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Morphological assessment of vasoconstriction and vascular hypertrophy in sustained hypertension in the rat
MICROVASCULAR
RESEARCH
3,4166425 (197 1)
Morphological Assessment of Hypertrophy in Sustained
Vasoconstriction Hypertension
SYDNEY M. FRIEDMAN, MIYOSHI NAKASHIMA Department
of Anatomy,
The University Received
of British April
and Vascular in the Rat
AND MARYETTE
Columbia,
Vancouver,
A. MAR B.C.,
Canada
8, 1971
Measurements of individual smooth muscle cells and of the media were made in blood vessels fast-frozen in viuo during norepinephrine administration, in active DOCA-induced hypertension, and in sustained post-DOCA (metacorticoid) hypertension. In acute vasoconstriction, the cells shorten and thicken without change in their surface envelope. Cell volume increases and this increase becomes pronounced as length decreases beyond 70%. The cross-sectional area of the media as a whole does not change. Active DOCA hypertension is dominated by an increase in the surface and volume of individual cells and no shortening was observed. The area of the media is increased in consequence and encroaches on the lumen. In metacorticoid hypertension, no cell hypertrophy was observed but cells were shortened. The area of the media was unchanged but the shortened cells narrowed the lumen in proportion to the degree of elevation of blood pressure. Whether this vasoconstriction is active or structural cannot be resolved. These studies demonstrate that vascular hypertrophy is not a necessary condition of the sustained hypertensive state.
INTRODUCTION Recent experiments with fast-frozen tissueshave shown that the structure of arteries can be examined in relation to specific physiological events (Van Citters, Wagner, and Rushmer, 1962; Giese, 1964). Up to now, however, the preparations have not been adequate for more than limited gross measurementsof the vesselwall during acute vasoconstriction and have not permitted any measurementsof cell dimensions in relation to the change of state (Phelps and Luft, 1969). Similarly, with regard to the sustained hypertensive state, no acceptable measurementsbearing on the morphological basisfor the observed increase of peripheral resistanceare available (Folkow, Grimby, and Thulesius, 1958; Sivertsson, 1970). We have recently found it possible to fast-freeze small blood vesselsin situ in less than 1 set and to prepare histological sectionsfrom thesewith sufficient detail to permit measurementsof the media and of individual smooth muscle cells. This report deals with thesemeasurementsin acute and sustained hypertensive states. MATERIALS
AND METHODS
The methods have been completely described elsewhere (Friedman, Scott, and Nakashima, 1971). In brief, malerats of an inbred Wistar strain (SPF, Woodlyn Farms) are anesthetized with a combination of intraperitoneal sodium pentobarbital (33 mg/ kg) and subcutaneoussodium phenobarbitone (60 mg/kg) at the time of termination of the experiments. The ventral caudal artery or the superficial epigastric neurovascular 416
VASCULAR
HYPERTROPHY
AND
HYPERTENSION
417
bundle are exposed and fast-frozen in lessthan 1 set by a cryosurgical probe continuously cooled by circulating liquid nitrogen. The frozen tissue is excised and freezesubstituted in absolute alcohol with 1y0 osmic acid maintained at -80” for lo-14 days before being slowly rewarmed to room temperature for routine paraffin embedding, sectioning, and staining with Mallory’s aniline blue, acid fuchsin method. With this stain, smooth musclecells are orange-red, and collagen is blue. Measurements of wall dimensions were made by photographing the histological section and then projecting the film transparency onto paper. The outlines of the artery wall layers were traced out as continuous envelopesand measuredby a precision map-reader. Planimeter measurementswere used at first but thesebecome meaningless if the artery is flattened by the pressureof the probe. Thus, in the limiting case,the area of the lumen of a fully flattened vesselis zero while its linear outline remains constant despite mechanical deformation. Color differentiation at the inner and outer outline of the media is sharp and no wrinkling was observed at any stage of constriction. The outline of the lumen also presented no problem except as wrinkling became extensive during the advance of acute vasoconstriction. In this case,the lumen envelope available to the column of blood was taken at the height of the endothelial folds. The outer adventitial outline could not be accurately defined and was not usedin this study. Measurements of cell dimensions were made with a precision ocular micrometer (Zeiss). Only cells whose outlines were sufficiently defined to permit both a transverse measurementacrossthe middle of the nucleus and a longitudinal measurementof the half length of the samecell were accepted. Both of these measurementswere facilitated by focusing through the thickness of the section. The linear measurementsobtained for the layers of the wall define the circumferences of a seriesof cylinders beginning first with the lumen and adding in successionfirst intima then media. Since circumference, C = 27~ and area, A = nr2; first the radius and then the area for each of the cylinders were readily derived. Estimates of cell dimensionswere made using asa cell model two right circular cones joined at their bases. Thus, let cell length = 2h and cell width = 2r then volume, V = 2/3nr2h and surface area, A = 2nr (r2 + h2)*. The cell dimensionsare such that h2 % r2 so that surface area can be simply approximated as A = 2mh. Since the contracting cell becomesprogressively more cylindrical, the conic model will underestimate changes in both area and volume during this process. In this study we do not, accordingly, consider these derived estimates in absolute terms but make use of our cell model only to provide reasonableapproxima-
418
FRIEDMAN,
NAKASHIMA,
AND
MAR
tions. Descriptions are thus confined to orders of magnitude and to an examination of the direction of change so that our conclusions do not depend upon the choice of model. The conventional procedures involved in producing acute vasoconstriction with norepinephrine and hypertension with deoxycorticosterone acetate and saline are summarised in connection with each experiment. RESULTS Vascular Dimensions in Simple Acute Vasoconstriction In this experiment, systemic blood pressure continuously measured in the left femoral artery was raised by the infusion of norepinephrine into the adjacent femoral CONTROL
NOREPINEPHRINE A=23+2
I
SUPERFICIAL EPIGASTRIC ARTERY
n=l3 A=0.5fO.I
A=O,x
n=g
1
ARTERIOLE n=7
FIG. I. Principal dimensions of relaxed blood vesselscompared with vessels constricted by systemically infused norepinephrine. Linear dimensions are in p; Area, A, is lo3 p2. Variability is shown where necessary as 5 standard error of the mean.
vein at the rate of 4 pg/O.Ol ml/min continued long enough (l-2 min) to obtain a plateau which was never lessthan +45 mm Hg diastolic. The previously exposed right neurovascular bundle was then fast-frozen, excised, and prepared as described. Histological measurementsof the superficial epigastric artery and of a large arteriole were obtained. The basic measurementsare presentedin Fig. 1. The signs of acute vasoconstriction, that is, wrinkling and folding of the intima, were observed in the superficial epigastric artery. Quantitatively, this change is characterized by two primary criteria, (1) reduction in the lumen, (2) absenceof change in the area of the media. It is apparent that the first criterion can be stated equally well in terms of area or radius. The second criterion, however, is only revealed by the measurementof area. Taken by itself a measuredincrease in wall/lumen ratio can as readily be produced by wall hypertrophy as by vasoconstriction. Similar observations were made on arterioles of the order of 20-30 p in these sections but the differences were not significant in the small groups examined.
VASCULAR
Cell Dimensions
HYPERTROPHY
in Simple Acute
AND
419
HYPERTENSION
Vasoconstriction
A large seriesof cells was measuredin the superficial epigastric artery in the relaxed and acutely constricted states defined by the presenceof wrinkling and folding of the endothelium. Vasoconstriction was termed “spontaneous” if no drug had been administered and “induced” if it followed the infusion of epinephrine. Cell measuring was continued until samplessufficiently large to define the average for each group with a reasonably small error had been accumulated. The basic measurementsare presented in Table 1. TABLE VASCULAR
SMOOTH-MUSCLE
Width (2~) (p) Length WI (14 Area of surface (p*) Volume (p3) nb
CELL
DIMENSIONS
I DURING
EXPOSURE
Relaxed
Control Spontaneous constriction
4.12 i 0.12 161 -h 4 1208 + 50 996 i 60 57
6.60 i 0.13” llo+4” 1143 *47 1288 i 71” 59
TO NOREPINEPHRINE
Norepinephrine Induced constriction 8.33 -i 0.18” 92 * 3” 1209i49 1735 f 109” 54
h n = number of cells measured.
Vasoconstriction, as expected, involves both widening and shortening of the cell. This was of greater degree in norepinephrine-induced than in spontaneous vasoconstriction. No change in the surface envelope, i.e., area, was involved in this change. Since our model underestimates the true area of the constricted cell, the area might actually have increased asthe cell becamemore spherical. There is apparently no great necessity for much infolding of the membrane to accompany vasoconstriction, however. A direct and important implication of the measurementsis that cell volume increasesduring vasoconstriction although this is of minor degreewhen cell shortening is of the order of 30 % or less.This means,in eftect, that water moves into cells. In measuringsmooth muscle cells, either relaxed or constricted, the random population necessarilyincludes shorter and longer cells and this was examined by regression analysis (Fig. 2). Within each group, cell volume is directly proportional to measuredcell length, i.e., the longer the cell, the larger its volume. Thus, either each cell population consists of bigger and smaller cells or, since the plane of section is not always central, of cells measuredas if they were of varying sizes. When the populations of relaxed and constricted cells are compared with one another they occupy different zones of length/ volume proportions and the volume of the cell increasessharply as the degree of vasoconstriction increases.In the regions where the distributions overlap, a cell of given length has a larger volume when constricted than when relaxed. Vascular
Dimensions
in Actice
DOCA-induced
Hypertension
Deoxycorticosterone acetate in a microcrystalline suspension, 25 mg/ml, was deposited subcutaneously in divided doses,0.5 ml in the first week and 0.25 in each of
420
FRIEDMAN, NAKASHIMA,
AND MAR
the next 3 weeks, in 24 animals beginning 1 week after unilateral nephrectomy. Saline, lx, was given as drinking water during this period of active treatment. Blood pressure was measured during the 8th week of exposure to the action of the steroid deposit and fast-frozen specimens of the tail artery and of the superficial epigastric neurovascular bundle were then obtained. A matched group of untreated uninephrectomized controls was similarly handled. The findings are presented in Fig. 3. No statistically significant reduction in lumen size was observed in either tail or superficial epigastric arteries or in a terminal arteriole. Although a reduction in lumen
r 3400
2600
200’
1. 30
n 40
, 50
60
80
90
100
II0
FIG. 2. The relation of cell volume to cell length in relaxed arteries compared with vessels with moderate constriction occurring spontaneously or with advanced vasoconstriction induced by norepinephrine. Regression lines and 98% confidence limits are shown for all three groups. Individual values are shown for the two extreme cases, relaxed (0) and norepinephrine treated (0).
size of small degree must exist in some vascular beds it is not conspicuous in these. In all three vessels there was a trend for the area of the media to increase; although this was not significant in any one instance the probability of such a combination occurring by chance was remote. Vascular Dimensionsin Post-DOCA Sustained(Metacorticoid) Hypertension
Permanent hypertension following cessation of a short intensive period of DOCAsaline treatment was produced in an initial pool of 50 rats (Friedman, Friedman, and Nakashima, 1951). The active treatment phase consisted of the administration of four 25-mg pellets of DOCA within a 2-week period and 1% saline as drinking water to previously uninephrectomized animals. Active treatment was terminated by removal
VASCULAR
HYPERTROPHY
AND
421
HYPERTENSION
TAIL ARTERY
SUPERFICIAL EPIGASTRIC
ARTERIOLE
FIG. 3. Principal vascular dimensions after 8 weeks’ exposure to DOCA-saline.
CONTROL
POST- DOCA “A”
POST-DOCA”B”
jUPERFlClA1 EPIGASTRIC ARTERY
jYSTOLlC
B.P
‘DIASTOLIC 1
i3.P
13at3 73?;2
l56+2 8221
194fS 113t4--I
FIG. 4. Principal vascular dimensions 4 months after cessation of DOCA-saline
treatment.
422
FRIEDMAN,
NAKASHIMA,
AND
MAR
of pellets and a return to tap water 10 weeks after the first implantation. Presence or absenceof hypertension wasassessed 4 months later on the basisof three direct femoral artery measurementsobtained under anesthesiaon 3 separatedays. Two groups of I2 animals each were selected, one from the lowest (subgroup A) and the other from the highest (subgroup B) blood pressuresof this series.A matched group of controls was examined similarly. The vascular dimensionsare presented in Fig. 4. A small but significant degree of hypertension was present in the lower pressure subgroup of post-DOCA animals and a neat correspondencein the radius of the lumen with this was observed. Thus, the lumen in both artery and arteriole was largest in controls, smallest in the high-pressure subgroup and in-between in the low-pressure subgroup. Since this constriction of the lumen was not associatedwith any increase in the area of the media the presenceof simple vasoconstriction is indicated. Cell Dimensionsin DOCA and Post- DOCA Sustained(Metacorticoid) Hypertension Cell measurementswere made in the superficial epigastric artery of the previously described animals. The results are summarized in Fig. 5 and compared with the dimensionsof cells in acute vasoconstriction. Cell diameter was increased in the animals with active DOCA-saline hypertension but this was not accompanied by cell shortening. An increaseof both surface area and volume of these cells, indicating the presence of hypertrophy, was calculated. The vascular smooth-musclecells in both groups of post-DOCA animals were significantly shorter and wider than in controls. The change in cell dimensionsindicates the presence of mild vasoconstriction and thus agreeswith the measurementsof the wall. The cell outlines were particularly sharp in these groups becauseof a considerable increasein the paracellular matrix (Fig. 6). CELL
RELAXED
DIMENSIONS-SUPERFICIAL
EPIGASTRIC
ARTERY
CONTROL 4.7f 04
NDREPINEPHRINE
DOCA
CONSTRICTION
I
HYPERTENSION 6.1 i
POST-DOCA
0.2’
---
2
HYPERTENSION
60
80
Cell
100
Length
120
p
140
160
500
O-Area
1000
p*
1500
KWVolumep’
FIG. 5. A comparison of vascular smooth muscle cell dimensions in acute and sustained hypertension. Observed data are shown in left part of diagram, derived estimates in right part.
VASCULAR
HYPERTROPHY
AND
HYPERTENSION
423
FIG. 6. A. Histological section, 6 p, of the superficial epigastric artery in active DOCA-saline hypertension of 8 weeks duration. B. Similar artery section in post-DOCA hypertension, 4 months after cessation of treatment.
Despite the cell constriction observed in this experiment, wrinkling of the endothelium in the arteries or in the arterioles was not apparent in the sections.
DISCUSSION From the foregoing analysis of vascular dimensionsa fairly precise picture of acute vasoconstriction has emerged to amplify the pioneer studies of Van Citters and his associates(Van Citters, 1966; Van Citters, Wagner, and Rushmer, 1962). In this process,sharply defined by wrinkling and folding of the intima, the lumen is narrowed by simple constriction of the media. Within the range of vasoconstriction studied here, the area of the media remains unchanged, a point which is not apparent in conventional wall/lumen ratio figures which consider only radius rather than area. These findings thus support the casewhich Short (1966) has made concerning the importance of area rather than the one dimensional measurementof wall thickness. In this laboratory, Holtby (1970) hasobserved a small decreasein the area occupied by either the wall or the media and no change in vessel length during severe vasoconstriction. This shrinkage, of the order of 10%, accords with measurementsof water content which is reduced to the same degree in vesselsmade to constrict under in vitro conditions (Daniel, 1965). It may be important that Holtby’s observations were also made on vesselsperfused with a physiological salt solution rather than under in vivo conditions. At all events, whether there is or is not somesmall degree of shrinkage during physiological vasoconstriction there is certainly no increase in the area of the media and consequently whenever an increasein the area of the media is measuredit can safely be
424
FRIEDMAN,
NAKASHIMA,
AND
MAR
ascribed to an increase in its mass whether this be of water, of cells, or of paracellular matrix. During simple vasoconstriction the smooth muscle cell shortens and thickens and, in moderate degrees of constriction not exceeding a reduction to about 70% of the original length, there is little or no change in the area of the cell envelope and only a small increase in the volume that this encloses. With further constriction the area of the cell envelope remains unchanged while volume increases steeply. Using the technique of infusion-fixation to fix arteries at the height of constriction induced by norepinephrine and then observing their ultrastructural appearance, Hatt, Berjal, and Bonvalet (1966) concluded that a significant inflow of water into the smooth muscle cells was involved. Since the volume of the media as a whole does not change, a translocation of water from extracellular space to cells is indicated. The morphologicai evidence thus tallies with our earlier observations which showed a decrease in the inulin space during acute vasoconstriction induced either by norepinephrine or by vasopressin (Friedman and Friedman, 1963). The morphological basis of sustained hypertension produced by active treatment with DOCA is less clear in the vessels examined. The individual smooth muscle cells are certainly hypertrophied as has generally been concluded from qualitative observation and this is readily measured (Giese, 1966). Quite unlike the pattern observed in acute vasoconstriction, these cells show an increase both in area and in volume with prominent cell widening not associated with measurable shortening. The hypertrophy so readily measured in cells is much less evident in the overall measurements of wall dimensions. It is evidently important that true cell hypertrophy may well exist and yet a sufficient number of cells “drop out” so that the overall dimensions of the wall may not be increased. The accuracy of the method is insufficient to allow us to decide whether active vasoconstriction is present in active DOCA hypertension or whether the encroachment of the hypertrophied wall on the lumen, especially in small vessels, could account for the rise in pressure. The pattern of vascular morphology changes again in the sustained hypertension observable months after cessation of active DOCA treatment (Friedman and Friedman, 1949). Here cell hypertrophy has apparently completely disappeared but the individual cells are moderately contracted to about 75 % of the normal length. The lumen was seen to be reduced in both artery and arteriole in proportion to the blood pressure elevation and this was not accompanied by any significant change in the area of the media. Independent measurements of both wall and cells accordingly show that vasocontriction is a cardinal feature of metacorticoid hypertension. The presence or absence of hypertension depends not only on the shortening of the individual cells but on the degree to which collectively this narrows the lumen. It is of interest in this connection that Short and Thomson (1959) have proposed, on the basis of studies in man, that prolonged arteriolar constriction leads to persistent shortening of the circular muscle. These experiments offer no support to the view that the hypertrophy of the media often described as a feature of the hypertensive state is either a necessary consequence or an invariable concomitant of the pressure rise (Giese, 1966). Two hypertensive states have here been described; in one hypertrophy dominates the pattern while in the other a previously established hypertrophy is not apparent despite the continuing high
VASCULAR HYPERTROPHY AND HYPERTENSION
425
pressure. The seemingparadox of the disappearance of hypertrophy in the face of a continuing intravascular pressureload can be ascribed to the increase in paracellular matrix, seenas a sharpening of cell boundaries, in the arteries of animals with metacorticoid hypertension. This interpretation is in line with Burton’s expectation of a work-sparing role for the “collagen net” and with the emphasiswhich we have previously given to the paracellular matrix in sustained hypertension (Burton, 1954; Friedman and Friedman, 1967). ACKNOWLEDGMENTS This work was supported by grants from the Medical Research Council of Canada and the British Columbia Heart Foundation. REFERENCES BURTON, A. C. (1954). Relation of structure to function of the tissues of the wall of blood vessels. Physiol.
Rev. 34, 619-642.
DANIEL, E. E. (1965). Effect of sympathomimetic amines and angiotensin on active ion transport in smooth muscles. Arch. Int. Phnrmacodyn. 158, 131-138. FOLKOW, B., GRIMBY, G., AND THULESIUS,0. (19%). Adaptive structural changes of the vascular walls in hypertension and their relation to the control of the peripheral resistance. Acre Physiol. Stand. 44,255-272.
FRIEDMAN, S. M., AND FRIEDMAN, C. L. (1949). Self-sustained hypertension in the albino rat: a hypothesis to explain it. Can. Med. Ass. J. 61,596-600. FRIEDMAN, S. M., AND FRIEDMAN, C. L. (1963). Effects of ions on vascular smooth muscle. In “Handbook of Physiology,” (W. F. Hamilton and P. Dow, Eds.), Vol. 2, pp. I 135-l 166. American Physiological Society, Washington, DC. FRIEDMAN, S. M., AND FRIEDMAN, C. L. (1967). The ionic matrix of vasoconstriction. Circ. Res. 20 and 21, Suppl. II, 147-155. FRIEDMAN, S. M., FRIEDMAN, C. L., AND NAKASHIMA, M. (1951). Sustained hypertension following the administration of desoxycorticosterone acetate. J. Exp. Med. 93,361-372. FRIEDMAN, S. M., Scorr, G. H., AND NAKASHIMA, M. (1971). Vascular morphology in hypertensive states in the rat. Anat. Rec. In press. GIESE, J. (1964). Acute hypertensive vascular disease. 2. Studies on vascular reaction patterns and permeability changes by means of vital microscopy and colloidal tracer technique. Acta Puthol. Microbial. Stand. 62,497-5 15. GIESE, J. (1966). “The Pathogenesis of Hypertensive Vascular Disease. Munksgaard, Copenhagen. HATS, P. Y., BERJAL, G., AND BONVALET, J. P. (1966). Structures arterielles et arteriolaires au tours de I’hypertension experimentale du rat. Etude au microscope Clectronique. In “Club International sur PHypertension Arterielle,” (P. Milliez and P. Tcherdakoff, Eds.) pp. 460-478. L’Expansion Scientifique Francaise, Paris. HOLTBY, M. E. (1970). Arterial hydration during vasoconstriction. Thesis, University of British Columbia. PHELPS, P. C., AND LUFT, J. H. (1969). Electron microscopical study of relaxation and constriction in frog arterioles. Amer. J. Anat. 125, 399428. SHORT, D. (1966). The vascular fault in chronic hypertension with particular reference to the role of medial hypertrophy. Luncet 1,1302-l 304. SHORT, D. S., AND THOMSON, A. D. (1959). The arteries of the small intestine in systemic hypertension. J. Pathol. Bacterial. 78, 321-334. SIVERTSSON, R. (1970). The hemodynamic importance of structural vascular changes in essential hypertension. Acta Physiol. Stand. suppl. 343. VAN CI~TERS, R. L. (1966). Occlusion of lumina in small arterioles during vasoconstriction. Circ. Res. 18,199-204.
VAN Crrre~~, R. L., WAGNER, B. M., AND RUSHMER,R. F. (1962). Architecture of small arteries during vasoconstriction. Circ. Res. 10,668-675.
RESEARCH
3,4166425 (197 1)
Morphological Assessment of Hypertrophy in Sustained
Vasoconstriction Hypertension
SYDNEY M. FRIEDMAN, MIYOSHI NAKASHIMA Department
of Anatomy,
The University Received
of British April
and Vascular in the Rat
AND MARYETTE
Columbia,
Vancouver,
A. MAR B.C.,
Canada
8, 1971
Measurements of individual smooth muscle cells and of the media were made in blood vessels fast-frozen in viuo during norepinephrine administration, in active DOCA-induced hypertension, and in sustained post-DOCA (metacorticoid) hypertension. In acute vasoconstriction, the cells shorten and thicken without change in their surface envelope. Cell volume increases and this increase becomes pronounced as length decreases beyond 70%. The cross-sectional area of the media as a whole does not change. Active DOCA hypertension is dominated by an increase in the surface and volume of individual cells and no shortening was observed. The area of the media is increased in consequence and encroaches on the lumen. In metacorticoid hypertension, no cell hypertrophy was observed but cells were shortened. The area of the media was unchanged but the shortened cells narrowed the lumen in proportion to the degree of elevation of blood pressure. Whether this vasoconstriction is active or structural cannot be resolved. These studies demonstrate that vascular hypertrophy is not a necessary condition of the sustained hypertensive state.
INTRODUCTION Recent experiments with fast-frozen tissueshave shown that the structure of arteries can be examined in relation to specific physiological events (Van Citters, Wagner, and Rushmer, 1962; Giese, 1964). Up to now, however, the preparations have not been adequate for more than limited gross measurementsof the vesselwall during acute vasoconstriction and have not permitted any measurementsof cell dimensions in relation to the change of state (Phelps and Luft, 1969). Similarly, with regard to the sustained hypertensive state, no acceptable measurementsbearing on the morphological basisfor the observed increase of peripheral resistanceare available (Folkow, Grimby, and Thulesius, 1958; Sivertsson, 1970). We have recently found it possible to fast-freeze small blood vesselsin situ in less than 1 set and to prepare histological sectionsfrom thesewith sufficient detail to permit measurementsof the media and of individual smooth muscle cells. This report deals with thesemeasurementsin acute and sustained hypertensive states. MATERIALS
AND METHODS
The methods have been completely described elsewhere (Friedman, Scott, and Nakashima, 1971). In brief, malerats of an inbred Wistar strain (SPF, Woodlyn Farms) are anesthetized with a combination of intraperitoneal sodium pentobarbital (33 mg/ kg) and subcutaneoussodium phenobarbitone (60 mg/kg) at the time of termination of the experiments. The ventral caudal artery or the superficial epigastric neurovascular 416
VASCULAR
HYPERTROPHY
AND
HYPERTENSION
417
bundle are exposed and fast-frozen in lessthan 1 set by a cryosurgical probe continuously cooled by circulating liquid nitrogen. The frozen tissue is excised and freezesubstituted in absolute alcohol with 1y0 osmic acid maintained at -80” for lo-14 days before being slowly rewarmed to room temperature for routine paraffin embedding, sectioning, and staining with Mallory’s aniline blue, acid fuchsin method. With this stain, smooth musclecells are orange-red, and collagen is blue. Measurements of wall dimensions were made by photographing the histological section and then projecting the film transparency onto paper. The outlines of the artery wall layers were traced out as continuous envelopesand measuredby a precision map-reader. Planimeter measurementswere used at first but thesebecome meaningless if the artery is flattened by the pressureof the probe. Thus, in the limiting case,the area of the lumen of a fully flattened vesselis zero while its linear outline remains constant despite mechanical deformation. Color differentiation at the inner and outer outline of the media is sharp and no wrinkling was observed at any stage of constriction. The outline of the lumen also presented no problem except as wrinkling became extensive during the advance of acute vasoconstriction. In this case,the lumen envelope available to the column of blood was taken at the height of the endothelial folds. The outer adventitial outline could not be accurately defined and was not usedin this study. Measurements of cell dimensions were made with a precision ocular micrometer (Zeiss). Only cells whose outlines were sufficiently defined to permit both a transverse measurementacrossthe middle of the nucleus and a longitudinal measurementof the half length of the samecell were accepted. Both of these measurementswere facilitated by focusing through the thickness of the section. The linear measurementsobtained for the layers of the wall define the circumferences of a seriesof cylinders beginning first with the lumen and adding in successionfirst intima then media. Since circumference, C = 27~ and area, A = nr2; first the radius and then the area for each of the cylinders were readily derived. Estimates of cell dimensionswere made using asa cell model two right circular cones joined at their bases. Thus, let cell length = 2h and cell width = 2r then volume, V = 2/3nr2h and surface area, A = 2nr (r2 + h2)*. The cell dimensionsare such that h2 % r2 so that surface area can be simply approximated as A = 2mh. Since the contracting cell becomesprogressively more cylindrical, the conic model will underestimate changes in both area and volume during this process. In this study we do not, accordingly, consider these derived estimates in absolute terms but make use of our cell model only to provide reasonableapproxima-
418
FRIEDMAN,
NAKASHIMA,
AND
MAR
tions. Descriptions are thus confined to orders of magnitude and to an examination of the direction of change so that our conclusions do not depend upon the choice of model. The conventional procedures involved in producing acute vasoconstriction with norepinephrine and hypertension with deoxycorticosterone acetate and saline are summarised in connection with each experiment. RESULTS Vascular Dimensions in Simple Acute Vasoconstriction In this experiment, systemic blood pressure continuously measured in the left femoral artery was raised by the infusion of norepinephrine into the adjacent femoral CONTROL
NOREPINEPHRINE A=23+2
I
SUPERFICIAL EPIGASTRIC ARTERY
n=l3 A=0.5fO.I
A=O,x
n=g
1
ARTERIOLE n=7
FIG. I. Principal dimensions of relaxed blood vesselscompared with vessels constricted by systemically infused norepinephrine. Linear dimensions are in p; Area, A, is lo3 p2. Variability is shown where necessary as 5 standard error of the mean.
vein at the rate of 4 pg/O.Ol ml/min continued long enough (l-2 min) to obtain a plateau which was never lessthan +45 mm Hg diastolic. The previously exposed right neurovascular bundle was then fast-frozen, excised, and prepared as described. Histological measurementsof the superficial epigastric artery and of a large arteriole were obtained. The basic measurementsare presentedin Fig. 1. The signs of acute vasoconstriction, that is, wrinkling and folding of the intima, were observed in the superficial epigastric artery. Quantitatively, this change is characterized by two primary criteria, (1) reduction in the lumen, (2) absenceof change in the area of the media. It is apparent that the first criterion can be stated equally well in terms of area or radius. The second criterion, however, is only revealed by the measurementof area. Taken by itself a measuredincrease in wall/lumen ratio can as readily be produced by wall hypertrophy as by vasoconstriction. Similar observations were made on arterioles of the order of 20-30 p in these sections but the differences were not significant in the small groups examined.
VASCULAR
Cell Dimensions
HYPERTROPHY
in Simple Acute
AND
419
HYPERTENSION
Vasoconstriction
A large seriesof cells was measuredin the superficial epigastric artery in the relaxed and acutely constricted states defined by the presenceof wrinkling and folding of the endothelium. Vasoconstriction was termed “spontaneous” if no drug had been administered and “induced” if it followed the infusion of epinephrine. Cell measuring was continued until samplessufficiently large to define the average for each group with a reasonably small error had been accumulated. The basic measurementsare presented in Table 1. TABLE VASCULAR
SMOOTH-MUSCLE
Width (2~) (p) Length WI (14 Area of surface (p*) Volume (p3) nb
CELL
DIMENSIONS
I DURING
EXPOSURE
Relaxed
Control Spontaneous constriction
4.12 i 0.12 161 -h 4 1208 + 50 996 i 60 57
6.60 i 0.13” llo+4” 1143 *47 1288 i 71” 59
TO NOREPINEPHRINE
Norepinephrine Induced constriction 8.33 -i 0.18” 92 * 3” 1209i49 1735 f 109” 54
h n = number of cells measured.
Vasoconstriction, as expected, involves both widening and shortening of the cell. This was of greater degree in norepinephrine-induced than in spontaneous vasoconstriction. No change in the surface envelope, i.e., area, was involved in this change. Since our model underestimates the true area of the constricted cell, the area might actually have increased asthe cell becamemore spherical. There is apparently no great necessity for much infolding of the membrane to accompany vasoconstriction, however. A direct and important implication of the measurementsis that cell volume increasesduring vasoconstriction although this is of minor degreewhen cell shortening is of the order of 30 % or less.This means,in eftect, that water moves into cells. In measuringsmooth muscle cells, either relaxed or constricted, the random population necessarilyincludes shorter and longer cells and this was examined by regression analysis (Fig. 2). Within each group, cell volume is directly proportional to measuredcell length, i.e., the longer the cell, the larger its volume. Thus, either each cell population consists of bigger and smaller cells or, since the plane of section is not always central, of cells measuredas if they were of varying sizes. When the populations of relaxed and constricted cells are compared with one another they occupy different zones of length/ volume proportions and the volume of the cell increasessharply as the degree of vasoconstriction increases.In the regions where the distributions overlap, a cell of given length has a larger volume when constricted than when relaxed. Vascular
Dimensions
in Actice
DOCA-induced
Hypertension
Deoxycorticosterone acetate in a microcrystalline suspension, 25 mg/ml, was deposited subcutaneously in divided doses,0.5 ml in the first week and 0.25 in each of
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the next 3 weeks, in 24 animals beginning 1 week after unilateral nephrectomy. Saline, lx, was given as drinking water during this period of active treatment. Blood pressure was measured during the 8th week of exposure to the action of the steroid deposit and fast-frozen specimens of the tail artery and of the superficial epigastric neurovascular bundle were then obtained. A matched group of untreated uninephrectomized controls was similarly handled. The findings are presented in Fig. 3. No statistically significant reduction in lumen size was observed in either tail or superficial epigastric arteries or in a terminal arteriole. Although a reduction in lumen
r 3400
2600
200’
1. 30
n 40
, 50
60
80
90
100
II0
FIG. 2. The relation of cell volume to cell length in relaxed arteries compared with vessels with moderate constriction occurring spontaneously or with advanced vasoconstriction induced by norepinephrine. Regression lines and 98% confidence limits are shown for all three groups. Individual values are shown for the two extreme cases, relaxed (0) and norepinephrine treated (0).
size of small degree must exist in some vascular beds it is not conspicuous in these. In all three vessels there was a trend for the area of the media to increase; although this was not significant in any one instance the probability of such a combination occurring by chance was remote. Vascular Dimensionsin Post-DOCA Sustained(Metacorticoid) Hypertension
Permanent hypertension following cessation of a short intensive period of DOCAsaline treatment was produced in an initial pool of 50 rats (Friedman, Friedman, and Nakashima, 1951). The active treatment phase consisted of the administration of four 25-mg pellets of DOCA within a 2-week period and 1% saline as drinking water to previously uninephrectomized animals. Active treatment was terminated by removal
VASCULAR
HYPERTROPHY
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421
HYPERTENSION
TAIL ARTERY
SUPERFICIAL EPIGASTRIC
ARTERIOLE
FIG. 3. Principal vascular dimensions after 8 weeks’ exposure to DOCA-saline.
CONTROL
POST- DOCA “A”
POST-DOCA”B”
jUPERFlClA1 EPIGASTRIC ARTERY
jYSTOLlC
B.P
‘DIASTOLIC 1
i3.P
13at3 73?;2
l56+2 8221
194fS 113t4--I
FIG. 4. Principal vascular dimensions 4 months after cessation of DOCA-saline
treatment.
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of pellets and a return to tap water 10 weeks after the first implantation. Presence or absenceof hypertension wasassessed 4 months later on the basisof three direct femoral artery measurementsobtained under anesthesiaon 3 separatedays. Two groups of I2 animals each were selected, one from the lowest (subgroup A) and the other from the highest (subgroup B) blood pressuresof this series.A matched group of controls was examined similarly. The vascular dimensionsare presented in Fig. 4. A small but significant degree of hypertension was present in the lower pressure subgroup of post-DOCA animals and a neat correspondencein the radius of the lumen with this was observed. Thus, the lumen in both artery and arteriole was largest in controls, smallest in the high-pressure subgroup and in-between in the low-pressure subgroup. Since this constriction of the lumen was not associatedwith any increase in the area of the media the presenceof simple vasoconstriction is indicated. Cell Dimensionsin DOCA and Post- DOCA Sustained(Metacorticoid) Hypertension Cell measurementswere made in the superficial epigastric artery of the previously described animals. The results are summarized in Fig. 5 and compared with the dimensionsof cells in acute vasoconstriction. Cell diameter was increased in the animals with active DOCA-saline hypertension but this was not accompanied by cell shortening. An increaseof both surface area and volume of these cells, indicating the presence of hypertrophy, was calculated. The vascular smooth-musclecells in both groups of post-DOCA animals were significantly shorter and wider than in controls. The change in cell dimensionsindicates the presence of mild vasoconstriction and thus agreeswith the measurementsof the wall. The cell outlines were particularly sharp in these groups becauseof a considerable increasein the paracellular matrix (Fig. 6). CELL
RELAXED
DIMENSIONS-SUPERFICIAL
EPIGASTRIC
ARTERY
CONTROL 4.7f 04
NDREPINEPHRINE
DOCA
CONSTRICTION
I
HYPERTENSION 6.1 i
POST-DOCA
0.2’
---
2
HYPERTENSION
60
80
Cell
100
Length
120
p
140
160
500
O-Area
1000
p*
1500
KWVolumep’
FIG. 5. A comparison of vascular smooth muscle cell dimensions in acute and sustained hypertension. Observed data are shown in left part of diagram, derived estimates in right part.
VASCULAR
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423
FIG. 6. A. Histological section, 6 p, of the superficial epigastric artery in active DOCA-saline hypertension of 8 weeks duration. B. Similar artery section in post-DOCA hypertension, 4 months after cessation of treatment.
Despite the cell constriction observed in this experiment, wrinkling of the endothelium in the arteries or in the arterioles was not apparent in the sections.
DISCUSSION From the foregoing analysis of vascular dimensionsa fairly precise picture of acute vasoconstriction has emerged to amplify the pioneer studies of Van Citters and his associates(Van Citters, 1966; Van Citters, Wagner, and Rushmer, 1962). In this process,sharply defined by wrinkling and folding of the intima, the lumen is narrowed by simple constriction of the media. Within the range of vasoconstriction studied here, the area of the media remains unchanged, a point which is not apparent in conventional wall/lumen ratio figures which consider only radius rather than area. These findings thus support the casewhich Short (1966) has made concerning the importance of area rather than the one dimensional measurementof wall thickness. In this laboratory, Holtby (1970) hasobserved a small decreasein the area occupied by either the wall or the media and no change in vessel length during severe vasoconstriction. This shrinkage, of the order of 10%, accords with measurementsof water content which is reduced to the same degree in vesselsmade to constrict under in vitro conditions (Daniel, 1965). It may be important that Holtby’s observations were also made on vesselsperfused with a physiological salt solution rather than under in vivo conditions. At all events, whether there is or is not somesmall degree of shrinkage during physiological vasoconstriction there is certainly no increase in the area of the media and consequently whenever an increasein the area of the media is measuredit can safely be
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ascribed to an increase in its mass whether this be of water, of cells, or of paracellular matrix. During simple vasoconstriction the smooth muscle cell shortens and thickens and, in moderate degrees of constriction not exceeding a reduction to about 70% of the original length, there is little or no change in the area of the cell envelope and only a small increase in the volume that this encloses. With further constriction the area of the cell envelope remains unchanged while volume increases steeply. Using the technique of infusion-fixation to fix arteries at the height of constriction induced by norepinephrine and then observing their ultrastructural appearance, Hatt, Berjal, and Bonvalet (1966) concluded that a significant inflow of water into the smooth muscle cells was involved. Since the volume of the media as a whole does not change, a translocation of water from extracellular space to cells is indicated. The morphologicai evidence thus tallies with our earlier observations which showed a decrease in the inulin space during acute vasoconstriction induced either by norepinephrine or by vasopressin (Friedman and Friedman, 1963). The morphological basis of sustained hypertension produced by active treatment with DOCA is less clear in the vessels examined. The individual smooth muscle cells are certainly hypertrophied as has generally been concluded from qualitative observation and this is readily measured (Giese, 1966). Quite unlike the pattern observed in acute vasoconstriction, these cells show an increase both in area and in volume with prominent cell widening not associated with measurable shortening. The hypertrophy so readily measured in cells is much less evident in the overall measurements of wall dimensions. It is evidently important that true cell hypertrophy may well exist and yet a sufficient number of cells “drop out” so that the overall dimensions of the wall may not be increased. The accuracy of the method is insufficient to allow us to decide whether active vasoconstriction is present in active DOCA hypertension or whether the encroachment of the hypertrophied wall on the lumen, especially in small vessels, could account for the rise in pressure. The pattern of vascular morphology changes again in the sustained hypertension observable months after cessation of active DOCA treatment (Friedman and Friedman, 1949). Here cell hypertrophy has apparently completely disappeared but the individual cells are moderately contracted to about 75 % of the normal length. The lumen was seen to be reduced in both artery and arteriole in proportion to the blood pressure elevation and this was not accompanied by any significant change in the area of the media. Independent measurements of both wall and cells accordingly show that vasocontriction is a cardinal feature of metacorticoid hypertension. The presence or absence of hypertension depends not only on the shortening of the individual cells but on the degree to which collectively this narrows the lumen. It is of interest in this connection that Short and Thomson (1959) have proposed, on the basis of studies in man, that prolonged arteriolar constriction leads to persistent shortening of the circular muscle. These experiments offer no support to the view that the hypertrophy of the media often described as a feature of the hypertensive state is either a necessary consequence or an invariable concomitant of the pressure rise (Giese, 1966). Two hypertensive states have here been described; in one hypertrophy dominates the pattern while in the other a previously established hypertrophy is not apparent despite the continuing high
VASCULAR HYPERTROPHY AND HYPERTENSION
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pressure. The seemingparadox of the disappearance of hypertrophy in the face of a continuing intravascular pressureload can be ascribed to the increase in paracellular matrix, seenas a sharpening of cell boundaries, in the arteries of animals with metacorticoid hypertension. This interpretation is in line with Burton’s expectation of a work-sparing role for the “collagen net” and with the emphasiswhich we have previously given to the paracellular matrix in sustained hypertension (Burton, 1954; Friedman and Friedman, 1967). ACKNOWLEDGMENTS This work was supported by grants from the Medical Research Council of Canada and the British Columbia Heart Foundation. REFERENCES BURTON, A. C. (1954). Relation of structure to function of the tissues of the wall of blood vessels. Physiol.
Rev. 34, 619-642.
DANIEL, E. E. (1965). Effect of sympathomimetic amines and angiotensin on active ion transport in smooth muscles. Arch. Int. Phnrmacodyn. 158, 131-138. FOLKOW, B., GRIMBY, G., AND THULESIUS,0. (19%). Adaptive structural changes of the vascular walls in hypertension and their relation to the control of the peripheral resistance. Acre Physiol. Stand. 44,255-272.
FRIEDMAN, S. M., AND FRIEDMAN, C. L. (1949). Self-sustained hypertension in the albino rat: a hypothesis to explain it. Can. Med. Ass. J. 61,596-600. FRIEDMAN, S. M., AND FRIEDMAN, C. L. (1963). Effects of ions on vascular smooth muscle. In “Handbook of Physiology,” (W. F. Hamilton and P. Dow, Eds.), Vol. 2, pp. I 135-l 166. American Physiological Society, Washington, DC. FRIEDMAN, S. M., AND FRIEDMAN, C. L. (1967). The ionic matrix of vasoconstriction. Circ. Res. 20 and 21, Suppl. II, 147-155. FRIEDMAN, S. M., FRIEDMAN, C. L., AND NAKASHIMA, M. (1951). Sustained hypertension following the administration of desoxycorticosterone acetate. J. Exp. Med. 93,361-372. FRIEDMAN, S. M., Scorr, G. H., AND NAKASHIMA, M. (1971). Vascular morphology in hypertensive states in the rat. Anat. Rec. In press. GIESE, J. (1964). Acute hypertensive vascular disease. 2. Studies on vascular reaction patterns and permeability changes by means of vital microscopy and colloidal tracer technique. Acta Puthol. Microbial. Stand. 62,497-5 15. GIESE, J. (1966). “The Pathogenesis of Hypertensive Vascular Disease. Munksgaard, Copenhagen. HATS, P. Y., BERJAL, G., AND BONVALET, J. P. (1966). Structures arterielles et arteriolaires au tours de I’hypertension experimentale du rat. Etude au microscope Clectronique. In “Club International sur PHypertension Arterielle,” (P. Milliez and P. Tcherdakoff, Eds.) pp. 460-478. L’Expansion Scientifique Francaise, Paris. HOLTBY, M. E. (1970). Arterial hydration during vasoconstriction. Thesis, University of British Columbia. PHELPS, P. C., AND LUFT, J. H. (1969). Electron microscopical study of relaxation and constriction in frog arterioles. Amer. J. Anat. 125, 399428. SHORT, D. (1966). The vascular fault in chronic hypertension with particular reference to the role of medial hypertrophy. Luncet 1,1302-l 304. SHORT, D. S., AND THOMSON, A. D. (1959). The arteries of the small intestine in systemic hypertension. J. Pathol. Bacterial. 78, 321-334. SIVERTSSON, R. (1970). The hemodynamic importance of structural vascular changes in essential hypertension. Acta Physiol. Stand. suppl. 343. VAN CI~TERS, R. L. (1966). Occlusion of lumina in small arterioles during vasoconstriction. Circ. Res. 18,199-204.
VAN Crrre~~, R. L., WAGNER, B. M., AND RUSHMER,R. F. (1962). Architecture of small arteries during vasoconstriction. Circ. Res. 10,668-675.