypertensive tail arteries was lower than that of its normotensive counterpart. It is not clear at this time, however, if these changes are real or the result of some consistent error propagated through the experimental and analysis procedures. The reason for the differences in the mechanical and chemical changes of the carotid and tail artery remain a matter of speculation. The carotid artery appeared to be more severely affected by the hypertension than the tail artery. One possible contributing factor is differences in the amount of sympathetic outflow to these two blood vessels. Bevan and co-workers have shown that post-ganglionic sympathetic innervation to blood vessels play an important trophic role in determining the functional and compositional properties of blood vessels. Since the tail artery is a heavily innervated blood vessel, this factor could play some role in the difference observed between these two sites with regard to their response to experimental hypertension. Another possibility relates to the rather specialized nature of the tail in the rat. This structure is employed primarily for temperature regulation. Increases in tail blood flow would aid heat loss by the animal. When animals are maintained in a normal laboratory environment (72 F) tail blood flow is very low. This can be witnessed by the fact that the animals must be "heated" in order to measure indirect tail blood pressures. Under these circumstances perhaps the arterial pressure in the tail vasculature may not be as severely affected as that of the carotid artery during hypertension. This suggestion remains for future evaluation. References 1 Bohr D, Berccek K' Relevance of vascular structural and smooth muscle sensitivity changes in hypertension. Aust N Z J Med 6: 26, Wolinsky H- Response of the rat aortic media to hypertension. Circ Res 26: 57, Brecher P, Chan C, Franzblau C, Fans B, Chobaman A Effects of hypertension and its reversal on aortic metabolism in the rat Ore Res 43: 561, Berry C. Hypertension and arterial development, long-term considerations Br Heart J 4: 79, Bevan R, Eggena P, Hume W, Marthens E, Bevan J. Transient and persistent changes in rabbit blood vessels associated with maintained elevation in arterial pressure Hypertension 2: 63, Jones A Altered ion transport in vascular smooth muscle from spontaneously hypertensive rats. Circ Res 33: 563, 198

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