Myogenic Tone Attenuates Pressure-Induced Gene Expression in Isolated Small Arteries
Abstract This study was designed to determine whether pressure-induced expression of early response genes in the arterial wall is dependent on an increase in cell stretch or an increase in wall stress. Mesenteric arteries (245 to 385 μm in diameter) were isolated from Wistar rats and subjected to static pressures of either 90 mm Hg (control), 140 mm Hg, or 165 mm Hg for a period of 3 hours. Arteries developed a range of myogenic tone such that wall stresses in the 140 and 165 mm Hg arteries (1.60 to 4.44×106 dynes/cm2) were equivalent in some cases to those of controls (1.76 to 2.63×106 dynes/cm2). Vessels subjected to 140 or 165 mm Hg intraluminal pressure had diameters ranging from 74% to 104% of their relaxed diameter at 90 mm Hg, whereas control vessel diameters ranged from 88% to 100%. At the end of each experiment, vessels were fixed in 10% formalin, embedded in paraffin, and sectioned for in situ hybridization. Wall stress significantly correlated with c-myc mRNA and 18S rRNA expression. Gene expression did not correlate with vessel diameter, expressed as a percentage of the relaxed diameter at 90 mm Hg, ie, cell stretch. The expression of β-actin mRNA did not differ between vessels and showed no correlation with wall stress, suggesting that the induction of c-myc mRNA and 18S rRNA was part of a specific response. These findings show that in an isolated artery, a pressure stimulus can be perceived as an increase in wall stress, independently of cell stretch. Therefore, wall stress may be the signaling parameter in hypertension where arteries are tonically constricted. The inhibition of gene expression by myogenic constriction may explain why hypertrophy takes place in large arteries during hypertension but not in arterioles where increased tone reduces wall stress.
In essential hypertension in humans, walls of large arteries hypertrophy, whereas arterioles remodel into vessels with smaller lumens without medial hypertrophy.1 Small arteries located at the transition from conduit to resistance vessels undergo both lumen reduction and hypertrophy, a process of inward hypertrophic remodeling.2 Wall hypertrophy is the dominant structural change in the SHR,3 4 5 but eutrophic inward remodeling of arterioles in renal hypertensive rats6 7 is very similar to that seen in essential hypertension. These structural changes result in an increase in the media to lumen ratio, which helps maintain hypertension by increasing peripheral resistance and responsiveness to vasoconstrictor stimuli.8 In the SHR, the use of antihypertensive drugs3 4 to lower blood pressure and the use of ligatures to protect parts of the vascular system from the increase in pressure5 attenuate these changes, suggesting that pressure itself may be a major stimulus for both hypertrophy and remodeling. The present study addresses the question of how a single mechanical signal of elevated pressure can lead to both hypertrophy of arteries and inward eutrophic remodeling of arterioles.
The second question is how a change in pressure is sensed by the VSMC. Cell stretch has been shown to stimulate second messenger production and growth responses in many different studies, especially those in isolated VSMC9 10 11 12 13 14 15 and segments of whole vessels.16 17 18 19 20 21 While these studies indicate that cell stretch is sufficient stimulus for a growth response, the mechanism by which these cells sense stretch is unclear. In these models, stretching of the cells is invariably associated with an increase in wall tension, and it has been suggested that this factor plays an important role in mechanotransduction.16 18 20 It is unlikely that VSMC in artery walls will actually be stretched during the development of hypertension, although they will experience an increase in circumferential wall stress. A recent study by Wilson et al15 shows that signal transduction of cyclical stretch is mediated through specific integrins recognizing the RGD binding motif. Considering this, stretch-activated phospholipase C19 or opening of ion channels22 by stretch of the cell membrane may not be necessary for signal transduction.
We have shown previously in an isolated pressurized mesenteric artery preparation that a 60% to 80% increase in wall stress with minimal cell stretch (<3%) was sufficient to stimulate proto-oncogene expression and rRNA production, suggesting that wall stress may indeed be the factor sensed in these vessels.23 This present study was therefore undertaken to completely dissociate wall stress from cell stretch by using smaller mesenteric arteries that develop myogenic tone, ie, they are able to constrict as pressure is increased. This made it possible to raise intraluminal pressures such that wall stresses were significantly elevated while the vessels were still constricted; therefore, VSMC were not stretched in these preparations. The results of this study clearly demonstrate a correlation between wall stress and changes in expression of c-myc and 18S rRNA in these arteries. This indicates that wall stress can be sensed independently of cell stretch and suggests that the detection of cell stretch may in fact be dependent on an increase in wall stress.
Isolation of Mesenteric Arteries
Twenty-three male Wistar rats weighing between 183 and 353 g were anesthetized with a subcutaneous injection of ketamine/xylazine (80/12 mg/kg) and killed by cervical dislocation. The experimental protocol was approved by the institutional animal care and use committee. An incision was made in the abdominal wall, and the mesentery was carefully dissected away from the intestine and placed in cold (4°C) dissection buffer (30 mmol/L MOPS, 1.2 mmol/L NaH2PO4, 5 mmol/L glucose, 2 mmol/L pyruvate, 0.02 mmol/L [disodium salt] EDTA, 145 mmol/L NaCl, 4.7 mmol/L KCl, 2 mmol/L CaCl2, 1.2 mmol/L MgSO4, 10 g/L BSA, pH 7.4, osmolarity 290 to 310). Two arteries per animal were dissected free of fat and excised. Mesenteric arteries of 224 to 385 μm were mounted on pipettes in a dual-vessel chamber. Vessels were superfused at 4 mL/min with HEPES-buffered Krebs (112 mmol/L NaCl, 11.5 mmol/L glucose, 25.5 mmol/L NaHCO3, 10 mmol/L HEPES, 1.2 mmol/L MgSO4, 2.5 mmol/L CaCl2, 1.2 mmol/L KH2PO4, 4.7 mmol/L KCl) gassed with air/CO2 (95%/5%). The medium was maintained at a temperature of 37°C and a pH of 7.4 throughout each experiment. The intraluminal pressure (with no flow) was then increased from 30 mm Hg in steps of 15 mm Hg per 15 minutes over a 1-hour period until a pressure of 90 mm Hg was reached. During this initial pressurization period, many of the vessels typically developed spontaneous myogenic tone. Each vessel was allowed to constrict until it reached a stable diameter at 90 mm Hg.
Experimental Treatment of Vessels
Vessels were assigned to either a control or one of two high-pressure groups. Intraluminal pressure was left at 90 mm Hg in controls; pressure in the high-pressure groups was initially raised to either 140 or 165 mm Hg. Control and high-pressure vessels were paired until 10 control vessels were obtained, and then both arteries were maintained at the elevated pressures, resulting in data from 20 arteries at 140 mm Hg and 10 at 165 mm Hg. Vessels were maintained at their designated experimental pressure for a period of 3 hours. At the end of each experiment, the pressure in all vessels was set to 90 mm Hg while each vessel was constricted with norepinephrine (2 μmol/L) and dilated with acetylcholine (2 μmol/L) to check that the vessels were still viable and the endothelium was intact. Vessels were immediately fixed in 10% buffered formalin, processed in graded alcohols and xylene, and embedded in paraffin for sectioning.
Measurement of Arterial Dimensions
Throughout each experiment, the vessels were observed with closed-circuit television microscopy using a ×10 objective on a Zeiss ACM microscope. Internal and external diameters of each vessel were measured with a video image shearer (Vista model 308) and used to calculate wall thickness. Wall tension was calculated using the La Place relation, which states that Wall Tension=Transmural Pressure×Radius. Wall stress in each vessel was derived by dividing wall tension by wall thickness.
In Situ Hybridization and Quantification
The protocols for in situ hybridization with 35S-labeled riboprobe and for quantification have been described previously.23 Briefly, paraffin sections of arteries (4 μm) were mounted on Superfrost/Plus slides. Experimentally paired vessels (4 to 5 sections of each) were mounted on the same slide so that hybridization conditions were identical. Slides were deparaffinized, and the vessels were rehydrated in graded alcohol and then washed with 0.5× SSC (sodium citrate, sodium chloride). Vessels were treated with proteinase K at room temperature, washed three times with PBS, and fixed in 4% paraformaldehyde for 15 minutes at 4°C. Slides were washed three times with PBS and allowed to air dry. The vessel sections were then covered with 200 μL prehybridization solution (10% dextran sulfate, 1× Denhardt’s solution, 1 mmol/L EDTA, 10 mmol/L Tris, 0.3 mol/L NaCl, 50% formamide, 0.5 mg/mL yeast tRNA, 10 mmol/L DTT) and incubated for 2 to 3 hours at 38°C to 42°C in a humidified hybridization box. Slides were briefly washed with 0.5× SSC, covered with 100 μL hybridization solution (prehybridization solution containing 107 cpm of probe per milliliter), and incubated overnight at the calculated melting temperature of the riboprobe (50°C to 55°C) in a humidified hybridization box. Slides were washed with 2× SSC containing 1 mmol/L EDTA and 10 mmol/L β-mercaptoethanol, then treated with RNase A for 30 minutes at room temperature. Slides were then washed in the following solutions: 2× SSC containing 1 mmol/L EDTA and 10 mmol/L β-mercaptoethanol, 0.1× SSC containing 1 mmol/L EDTA and 10 mmol/L β-mercaptoethanol at 55°C, and 0.5× SSC. Finally, the vessel sections were dehydrated in graded alcohols and air dried.
Results were quantified by densitometric analysis of the slides with a Molecular Dynamics PhosphorImager SF. Slides were exposed on the phosphorimager screen for 5 to 7 days before analysis. Using a volume integration function, the total density minus background was determined for each vessel cross section. The mean value in arbitrary units was calculated to obtain a single value for each vessel. For c-myc, any values for sense were subtracted from antisense to obtain specific binding.
The cDNA for c-myc was a 1.4-kb fragment of exons 2 and 3 obtained from Michael Cole (Princeton University). Human 18S rRNA and β-actin templates were obtained from Ambion Inc. Radiolabeled CTP was obtained from Du Pont. All other chemicals or biochemicals were obtained from Sigma Chemical Co, Fisher Scientific, Gibco BRL, or Promega.
Data are presented as mean±SEM. Statistical analysis was performed by ANOVA and linear regression analysis with rejection of the null hypothesis at a value of P<.05.
Diameters and Wall Stresses
Fig 1⇓ shows the group data for internal diameter of the arteries throughout the experiment. As the pressure was raised stepwise to 70 mm Hg, the diameter increased in all groups. After that, the mean diameter was maintained in the 90 and 140 mm Hg groups and decreased in the 165 mm Hg group because of myogenic constriction. Four of the 10 arteries in the control group constricted to increasing pressure, whereas 12 of 20 arteries in the 140 mm Hg group and 7 of the 10 arteries in the 165 mm Hg group showed myogenic tone. Myogenic tone was greatest in the latter group but tended to decrease during the 3-hour period of elevated pressure. The myogenic constrictions of the vessels varied such that the ranges of wall stresses for each of the groups were from 1.76 to 2.63, 1.60 to 4.44, and 2.52 to 3.62×106 dynes/cm2 for control (90 mm Hg), 140 mm Hg, and 165 mm Hg, respectively.
There were no significant differences in the responses of any of the different groups to treatment with norepinephrine (2 μmol/L) or acetylcholine (2 μmol/L), showing that vessels without tone were equally viable and capable of constricting. The response to acetylcholine indicates the presence of a functional endothelium and provided a measurement of relaxed diameter that did not differ between the groups.
Expression of c-myc mRNA
Quantitation of in situ hybridization by phosphorimager is an unusual application that is suitable in this case, as previously shown,23 because expression of proto-oncogenes and 18S rRNA is uniform across the vessel media, there are no adjoining tissues in the samples, and the density measurements correlate with quantitation by autoradiography using grain counting.
Fig 2⇓ shows the expression of c-myc plotted as a function of pressure groups (A), mean wall stress throughout the experiment (B), and the experimental diameter (C), shown as a percentage of the relaxed diameter at 90 mm Hg. The group comparisons show a highly significant increase in c-myc expression in the 140 mm Hg group but not in the 165 mm Hg group, possibly because of the greater myogenic tone in that group. There was a direct correlation between wall stress and c-myc expression in these vessels, with a correlation coefficient of .616 (Fig 2B⇓). As can be seen in this figure, most of the arteries with high wall stress are from the 140 mm Hg group.
The correlation of c-myc expression with experimental diameter (Fig 2C⇑) is not statistically significant. Experimental diameter shown as a percentage of relaxed diameter at 90 mm Hg is an indicator of cell length rather than vessel size. Thus, if cell stretch were the important parameter, a strong correlation should be present in this graph.
Expression of 18S rRNA
The group data for 18S rRNA expression (Fig 3A⇓) show significant elevations in both of the high-pressure groups, but raising the pressure to 165 mm Hg did not increase expression above that of the 140 mm Hg group. The significant correlation with wall stress (Fig 3B⇓) again suggests that this is the important parameter for a pressure increase to activate a growth response. The lack of any correlation with experimental diameter (Fig 3C⇓) suggests once again that cell stretch is not required for this response to occur.
Expression of β-Actin mRNA
Fig 4⇓ shows expression of β-actin mRNA again as a function of pressure group (A), wall stress (B), and experimental diameter (C). There were no differences in β-actin expression among the three pressure groups and no correlation between β-actin expression and wall stress or experimental diameter. This suggests that the increases in c-myc and 18S rRNA seen with increased wall stress represent a specific pattern of gene expression rather than a generalized upregulation of all genes.
In hypertension, many mechanisms may contribute to hypertrophy of the vascular wall. In all cases, however, there is an increase in intraluminal pressure that in itself may play an important role in hypertrophy. Interventions that lower blood pressure in the SHR, either by drug treatment3 4 or use of a ligature to protect a part of the vascular system,5 normalize arterial structure, suggesting that pressure itself may be one of the dominant factors influencing vessel-wall hypertrophy. The use of in vivo models, however, is confounded by the presence of hormonal as well as mechanical effects. Mechanical effects can be studied separately by use of in vitro approaches to the problem. Studies using cultured VSMC subjected to stretch have produced considerable amounts of information,9 10 11 12 13 14 15 but because of possible phenotypic changes,24 25 some caution must be used when interpreting these results. Stretching cells 10% to 20%, either cyclically or statically, has been shown to result in an increase in phosphoinositide turnover,13 an increase in adenylate cyclase activity,11 and proto-oncogene induction.12 13 These early signaling events are followed by increases in protein9 10 and DNA12 14 15 synthesis. The autocrine release of PDGF-AA has been implicated in this model, since antibodies to PDGF-AA significantly blocked the mitogenic effects of stretch.14 In this model, it has also been demonstrated that stretch is sensed through specific matrix/integrin interactions because treatment with RGD peptide or antibodies to β3 or αvβ5 integrins also abolished the mitogenic effects of stretch.15 Another approach taken to investigate the effects of stretch was the use of stretched whole-vessel preparations, which have the advantage over isolated cell cultures in that cell-cell and cell-matrix interactions are maintained. Stretched vessels showed increased phospholipase C activity19 and DNA16 18 20 and protein17 18 synthesis, showing that stretch is also a stimulus for growth in whole vessels.
The stimulus used in these whole vessels and cultured smooth muscle cells (ie, cell stretch) may not be the same as the stimulus occurring in hypertension. In vivo, vessels maintain tone and the VSMC are embedded in an extracellular matrix where they are unlikely to experience an increase in cell length with developing hypertension. Both cultured cells and cells in situ, however, are anchored through their integrins, making it possible that the mechanotransduction pathway has similar features whether the cell is stretched or responds to an increase in pressure, since both will experience an increase in stress conducted through the adhesion molecules.26 Indeed, in isolated arteries, an increase in intraluminal pressure results in the production of second messengers inositol 1,4,5-trisphosphate and 1,2-diacylglycerol21 and also protein synthesis.27 We have shown previously that in mesenteric arteries subjected to high pressures, proto-oncogene and 18S rRNA expression are specifically increased,23 demonstrating that these vessels are able to sense the increase in pressure and elicit the signaling pathways that immediately precede a growth response.28 Those vessels were largely passive, thereby subjecting the high-pressure vessels to a small degree of circumferential stretch (<3%). This amount is much smaller than is typically applied in cell culture (10% to 20%), but it may have contributed to signal transduction. In addition to this increase in cell stretch, there was also a significant increase in wall stress (60% to 80%) in the high-pressure vessels,23 which may been the stimulus for growth.
The present study was an attempt to differentiate between cell stretch and wall stress more completely in the same isolated pressurized mesenteric artery preparation. In these experiments, many of the vessels showed some degree of myogenic constriction as the pressure was increased, resulting in a decrease in vessel diameter. At high pressures (140 or 165 mm Hg), vessels showed a range of myogenic constriction such that the resulting wall stresses ranged from equal to approximately double those seen in controls (90 mm Hg). On the basis of results of our earlier study,23 we selected the 3-hour time point at which increased expression of both c-myc and the very abundant 18S rRNA could be detected. Phosphorimages showed that gene expression was uniform throughout the media of the arteries, and this was confirmed by autoradiography,23 but detection with the phosphorimager does not rule out the possibility that expression also occurs in endothelial cells, adventitia, or any neural tissue in the vascular wall. In the present experiments, the induction of c-myc and 18S rRNA was attenuated in mesenteric arteries showing myogenic responses in which vessel wall stresses were normalized (Figs 2B⇑ and 3B⇑). The stimulus for proto-oncogene and rRNA expression was therefore most likely the increase in wall stress, since this correlated significantly with gene expression. Cell stretch would not appear to be important in this model because there was no correlation between normalized vessel diameter, ie, cell stretch, and gene expression (Fig 2C⇑ and 3C⇑). Increased gene expression was observed in constricted vessels where the cells were shorter than those in control vessels, but wall stress was elevated by an increase in the pressure. This also shows that the myogenic response did not directly reduce gene expression through a signaling pathway but only by virtue of the reduced wall stress.
These results therefore suggest that wall stress and not cell stretch may be the important factor sensed by the vasculature during hypertensive disease. The finding that vessels with myogenic responses and normalized wall stresses do not show increased gene expression may explain why the small arterioles in the hypertensive vasculature respond differently than the arteries. Large arteries in the hypertensive vasculature undergo hypertrophy, leading to an increased media to lumen ratio.3 29 In contrast, the arterioles (<100 μm) that show the largest myogenic constrictions are therefore the most likely to normalize wall stress by lumen reduction during hypertension. This is accomplished first by active vasoconstriction followed by structural changes, ie, inward eutrophic remodeling.6 7 In between are the small arteries (100 to 300 μm) that develop some tone and undergo a combination of hypertrophy and lumen reduction.2 30 These findings suggest that arteries regulate wall stress through a gradient of responses, with hypertrophy decreasing from large arteries to the smallest and lumen reduction varying in the reverse direction. The hypertrophic response varies inversely with the myogenic ability of the vessel. In the terminal arterioles, where pressure has been normalized by upstream resistance, there appears to be little difference between hypertensive and normotensive vessels.6 7 31 32
Although constriction of an artery may inhibit the growth response as measured by expression of early response genes, it is possible that maintained constriction initiates another signaling pathway with different end points. An isolated VSMC constricts in a corkscrewlike fashion33 because of the arrangement of the actin filaments and focal adhesion sites. In the vascular wall, such a constriction reduces circumferential wall stress, but it may result in tangential shearing stress between focal adhesion sites and the matrix. Focal adhesion sites on endothelial cells undergo continuous remodeling, which becomes directed in the line of flow when the cells are exposed to a shear stress.34 A similar realignment of adhesion sites on VSMC may be part of the remodeling process of the arteriolar wall whereby the lumen becomes structurally reduced without hypertrophy.
The isolated mesenteric vessel preparation therefore appears to be an advantageous model for investigating early mechanotransduction events. This model may complement cell stretch experiments because both cell stretch and increased wall stress, with and without cell stretch, most likely result in an increase in stress in the cytoskeleton and focal adhesions. The integrins and associated molecules, particularly protein kinases and mediators of phospholipid metabolism,35 may therefore play an important initial role in mechanotransduction events, as has been suggested by Wilson et al.15
Selected Abbreviations and Acronyms
|PDGF||=||platelet-derived growth factor|
|SHR||=||spontaneously hypertensive rat(s)|
|VSMC||=||vascular smooth muscle cells|
This study was supported in part by National Institutes of Health grants HL-36551 and HL-54810 and the Thomas F. Jeffress and Kate Miller Jeffress Memorial Trust. Dr Allen is the recipient of a Fellowship from the American Heart Association, Virginia Affiliate.
- Received June 21, 1996.
- Revision received July 18, 1996.
- Accepted January 21, 1997.
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