Hypoxic Contraction of Cultured Pulmonary Vascular Smooth Muscle Cells Thomas R. Murray, Linda Chen, Bryan E. Marshall, and Edward J. Macarak Center for Research in Anesthesia, School of Medicine, and Department of Anatomy and Histology, School of Dental Medicine, University of Pennsylvania; Anesthesia Department, The Graduate Hospital; and Connective Tissue Research Center, University City Science Center, Philadelphia, Pennsylvania
The cellular events involved in generating the hypoxic pulmonary vasoconstriction response are not clearly understood, in part because of the multitude of factors that alter pulmonary vascular tone. The goal of the present studies was to determine if a cell culture preparation containing vascular smooth muscle (VSM) cells could be made to contract when exposed to a hypoxic atmosphere. Cultures containing only fetal bovine pulmonary artery VSM cells were assessed for contractile responses to hypoxic stimuli by two methods. In the first, tension forces generated by cells grown on a flexible growth surface (polymerized polydimethyl siloxane) were manifested as wrinkles and distortions of the surface under the cells. Wrinkling of the surface was noted to progressively increase with time as the culture medium bathing the cells was made hypoxic (Po 2 ::::: 25 mmHg). The changes were sometimes reversible upon return to normoxic conditions and appeared to be enhanced in cells already exhibiting evidence of some baseline tone. Repeated passage in culture did not diminish the hypoxic response. Evidence for contractile responses to hypoxia was also obtained from measurements of myosin light chain (MLC) phosphorylation. Conversion of MLC to the phosphorylated species is an early step in the activation of smooth muscle contraction. Lowering the Po2 in the culture medium to 59 mmHg caused a 45 % increase in the proportion ofMLC in the phosphorylated form as determined by two-dimensional gel electrophoresis. Similarly, cultures preincubated for 4 h with 32p and then exposed to normoxia or hypoxia for a 5-min experimental period showed more than twice as much of the label in MLCs of the hypoxic cells. VSM cell cultures were shown to be devoid of endothelium by negative immunohistochemical staining for the endothelialderived von Willebrand factor. These results demonstrate that cultured pulmonary artery VSM cells exhibit a contractile response to hypoxia in cell culture and that the response is not dependent on any nonmuscle cells.
Decreases in oxygen tension in the lungs cause rises in pulmonary arterial pressure and changes in the distribution of blood flow, a process known as hypoxic pulmonary vasoconstriction (HPV) (1-3). It is a prominent mechanism for the active regulation of regional blood flow in the lungs and an important control mechanism for matching ventilation and perfusion in diseased (4) and atelectatic lungs (5). Despite extensive investigation in this area, it has neither been established how hypoxia is sensed in the lung nor how this information is used to increased pulmonary vascular tone and alter regional blood flow. It has been postulated that diffusible (Received in original form February 15, 1990 and in revised form May 22, 1990) Address correspondence to: Thomas R. Murray, M.D., Ph.D., Connective Tissue Research Institute, 3624 Market Street, Philadelphia, PA 19104. Abbreviations: endothelial-derived relaxing factor, EDRF; fetal bovine serum, PBS; hypoxic pulmonary vasoconstriction, HPV; Medium 199, MI99; myosin light chain, MLC; pulmonary artery, PA; vascular smooth muscle, VSM. Am. J. Respir. Cell Mol. BioI. Vol. 3. pp, 457-465, 1990
mediator substances released by nonmuscle cells cause the increased pulmonary vascular smooth muscle (VSM) tone (2, 3, 6), but this has not been proved. Efforts to elucidate the mechanism underlying the hypoxic pressor response have made it apparent that pulmonary vascular tone and HPV are influenced by a multitude of factors. HPV in vivo is modified by both alveolar and mixed venous Po 2 and Pco., and by pH, cardiac output, temperature, age, sex, and preexisting hypertensive disease (2,3, 7). Also, diverse stimuli including hyperinflation, increases in vascular sheer stress, endotoxins, and embolization promote the synthesis and release of vasoactive compounds by the lung (8, 9). Failure to recognize and control for these varied factors appropriately accounts for many of the controversies and inconsistencies found in research reports on this subject (10). Several in vitro models have been developed in an attempt to reduce the variables in the experimental preparations, including isolated perfused lungs and pulmonary vascular muscle strips or vessel loops. These approaches have proved to be fruitful in providing better control over many of the fac-
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tors influencing vascular tone and they have yielded significant insights into the HPV process. However, the presence of nonmuscle cells and diffusion barriers in these preparations still complicates the study of the effects of hypoxia on VSM cells. In particular, the interaction between pulmonary vascular endothelium and smooth muscle cells is of concern in light of the ability of endothelium to release both vasoconstricting and vasodilating substances (11, 12). Evidence both for and against the endothelium playing an obligatory role in hypoxic vasoconstriction has been provided by experimentswith pulmonary vascular strips and loops. Holden and McCall concluded that hypoxia-induced contractions of porcine pulmonary arteries were largely dependent on the presence of an intact endothelium (13). Conversely, BurkeWolin and Wolin (14) and Hottenstein and coworkers (15) observed hypoxic contractile responses in the absence of endothelium. Others have reported a 48 to 80 % reduction of maximal hypoxic contraction of pulmonary artery (PA) vessel loops following removal of endothelium (16). Likewise, DeMay and Vanhoutte (17) noted a decreased but not absent anoxic facilitation of PA vascular loop contraction in response to norepinephrine after endothelial removal. Explanations for the variability in attempts to define the role of endothelium result from differences in the source of the experimental tissue and in its preparation. The methods for removing endothelium have the potential to alter vascular tone by virtue of their invasive nature. It has been suggested that vasoactive compounds are liberated by the tissue during surgery and manipulation of the lungs that could alter subsequent pressor responses to hypoxia (18-20). We therefore undertook efforts to develop a cell culture system in which the responses of VSM cells to hypoxia could readily be studied in either the presence or absence of endothelium. Recent results from our laboratory demonstrated that isolated VSM cells could be made to display appropriate responses to vasoconstrictor and vasodilator substances in long-term culture (21). The goals of the present studies were to determine if: (1) VSM cells in culture could be made to contract to hypoxia and (2) if there is an obligatory role for any nonmuscle cells in the response.
Materials and Methods Cell Culture Cultures of PA smooth cells were isolated and maintained as previously described (21). Vessels (main PA and first branch PA) from second- or third-trimester fetal calves were obtained fresh from a local abattoir (Moyer Packing Co., Souderton, PA) immediately after killing. Strips of tissue from the medial portion of the vessel wall (which contains only smooth muscle cells [22]) were isolated by dissection under aseptic conditions. Single VSM cells were liberated from the muscle strips by first mincing the tissue into 1-mm pieces and then digesting the extracellular matrix using proteolytic enzymes (23). The resulting cell suspension was washed with Medium 199 (M199; Hazelton, Lenexa, KS) and seeded into culture vessels. Cells were fed twice weekly with M199 supplemented with 10% fetal bovine serum (FBS) (Sigma Chemical Co., St. Louis, MO), L-glutamate (0.29 glliter; Sigma), glucose (3 g/liter; Sigma), bactopeptone (0.5 glliter; Difco, Detroit, MI), BME amino acids (Hazelton), BME vitamins
(Flow Laboratories, McLean, VA), and Hepes (15 mM; Research Organics, Cleveland, OH). Cells were passaged at weekly intervals and maintained in a humidified atmosphere of 5 % CO 2 in air at 37 C. Endothelial cells from pulmonary arteries were isolated and cloned using modifications of previously published methods (24). The intimal surface of fetal bovine PAs were scraped lightly with a scalpel blade. Cells collected on the blade by a single pass over each area of the vessel wall were washed and resuspended in M199 supplemented with thymidine (10-6 M), Hepes (15 mM), and 17% FBS. After cells were allowed to attach overnight, cultures were washed once and refed with M199 supplemented as above and also containing a 1:1 dilution of "conditioned medium." The conditioned medium was prepared by incubating confluent cultures of fetal bovine PA VSM cells with supplemented serumless medium for 2 to 3 d. Primary cultures of endothelium were allowed to proliferate for 5 to 7 d and then treated with 0.03% trypsin-0.05 % ethylenediaminetetraacetic acid (EDTA) in Tyrode's solution at pH 7.4. Cells were washed with serumless medium and a suspension containing an average of one endothelial cell per 200-,.d drop of culture medium was then made. Ninety-six well culture plates were seeded with 1 drop/well of the suspension. Culture medium was added and cells were allowed to proliferate. Clones having the typical cobblestone appearance of endothelium were progressively transferred to larger culture vessels. Further characterization of clones as endothelial cells was obtained from immunohistochemical staining for the presence of von Willebrand factor (25). 0
Monitoring Cellular Contraction Contraction of cultured PAVSM cells in response to hypoxia was evaluated using a modification of the polymerized polydimethyl siloxane technique of Harris and colleagues (26) as previously described (21). A thin layer of the silicone fluid (30,000 centipoise; Hopkins & Williams, Essex, UK) was spread on pieces of microscope coverslips. These were then inverted and briefly heated over a low Bunsen burner flame. The heating cross-linked the outermost surface and formed a polymerized "skin" overlying the non-cross-linked fluid below (which then served as a lubricant and allowed the surface to move independently of the coverslip). Tension forces exerted by the cells attached to this thin rubber "skin" caused wrinkles in the surface, which were followed by phase-contrast photomicroscopy. Coated coverslips were placed on the bottom of T 25 culture flasks and seeded with VSM cells (5 to 20 X 103 cells/em') and grown as described above. T25 flasks were chosen because the opening could readily be sealed by placing a rubber stopper in the neck of the flask. Two 19-9auge needles were inserted through the rubber stopper to serve as inflow and outflow ports for the gases. A 7-cm length of silastic tubing was attached to the portion of the inflow needle protruding through the stopper so that gas flow through the needle could be introduced into the flask at the end opposite to the exit port, facilitating more rapid equilibration of the infused gas. Cultures of cells on silicone rubber were exposed to either normoxia (95 % air/5 % CO2) or hypoxia for varying lengths of time while on the heated (37 0 C) micro-
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Figure 1. Contractile response of PA VSM cells to hypoxia. Cells from primary cultures of VSM cells were passaged onto microscope coverslips coated with polymerized polydimethyl siloxane. Photomicrographs were taken before (a), 5 min after (b), and 15 min after (c) starting to expose cells to a hypoxic atmosphere. Tension forces generated by the cells are manifested as growth surface wrinkles that are progressively increased by the hypoxic stimulation. Bar = 50 JLffi.
scope stage, and responses to changes in oxygen tension were then photographed. Gas flow rate was approximately 0.65 liters/min, and the total flask volume was 55 ml, of which 5 rnl was occupied by the culture media. The use of 95 % N2/5% CO2 gas mixtures for the hypoxic condition delivered as described above resulted in Po.s in the culture medium of 20 to 30 mmHg. These were determined by oxygen measurements using a Corning blood gas analyzer (model 168) on aliquots of medium drawn from cultures. Oxygen tension in the culture medium bathing the cells did not drop below 20 mmHg, suggesting that a small amount of oxygen was able to diffuse back into the culture vessels. Myosin Light Chain (MLC) Phosphorylation Culture medium from confluent 100-rom cultures of PA VSM cells was removed and replaced with 5 ml of serumless medium. Lids from the standard plastic culture dishes were replaced by modified glass lids. Three glass nipples were incorporated into these lids, which served as ports for inflow and outflow of gases and for sampling culture medium. Cultures were then exposed to normoxia or hypoxia (as indicated) by flowing appropriate combinations of 5 % CO2, O2, and balance N2 through the sealed culture dishes at 2 liters/min for 5 min. Oxygen levels in the gas phase were monitored by mass spectroscopy (Perkin-Elmer Norwalk, CT). Fluid-phase oxygen tensions were determined by drawing samples of medium from each dish into glass syringes and measured using a blood-gas analyzer. Incubation periods were halted, and afterwards the medium was sampled and cultures were rapidly frozen and harvested (21). Two-dimensional gel electrophoresis of sonicated aliquots of cellular homogenates was performed using the modified method of O'Farrell (27) as previously described (21). Isoelectric focusing in the first dimension followed by sodium dodecyl sulfate-polyacrylamide (12%) gel elec-
trophoresis in the second dimension was used to isolate phosphorylated and unphosphorylated forms of VSM MLCs. The two-dimensional gels were stained with Coomassie blue (Sigma) and then scanned on an LKB laser densitometer to quantitate the proportion of the total amount of MLC that was in the phosphorylated form.
32p Incorporation into MLCs Culture medium was removed from confluent T25 flasks of VSM cells and 2 ml of serum-free M199 containing 50 jtCi 32p was added. Cultures were then preincubated for 4 h at 37° C in the presence of 32p prior to a 5-min experimental period during which cells were exposed to either normoxia (Po 2 = 145 mmHg) or hypoxia (Po 2 ::::: 25 mmHg). Cultures were rapidly frozen, and aliquots of cellular homogenates containing equivalent amounts of protein were subjected to two-dimensional gel electrophoresis to isolate MLCs. Gels were then stained with Coomassie blue and dried, and autoradiographs were made that indicated which proteins had incorporated the 32P. Portions of the gels containing the MLCs were excised, solubilized in 30 % H20 2, and placed in scintillant for determination of the amount of incorporated 32P. Phosphorylation of MLCs in normoxic versus hypoxic cells was assessed by comparing the amount of radiolabeled phosphorous incorporated into the MLC bands.
Results Hypoxia-induced Contraction of PA VSM Cells Documentation of PA VSM cell contraction was obtained using a flexible, heat-polymerized polydimethyl siloxane growth surface such that tension forces exerted by the cells caused wrinkling and distortion of this polymerized silicone rubber "skin." Examination of cultures under standard maintenance conditions and a normoxic environment (Po 2 =
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Figure 2. Hypoxia-induced changes in VSM tension are reversible. Cells were isolated and tested for responses to hypoxia as noted in MATERIALS AND METHODS. Cells were allowed to grow to a higher density than those in Figure 1. Photographs were taken before (a) and 10 min after (b) starting to make the culture medium hypoxic. Much of the increased tension noted during the hypoxic period (b) is reversed during a subsequent 17-min period of normoxic gas flow (c). Bar = 50 ILm.
145 mmHg) revealed a small degree of baseline wrinkling, indicating that the PA VSM cells had some resting tone in the unstimulated state (Figure la). Exposure to hypoxia (Paz::::: 25 mmHg) for 5 (Figure lb) or 15 min (Figure lc) caused progressive increases in cell tone reflected by changes in wrinkling noted under these same cells. Hypoxic contractions of this type were seen in 16 of 17 cultures examined. Also, the capacity to contract in response to hypoxia was found to be well-maintained through several passages in culture. Cells that had been passaged up to 5 times at weekly intervals continued to demonstrate hypoxic contractile responses indistinguishable from those of primary cultures. Oxygen tension in the cell culture flasks was lowered by flowing oxygen-deficient gas mixtures through the flasks. The wrinkling was not artifactually due to effects of the gas flow because cultures exposed to comparable flow rates of normoxic gas for 10 min showed no change in tension. However, when these same cultures were subsequently switched to a hypoxic atmosphere, a marked increase in wrinklng occurred (data not shown). The changes in tension brought about by hypoxia could sometimes be readily reversed simply by returning the cells to a normoxic environment (relaxation of hypoxic contraction upon return to normoxia was seen in six of 13 cultures examined). Figure 2a shows a rather densely populated culture surface in which some resting tone is apparent. Increased distortion of the growth surface was noted within 2 min of beginning the flow of hypoxic gas into the culture flask. The wrinkling of the surface progressively increased with time and was quite prominent by 10 min of hypoxic gas flow (Figure 2b). Replacing the hypoxic gas mixture with a
normoxic one (5% COz/95% air) brought about a return to the resting level of tension seen earlier in these cells (Figure 2c versus Figure 2a). Also apparent in this sequence is a small island of cells (upper right-hand corner) that shrinks in size with hypoxia and then relaxes with a return to normoxia. (No wrinkles are seen here because these cells are off the polymerized portion to the silicone, which ends at the edge of the confluent area below.) Exposing cells to a repeated hypoxic challenge was found to increase the intensity of the contractile response in six of 13 cultures tested. Figure 3a shows the baseline level of tension in a sparse, normoxic culture that is modestly increased by an initial hypoxic challenge (Figure 3b). Little change occurred during a subsequent normoxic period (Figure 3c). However, a second hypoxic challenge caused a marked increase in contraction of these same cells (Figure 3d). Stimulation of MLC Phosphorylation by Hypoxia Phosphorylation of the 20,000-D light chains on the contractile protein myosin was analyzed by a two-dimensional gel electrophoresis technique that readily resolved MLCs from other cellular proteins. MLCs from normoxic cultures (Figure 4a) were predominantly in the unphosphorylated form (pI = 5.1), with only about 20% of the light chains phosphorylated. Exposure to hypoxia during the brief experimental period resulted in a marked increase in the proportion of the MLCs in the phosphoryated form (Figure 4b). Quantification of these hypoxia-induced changes in the phosphorylation ofMLCs was obtained from densitometric scans of the stained two-dimensional gels. Evaluation of VSM cultures exposed to a Paz of 145 mmHg during a 6-min study
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Figure 3. Effect of repeated hypoxic challenges on PA VSM cells. Cells were photographed before (a) and after (b) exposure to hypoxic gas flows for 25 min. Normoxic gas was then infused for 11 min (c) followed by a second hypoxic challenge for 17 min (d). Arrowheads indicate areas where changes in tension were most prominent. Bar = 20 JLm.
period revealed that 22 % of the MLCs were in the phosphorylated form under these conditions (Table 1). This is comparable to the level of phosphorylation of MLCs seen in resting cultures of PAVSM cells under standard growth conditions in our laboratory (21). Exposure to hypoxia during the experimental period brought about significant increases (45%) in the amount of phosphorylated MLCs.
32p Incorporation into MLCs Autoradiographs of two-dimensional gels illustrated the baseline level of 32p incorporation into the MLCs of cultures first preincubated with the label and then exposed to normoxia during the 5-min experimental period (data not shown). A marked increase in 32p incorporation into MLCs was observed when cultures were exposed to hypoxia during the 5-min experimental period prior to harvesting and this was readily apparent as darker MLC bands on autoradiographs. Quantitative analysis of these changes revealed that hypoxia caused the amount of 32p incorporated into the MLCs to more than double (Table 2). Immunofluorescence Staining for von Willebrand Factor The hypoxia-induced contraction studies described herein
were conducted on cultures of VSM cells isolated from the medial layer of pulmonary arteries. The absence of endothelial cell contamination of these VSM preparations was confirmed using an immunohistochemical technique (25) to detect the presence of the endothelial-derived protein von Willebrand factor. Endothelial cells in culture continue to synthesize and store von Willebrand factor, whereas neither VSM cells nor other vessel wall cells produce this protein. A primary rabbit antibody to human von Willebrand factor was first reacted with the cells, followed by incubation with rhodamine-conjugated anti-rabbit IgG antibody. Endothelial cells show the typical punctate staining appearance as expected (Figure 5a). Conversely, endothelial cells exposed only to the conjugated anti-IgG antibody (and not the antivon Willebrand antibody) lack the specific staining pattern (Figure 5c). The smooth muscle cell preparation (Figure 5e) treated with both antibodies (like the cells in Figure 5a) revealed only nonspecific background fluorescence just as the endothelium exposed only to the second antibody (Figure 5c). Thus, the absence of any cells with the specific binding pattern of endothelium indicated that the VSM cell studies were conducted in cultures free from the influence of endothelium.
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TABLE 2
Incorporation of 32P into myosin light chains Condition
32p Incorporated*
Normoxia Hypoxia
1,729 3,821
± 607
± 693
* Numbers represent mean counts per minute per milligram of cellular protein ± SD for n = 3. The difference between conditions was statistically significant at the P = 0.02 level using Student's t test.
Figure 4. Hypoxia-induced increases in MLC phosphorylation. Confluent cultures of PA VSM cells were exposed to normoxic (a) or hypoxic (b) atmospheres during a 6-min experimental period prior to harvesting. Homogenates of the cultures were subjected to two-dimensional electrophoresis. Isoelectric focusing (pH 4.0 to 6.5) was followed by electrophoresis in 12 % polyacrylamide gels in the second dimension. Arrows indicate the bands corresponding to the MLC protein in the unphosphorylated (D) and phosphorylated (P) forms.
Discussion It is apparent in observations from several investigators that the strength of the hypoxic pressor response is modified and modulated by the complex interaction of numerous variables
that affect pulmonary vascular tone. In order to understand these interactions at the cellular and ultimately at the molecular level, it has been advantageous to reduce the complexity of the systems being analyzed. As a result, there has been a steady progression in refinement in experimental systems aimed at simplifying the preparation to the essential components (28-30) while still maintaining the basic aspects of the mechanism being studied. The present studies represent the beginnings of an attempt to extend this progression to the cellular level. A previously described model system of cultured smooth muscle cells that demonstrated appropriate responses to a variety of vasoactive agents in long-term culture was employed here (21). Results demonstrate that isolated PAVSM cells contract when challenged with hypoxic stimuli and do so in the absence of any other cell types. Hypoxic contraction was documented through two distinct lines of evidence. First, hypoxia-induced increases in tension forces generated by the VSM cells resulted in wrinkling and distortion of a flexible growth surface, which was followed by photomicroscopy. The results of these studies were corroborated and complemented by measurements of increases in MLC phosphorylation, a biochemical marker for activation of VSM cell contraction (31). The cross-linked polydimethyl siloxane growth surface provides a very sensitive indicator of cellular traction forces and is capable of detecting small changes in tension generated by the cultured cells (26). It was employed here to demonstrate that hypoxia stimulates increases in smooth muscle cell tone and that these changes are sometimes reversible with a return to normoxic atmospheres. Further, the hypoxic contractile response was shown to be maintained through several passages in culture without any apparent diminution during prolonged culture periods (analogous to the preservation of responsiveness to pharmacologic agonists of contraction in long-term culture reported previously for this system [21]). The difficulty with quantifying the response using this technique led us to choose measurements of MLC phosphorylation as a means to corroborate these findings and to provide a more quantitative assessment of the changes.
TABLE 1
Hypoxia-induced increases in MLC phosphorylation
Normoxia Hypoxia-l Hypoxia-2
Oxygen in Gas Phase
P02 of Culture Medium
(%)
(mmHg)
20
145 59 25
6
o
* Statistically significant at P < 0.02
Percentage of MLC Phosphorylated (± SD [nD
22 32 32
compared with nonnoxic condition in Student's t test.
± 5 (6) ± 6 (5)* ± 5 (5)*
Percent Increase versus Normoxic Control
o 45 45
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Figure 5. Immunohistochemical staining for von Willebrand factor in PA VSM and endothelial cell cultures. Cultures of PA VSM and endothelial cells were isolated independently and maintained as described in MATERIALS AND METHODS. Endothelial clones treated first with rabbit IgG antibodies to von Willebrand factor and then rhodamine-labeled goat anti-rabbit IgG show a punctate staining appearance (a). Light (b) and fluorescent (c) photographs of endothelial cells treated only with the second antibody show background, nonspecific staining, which is not related to von Willebrand factor. A VSM culture representative of those used in the contraction studies that was treated with both antibodies is shown under light (d) and fluorescent illumination (e). Bar = 50 JLm.
Conversion of the light chains of myosin from an unphosphorylated to a phosphorylated form facilitates the interaction of actin with myosin, which leads to VSM contraction. It has been shown to correlate well with initial force development for several smooth muscle preparations (31). Brief exposure of PA VSM to moderate hypoxia (Po, = 59 mmHg) is sufficient to activate this conversion and leads to significant increases over baseline levels in the proportion of
the MLCs that are in the phosphorylated form. Decreasing O2 in the culture media from 59 to 25 mmHg did not cause significant increases in phosphorylaton. It is not known if further decreases in Po, of the media bathing the cells would cause higher levels ofMLC phosphorylation. Oxygen partial pressure in the medium was lowered by flowing hypoxic gas through the space above the thin layer of media bathing the cells in covered culture dishes. Po, measured in the
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liquid phase dropped quickly during the first 60 to 75 s after hypoxic gas flow was begun, but then plateaued and stayed relatively constant for the remainder of the 5- or 6-min hypoxic periods. Oxygen levels in the culture media overlying the cells consistently remained higher than those of the gas phase and only decreased to an average Po2 of 25 mmHg when gas containing 95 % N 2/5 % CO 2 was employed. Thus, some modification of the hypoxic chamber will be needed in order to assess the responses of isolated VSM to more severe levels of hypoxia. The two-dimensional electrophoresis gels of MLCs revealed about 22 % of the light chains to be in the phosphorylated form under normoxic conditions. This is comparable to the levels reported earlier for unstimulated PA VSM cells (21) The relatively high levels of basal phosphorylation in cultured cells seen here and by others (32) compared with that of arterial muscle strips (33) is likely a reflection of basal tone in VSM cells in culture where they are attached to a growth surface. Consistent with this concept are the findings by Anderson and associates (32), who noted that cultures of mesenteric artery VSM cells had significant amounts of basal phosphorylation of MLCs that they were able to markedly reduce by treating cultures with dibutyryl cyclic AMP or chlorpromazine, agents that can cause vasodilation. The 32p incorporation studies and the presence of wrinkles under some unstimulated cells on the polymerized polydimethyl siloxane are also consistent with the presence of basal tone. In the former case, there is some 32p incorporated into the light chains of the control cultures not stimulated to contract with hypoxia (Table 2). Cross-bridge cycling between actin and myosin in cells with basal tone is a likely explanation for much of the 32p incorporation. Under conditions of constant stress, there continues to be a steady turnover of actin-myosin cross-bridges and MLCs undergo associated phosphorylation/dephosphorylation reactions (34). 32p taken up into the VSM cells during the 4-h pre-incubation used for these experiments could readily have exchanged with the unlabeled pool of intracellular phosphate and become attached to MLCs during turnover of the phosphorylated species of the protein. The response to hypoxia in individual cells is somewhat variable. .Photomicrographs of cells on the polymerized polydimethyl siloxane demonstrate that not all cells contract when challenged. Typically, the best responses were seen in areas of the cultures where there was already some baseline distortion of the growth surface prior to testing with hypoxia. The reason for this is uncertain at present, but it suggests that the response may be affected by the basal tone of the muscle. It has been shown with PA vessel loops and isolated lung preparations that hypoxic contractions are potentiated by prestimulating the tissue with various vasoconstrictors (16, 28,35). Rodman and colleagues (16) have suggested that a "threshold synergy" (36) between hypoxia and other vasoactive agents may be responsible for the potentiation of hypoxic contraction. This concept was based on observations by Stupecky and coworkers (36), who suggested that addition of threshold concentrations of an agonist converted resting muscle to an activated state that resulted in greater than additive contraction upon addition of a second agonist. Our observations with the flexible growth surface, while not designed to test this, are consistent with the proposal that
hypoxia acts as a more potent stimuli for VSM contraction in cells with elevated basal tone. The concept of an "oxygen sensor" has been discussed in recent reviews of HPV and a variety of different locations in the lungs have been proposed for it (2, 3, 37). However, the anatomic site and mechanism by which hypoxia is sensed and coupled to VSM cell excitation and contraction remain to be identified. Studies by Marshall and Marshall (38) with isolated rat lungs in which alveolar and mixed venous oxygen levels were varied independently suggested that the oxygen sensor behaved as ifit were located within the medial portion of the vessel wall. A significant finding of the present work is that VSM cells isolated from the PA medial layer are able to sense hypoxia and initiate a contractile response to it in the absence of other cell types. Thus, PA VSM cells are not dependent upon nonmuscle cells to initiate a hypoxia-induced contraction, although a role for other cells in modulating basal tone and modifying the hypoxic responses seems likely. The contribution of endothelium to the HPV response has been an area of ongoing controversy. An obligatory role for them has been reported in some cases (13) but not in others (14, 15). Recently, it has been shown (16, 39) that the response to hypoxia of PA vessel loops denuded of endothelium was diminished, but not abolished, while antagonists of endothelial-derived relaxing factor (EDRF) were shown to augment the HPV response in isolated rat lung (40-42). Some of the confusion undoubtedly lies in the ability of endothelium to synthesize several factors that can cause VSM relaxation or constriction. For example, endothelia exposed to hypoxia continue to release prostacyclin, a potent vasodilator (43), as well as endothelium-derived constricting factors (6). The various conditions that lead to release of these endothelial-derived products are not all understood and therefore may not always be appropriately controlled for in the different experimental preparations used. Results presented here show hypoxia-induced contractions of isolated PA VSM cells in cultures devoid of endothelium, findings more consistent with a modulatory rather than obligatory role for endothelium. A relaxation toward baseline levels of tension in cells first contracted with hypoxia and then returned to normoxia was noted (Figure 2). However, this response time was slower than that previously reported for cultured cells treated with the smooth muscle relaxant isoproterenol (21), as well as that for vessel loops and isolated lungs (which contained intact endothelium) following termination of a hypoxic challenge. It has been suggested that the pulmonary circulation behaves like the systemic circulation by generating vasodilatory prostaglandins in response to vasoconstrictive stimuli and these act to attenuate the constriction (44). Endothelia are known to be the source of multiple vasodilatory factors, including prostacyclin and EDRF. Further, hypoxia promotes increased synthesis of prostacyclin from animal lungs (45, 46) and prostacyc1in continues to be produced by cultured bovine jlA endothelial cells during hypoxic stimulation (43). Inhibitors of these compounds have been shown to augment the HPV response in some in vitro (28, 40) and in vivo (47) preparations. It is not clear at this time what role if any the absence of endothelium may play in the somewhat slower return toward basal tone seen in our cultures.
Murray, Chen, Marshall et al.: Hypoxic Contraction of Cultured Vascular Smooth Muscle Cells
In summary, the present work has demonstrated hypoxic contraction of isolated pulmonary artery smooth muscle cell cultures that occurs in the absence of nonmuscle cells. The applicability of these findings to the in vivo phenomenon of hypoxic pulmonary vasoconstriction remains to be established. However, if analogous, they suggest that the presence of endothelium and other lung cells in vivo serve a modulator rather than mediator role in this important physiologic response. Ackno:vledg"!ents: It is a pleasure to acknowledge Robert Rogers for his valuable technical assistance. We thank Drs. Albert K. Harris, Mike A. Clarke, Thomas M. Butler, Robert S. Moreland, Stephen F. Gorfien, and Pamela S. Howard for their assistance and advice and Dr. Phyllis M. Sampson for the use of her laser densitometer. A preliminary report of this work was presented at the 1988 meeting of the American Society of Anesthesiologists, San Francisco, California. This work was supported in part by grants from the Pennsylvania Thoracic Society (TRM) , the American Society of Anesthesiologists (TRM) , and Grants 5T32GM-07612 (TRM), GM-29629 (BEM), and HL-34005 (EJM) from the National Institutes of Health.
References I. von Euler, U. S., and G. Liljestrand. 1946. Observations on the pulmonary arterial blood pressure in the cat. Acta Physiol. Scand. 12:301-320. 2. Fishman, A. P. 1976. Hypoxia on pulmonary circulation: how and where it acts. Circ. Res. 38:221-231. 3. Voelkel, N. F. 1986. Mechanisms of hypoxic pulmonary vasoconstriction. Am. Rev. Respir. Dis. 133:1186-1195. 4. Hales, C. A., and D. Westphal. 1978. Hypoxemia following the administration of sublingual nitroglycerine. Am. J. Med. 65:911-917. 5. Thomas, H. M., III, and R. C. Garrett. 1982. Strength of hypoxic vasoconstriction determines shunt fraction in dogs with atelectasis. J. Appl. Physiol. 53:44-51. 6. Rubanyi, G. M., and P. M. Vanhoutte. 1985. Hypoxia releases a vasoconstrictor substance from the canine vascular endothelium. J. Physiol. 364:45-56. 7. Guazzi, M. D., M. Alimento, M. Berti, C. Fiorentini, C. Galli, and G. Tamborini. 1989. Enhanced hypoxic pulmonary vasoconstriction in hypertension. Circulation 79:337-343. 8. Voelkel, N. F., M. Morganroth, and O. C. Fedderson. 1985. Potential role of arachidonic acid metabolites in hypoxic pulmonary vasoconstriction. Chest 88:245S-248S. 9. Fishman, ~. P. 1985. Pulmonary circulation. In Handbook of Physiology. The Respiratory System I. A. P. Fishman and A. B. Fisher, editors. Waverly Press, Baltimore, MD. 93-165. 10. Marshall, B. E., and C. Marshall. 1988. A modelfor hypoxic constriction of the pulmonary circulation. J. Appl. Physiol. 64:68-77. II. O'Brien, R. F., R. J. Robbins, and I. F. McMurtry. 1987. Endothelial cells in culture produce a vasoconstrictor substance. J. Cell. Physiol. 132:263-270. 12. Furchgott, R. F. 1983. Role of endothelium in responses of vascular smooth muscle. Circ. Res. 53:557-573. 13. Holden, w. E., and E. McCall. 1984. Hypoxia-induced contractions of porcine pulmonary artery strips depends on intact endothelium. Exp. Lung Res. 7:101-112. 14. Burke-Wolin, T., and M. S. Wolin. 1989. H20 2 and cGMP may function as an O 2 sensor in the pulmonary artery. J. Appl. Physiol. 66:167-170. 15. Hottenstein, 0., W. Mitzner, and G. G. Bierkamper, 1982. Hypoxia alters membrane potentials in rat main pulmonary artery smooth muscle: a possible calcium mechanism. Physiologist 25:276. (Abstr.) 16. Rodman, D. M., T. Yamaguchi, R. F. O'Brien, and I. F. McMurtry. 1989. Hypoxic contraction of isolated rat pulmonary artery. J. Pharmacol. Exp. Ther. 248:952-959. 17. DeMay, J. G., and P. M. Vanhoutte. 1982. Heterogeneous behavior of the canine arterial and venous wall. Circ. Res. 51:439-447. 18. Grover, R. F. 1985. The fascination of the hypoxic lung. Anesthesiology 63:580-582. 19. Ishibe, Y., C. Marshall, and B. E. Marshall. 1988. Hypoxic pulmonary vasoconstriction inhibited by lung manipulation in rabbits. Anesthesiology 69:A139. (Abstr.) 20. Shams, H., H. Schulz, M. Mohr et al. 1989. Cyclooxygenase inhibition and effects of hypoxia on pulmonary circulation and gas exchange in
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anesthetized dogs. Respir. Physiol. 75:39-50. Murray, T. R., B. E. Marshall, and E. J. Macarak. 1990. Contraction of vascular smooth muscle in cell culture. J. Cell. Physiol. 143:26-38. 22. ~ease, D. C., a~d W. J. Paule. 1960. Electron microscopy of elastic arteries; the thoracic aorta of the rat. J. Ultrast. Res. 3:469-483. 23. Van Dijk, A. M., and J. D. Laird. 1984. Characterization of single isolated vascular smooth muscle cells from bovine coronary artery. Blood Vessels 21:267-278. 24. Macarak, E. J. 1984. Collagen synthesis by cloned pulmonary artery endothelial cells. J. Cell. Physiol. 119:175-182. 25. Wagner, D. D., J. B. Olmsted, and V. J. Marder. 1982. Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human endothelial cells. J. Cell Biol. 95:355-360. 26. Harris, A: K., ~. Wild, and D. Stopic. 1980. Silicon rubber substrata; a new wnnkle m the study of cell locomotion. Science 208:177-179. 27. O'Farrell, P. H. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 26:14740-14745. 28. McMurtry, I. F. 1984. Angiotensin is not required for hypoxic constriction in salt solution-perfused rat lungs. J. Appl. Physiol. 5.6:375-380. 29. Marshall, C. 1984. A method for perfusing and ventilating rat lungs in vitro. Methods Find. Exp. Clin. Pharmacol. 6:281-285. 30. Harder, D. R., J. A. Madden, and C. Dawson. 1985. Hypoxic induction of Ca + + -dependent action potentials in small pulmonary arteries of the cat. J. Appl. Physiol. 59: 1389-1393. 31. Silver, P. J. 1986. Pharmacological modulation of cardiac and vascular contractile protein function. J. Cardiovascular Pharmacol. 8(Suppl. 9):S34-S46. 32. Anderson, J. M., M. A. Gimbrone, and R. W. Alexander. 1981. Angiotensin II stimulates phosphorylation of the myosin light chain in cultured vascular smooth muscle cells. J. Biol. Chem. 256:4693-4696. 33. Driska, S. P., M. O. Aksoy, andR. A. Murphy. 1981. Myosin light chain phosphorylation associated with contraction in arterial smooth muscle. Am. J. Physiol. 240:C222-C233. 34. Hai, C.-M., and R. A. Murphy. 1988. Cross-bridge phosphorylation and regulation of latch state in smooth muscle. Am. J. Physiol. 254: C99-CI06. 35. He~get, J., and ~. ~cMurtry ..1987. Dexamethasone potentiates hypOXIC vasoconstriction m salt solution-perfused rat lungs. Am. J. Physiol. 253:H574-H581. 36. Stupecky, G. L., D. L. Murray, and R. E. Purdy. 1986. Vasoconstrictor threshold synergism and potentiation in the thoracic aorta. J. Pharmacol. Exp. Ther. 238:801-808. 37. Hales, C. A. 1985. The site and mechanism of oxygen sensing for the pulmonary vessels. Chest 88:S235-S24O. 38. ~arshall, C., a~d B. E. Marshall. 1983. Site and sensitivity for stimulation of hypOXIC pulmonary vasoconstriction. J. Appl. Physiol. 55: 711-716. 39. Johns, R. A., J. M. Linden, and M. J. Peach. 1989. Endotheliumdependent relaxation and cyclic GMP accumulation in rabbit pulmonary artery are selectively impaired by moderate hypoxia. Circ. Res. 65: 1508-1515. L., M. J. Peach, and C. E. Rose, Jr. 1988. Augmentation 40. Brashers, of ~y~XlC pulmon~ry vasoconstri~tion in the isolated perfused rat lung by In vuro antagomsts of endothelium-dependent relaxation. J. Clin. Invest. 82:1495-1502. 41. Archer, S. L., J. P. Tolins, L. Raij, and E. K. Weir. 1989. Hypoxic pulmonary vasoconstriction is enhanced by inhibition of the synthesis of an endothelium-derived relaxing factor. Biochem. Biophys. Res. Commun. 164:1198-1205. 42. Mazatl?anian, G.-M., B. Baudet, C. Brink et al. 1989. Methylene blue potentiates vascular reactivity in isolated rat lungs. J. Appl. Physiol. 66: 1040-1045. 43. Madden, M. C., R. L. Vender, and M. Friedman. 1986. Effect of hypoxia on prostacyclin production in cultured pulmonary artery endothelium. Prostaglandins 31: 1049-1062. 44. Gerber, J. G., N. Voelkel, A. S. Nies, I. F. McMurtry, and J. T. Reeves. 1980. Moderation of hypoxic vasoconstriction by infused arachidonic acid: role of P0I 2 • J. Appl. Physiol. 149:107-112. 45. Voelkel, N. F., J. G. Gerber, I. F. McMurtry, A. S. Niles, and J. T. Reeves. 1981. Release of vasodilator prostaglandin, PGI 2 , from isolated rat lung during vasoconstriction. Circ. Res. 48:207-213. 46. Hanasaki, Y., H.-H. Tai, and S. I. Said. 1982. Hypoxia stimulates prostacyclin generation by dog lung in vitro. Prostglandins Leukotrienes Med. 8:311-316. 47. Weir, E. K., I. F. McMurtry, A. Tucker, J. T. Reeves, and R. F. Grover. 1976. Prostaglandin synthetase inhibitors do not decrease hypoxic pulmonary vasoconstriction. J. Appl. Physiol. 41:714-718. 21.
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y.
The cellular events involved in generating the hypoxic pulmonary vasoconstriction response are not clearly understood, in part because of the multitude of factors that alter pulmonary vascular tone. The goal of the present studies was to determine if a cell culture preparation containing vascular smooth muscle (VSM) cells could be made to contract when exposed to a hypoxic atmosphere. Cultures containing only fetal bovine pulmonary artery VSM cells were assessed for contractile responses to hypoxic stimuli by two methods. In the first, tension forces generated by cells grown on a flexible growth surface (polymerized polydimethyl siloxane) were manifested as wrinkles and distortions of the surface under the cells. Wrinkling of the surface was noted to progressively increase with time as the culture medium bathing the cells was made hypoxic (Po 2 ::::: 25 mmHg). The changes were sometimes reversible upon return to normoxic conditions and appeared to be enhanced in cells already exhibiting evidence of some baseline tone. Repeated passage in culture did not diminish the hypoxic response. Evidence for contractile responses to hypoxia was also obtained from measurements of myosin light chain (MLC) phosphorylation. Conversion of MLC to the phosphorylated species is an early step in the activation of smooth muscle contraction. Lowering the Po2 in the culture medium to 59 mmHg caused a 45 % increase in the proportion ofMLC in the phosphorylated form as determined by two-dimensional gel electrophoresis. Similarly, cultures preincubated for 4 h with 32p and then exposed to normoxia or hypoxia for a 5-min experimental period showed more than twice as much of the label in MLCs of the hypoxic cells. VSM cell cultures were shown to be devoid of endothelium by negative immunohistochemical staining for the endothelialderived von Willebrand factor. These results demonstrate that cultured pulmonary artery VSM cells exhibit a contractile response to hypoxia in cell culture and that the response is not dependent on any nonmuscle cells.
Decreases in oxygen tension in the lungs cause rises in pulmonary arterial pressure and changes in the distribution of blood flow, a process known as hypoxic pulmonary vasoconstriction (HPV) (1-3). It is a prominent mechanism for the active regulation of regional blood flow in the lungs and an important control mechanism for matching ventilation and perfusion in diseased (4) and atelectatic lungs (5). Despite extensive investigation in this area, it has neither been established how hypoxia is sensed in the lung nor how this information is used to increased pulmonary vascular tone and alter regional blood flow. It has been postulated that diffusible (Received in original form February 15, 1990 and in revised form May 22, 1990) Address correspondence to: Thomas R. Murray, M.D., Ph.D., Connective Tissue Research Institute, 3624 Market Street, Philadelphia, PA 19104. Abbreviations: endothelial-derived relaxing factor, EDRF; fetal bovine serum, PBS; hypoxic pulmonary vasoconstriction, HPV; Medium 199, MI99; myosin light chain, MLC; pulmonary artery, PA; vascular smooth muscle, VSM. Am. J. Respir. Cell Mol. BioI. Vol. 3. pp, 457-465, 1990
mediator substances released by nonmuscle cells cause the increased pulmonary vascular smooth muscle (VSM) tone (2, 3, 6), but this has not been proved. Efforts to elucidate the mechanism underlying the hypoxic pressor response have made it apparent that pulmonary vascular tone and HPV are influenced by a multitude of factors. HPV in vivo is modified by both alveolar and mixed venous Po 2 and Pco., and by pH, cardiac output, temperature, age, sex, and preexisting hypertensive disease (2,3, 7). Also, diverse stimuli including hyperinflation, increases in vascular sheer stress, endotoxins, and embolization promote the synthesis and release of vasoactive compounds by the lung (8, 9). Failure to recognize and control for these varied factors appropriately accounts for many of the controversies and inconsistencies found in research reports on this subject (10). Several in vitro models have been developed in an attempt to reduce the variables in the experimental preparations, including isolated perfused lungs and pulmonary vascular muscle strips or vessel loops. These approaches have proved to be fruitful in providing better control over many of the fac-
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tors influencing vascular tone and they have yielded significant insights into the HPV process. However, the presence of nonmuscle cells and diffusion barriers in these preparations still complicates the study of the effects of hypoxia on VSM cells. In particular, the interaction between pulmonary vascular endothelium and smooth muscle cells is of concern in light of the ability of endothelium to release both vasoconstricting and vasodilating substances (11, 12). Evidence both for and against the endothelium playing an obligatory role in hypoxic vasoconstriction has been provided by experimentswith pulmonary vascular strips and loops. Holden and McCall concluded that hypoxia-induced contractions of porcine pulmonary arteries were largely dependent on the presence of an intact endothelium (13). Conversely, BurkeWolin and Wolin (14) and Hottenstein and coworkers (15) observed hypoxic contractile responses in the absence of endothelium. Others have reported a 48 to 80 % reduction of maximal hypoxic contraction of pulmonary artery (PA) vessel loops following removal of endothelium (16). Likewise, DeMay and Vanhoutte (17) noted a decreased but not absent anoxic facilitation of PA vascular loop contraction in response to norepinephrine after endothelial removal. Explanations for the variability in attempts to define the role of endothelium result from differences in the source of the experimental tissue and in its preparation. The methods for removing endothelium have the potential to alter vascular tone by virtue of their invasive nature. It has been suggested that vasoactive compounds are liberated by the tissue during surgery and manipulation of the lungs that could alter subsequent pressor responses to hypoxia (18-20). We therefore undertook efforts to develop a cell culture system in which the responses of VSM cells to hypoxia could readily be studied in either the presence or absence of endothelium. Recent results from our laboratory demonstrated that isolated VSM cells could be made to display appropriate responses to vasoconstrictor and vasodilator substances in long-term culture (21). The goals of the present studies were to determine if: (1) VSM cells in culture could be made to contract to hypoxia and (2) if there is an obligatory role for any nonmuscle cells in the response.
Materials and Methods Cell Culture Cultures of PA smooth cells were isolated and maintained as previously described (21). Vessels (main PA and first branch PA) from second- or third-trimester fetal calves were obtained fresh from a local abattoir (Moyer Packing Co., Souderton, PA) immediately after killing. Strips of tissue from the medial portion of the vessel wall (which contains only smooth muscle cells [22]) were isolated by dissection under aseptic conditions. Single VSM cells were liberated from the muscle strips by first mincing the tissue into 1-mm pieces and then digesting the extracellular matrix using proteolytic enzymes (23). The resulting cell suspension was washed with Medium 199 (M199; Hazelton, Lenexa, KS) and seeded into culture vessels. Cells were fed twice weekly with M199 supplemented with 10% fetal bovine serum (FBS) (Sigma Chemical Co., St. Louis, MO), L-glutamate (0.29 glliter; Sigma), glucose (3 g/liter; Sigma), bactopeptone (0.5 glliter; Difco, Detroit, MI), BME amino acids (Hazelton), BME vitamins
(Flow Laboratories, McLean, VA), and Hepes (15 mM; Research Organics, Cleveland, OH). Cells were passaged at weekly intervals and maintained in a humidified atmosphere of 5 % CO 2 in air at 37 C. Endothelial cells from pulmonary arteries were isolated and cloned using modifications of previously published methods (24). The intimal surface of fetal bovine PAs were scraped lightly with a scalpel blade. Cells collected on the blade by a single pass over each area of the vessel wall were washed and resuspended in M199 supplemented with thymidine (10-6 M), Hepes (15 mM), and 17% FBS. After cells were allowed to attach overnight, cultures were washed once and refed with M199 supplemented as above and also containing a 1:1 dilution of "conditioned medium." The conditioned medium was prepared by incubating confluent cultures of fetal bovine PA VSM cells with supplemented serumless medium for 2 to 3 d. Primary cultures of endothelium were allowed to proliferate for 5 to 7 d and then treated with 0.03% trypsin-0.05 % ethylenediaminetetraacetic acid (EDTA) in Tyrode's solution at pH 7.4. Cells were washed with serumless medium and a suspension containing an average of one endothelial cell per 200-,.d drop of culture medium was then made. Ninety-six well culture plates were seeded with 1 drop/well of the suspension. Culture medium was added and cells were allowed to proliferate. Clones having the typical cobblestone appearance of endothelium were progressively transferred to larger culture vessels. Further characterization of clones as endothelial cells was obtained from immunohistochemical staining for the presence of von Willebrand factor (25). 0
Monitoring Cellular Contraction Contraction of cultured PAVSM cells in response to hypoxia was evaluated using a modification of the polymerized polydimethyl siloxane technique of Harris and colleagues (26) as previously described (21). A thin layer of the silicone fluid (30,000 centipoise; Hopkins & Williams, Essex, UK) was spread on pieces of microscope coverslips. These were then inverted and briefly heated over a low Bunsen burner flame. The heating cross-linked the outermost surface and formed a polymerized "skin" overlying the non-cross-linked fluid below (which then served as a lubricant and allowed the surface to move independently of the coverslip). Tension forces exerted by the cells attached to this thin rubber "skin" caused wrinkles in the surface, which were followed by phase-contrast photomicroscopy. Coated coverslips were placed on the bottom of T 25 culture flasks and seeded with VSM cells (5 to 20 X 103 cells/em') and grown as described above. T25 flasks were chosen because the opening could readily be sealed by placing a rubber stopper in the neck of the flask. Two 19-9auge needles were inserted through the rubber stopper to serve as inflow and outflow ports for the gases. A 7-cm length of silastic tubing was attached to the portion of the inflow needle protruding through the stopper so that gas flow through the needle could be introduced into the flask at the end opposite to the exit port, facilitating more rapid equilibration of the infused gas. Cultures of cells on silicone rubber were exposed to either normoxia (95 % air/5 % CO2) or hypoxia for varying lengths of time while on the heated (37 0 C) micro-
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Figure 1. Contractile response of PA VSM cells to hypoxia. Cells from primary cultures of VSM cells were passaged onto microscope coverslips coated with polymerized polydimethyl siloxane. Photomicrographs were taken before (a), 5 min after (b), and 15 min after (c) starting to expose cells to a hypoxic atmosphere. Tension forces generated by the cells are manifested as growth surface wrinkles that are progressively increased by the hypoxic stimulation. Bar = 50 JLffi.
scope stage, and responses to changes in oxygen tension were then photographed. Gas flow rate was approximately 0.65 liters/min, and the total flask volume was 55 ml, of which 5 rnl was occupied by the culture media. The use of 95 % N2/5% CO2 gas mixtures for the hypoxic condition delivered as described above resulted in Po.s in the culture medium of 20 to 30 mmHg. These were determined by oxygen measurements using a Corning blood gas analyzer (model 168) on aliquots of medium drawn from cultures. Oxygen tension in the culture medium bathing the cells did not drop below 20 mmHg, suggesting that a small amount of oxygen was able to diffuse back into the culture vessels. Myosin Light Chain (MLC) Phosphorylation Culture medium from confluent 100-rom cultures of PA VSM cells was removed and replaced with 5 ml of serumless medium. Lids from the standard plastic culture dishes were replaced by modified glass lids. Three glass nipples were incorporated into these lids, which served as ports for inflow and outflow of gases and for sampling culture medium. Cultures were then exposed to normoxia or hypoxia (as indicated) by flowing appropriate combinations of 5 % CO2, O2, and balance N2 through the sealed culture dishes at 2 liters/min for 5 min. Oxygen levels in the gas phase were monitored by mass spectroscopy (Perkin-Elmer Norwalk, CT). Fluid-phase oxygen tensions were determined by drawing samples of medium from each dish into glass syringes and measured using a blood-gas analyzer. Incubation periods were halted, and afterwards the medium was sampled and cultures were rapidly frozen and harvested (21). Two-dimensional gel electrophoresis of sonicated aliquots of cellular homogenates was performed using the modified method of O'Farrell (27) as previously described (21). Isoelectric focusing in the first dimension followed by sodium dodecyl sulfate-polyacrylamide (12%) gel elec-
trophoresis in the second dimension was used to isolate phosphorylated and unphosphorylated forms of VSM MLCs. The two-dimensional gels were stained with Coomassie blue (Sigma) and then scanned on an LKB laser densitometer to quantitate the proportion of the total amount of MLC that was in the phosphorylated form.
32p Incorporation into MLCs Culture medium was removed from confluent T25 flasks of VSM cells and 2 ml of serum-free M199 containing 50 jtCi 32p was added. Cultures were then preincubated for 4 h at 37° C in the presence of 32p prior to a 5-min experimental period during which cells were exposed to either normoxia (Po 2 = 145 mmHg) or hypoxia (Po 2 ::::: 25 mmHg). Cultures were rapidly frozen, and aliquots of cellular homogenates containing equivalent amounts of protein were subjected to two-dimensional gel electrophoresis to isolate MLCs. Gels were then stained with Coomassie blue and dried, and autoradiographs were made that indicated which proteins had incorporated the 32P. Portions of the gels containing the MLCs were excised, solubilized in 30 % H20 2, and placed in scintillant for determination of the amount of incorporated 32P. Phosphorylation of MLCs in normoxic versus hypoxic cells was assessed by comparing the amount of radiolabeled phosphorous incorporated into the MLC bands.
Results Hypoxia-induced Contraction of PA VSM Cells Documentation of PA VSM cell contraction was obtained using a flexible, heat-polymerized polydimethyl siloxane growth surface such that tension forces exerted by the cells caused wrinkling and distortion of this polymerized silicone rubber "skin." Examination of cultures under standard maintenance conditions and a normoxic environment (Po 2 =
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Figure 2. Hypoxia-induced changes in VSM tension are reversible. Cells were isolated and tested for responses to hypoxia as noted in MATERIALS AND METHODS. Cells were allowed to grow to a higher density than those in Figure 1. Photographs were taken before (a) and 10 min after (b) starting to make the culture medium hypoxic. Much of the increased tension noted during the hypoxic period (b) is reversed during a subsequent 17-min period of normoxic gas flow (c). Bar = 50 ILm.
145 mmHg) revealed a small degree of baseline wrinkling, indicating that the PA VSM cells had some resting tone in the unstimulated state (Figure la). Exposure to hypoxia (Paz::::: 25 mmHg) for 5 (Figure lb) or 15 min (Figure lc) caused progressive increases in cell tone reflected by changes in wrinkling noted under these same cells. Hypoxic contractions of this type were seen in 16 of 17 cultures examined. Also, the capacity to contract in response to hypoxia was found to be well-maintained through several passages in culture. Cells that had been passaged up to 5 times at weekly intervals continued to demonstrate hypoxic contractile responses indistinguishable from those of primary cultures. Oxygen tension in the cell culture flasks was lowered by flowing oxygen-deficient gas mixtures through the flasks. The wrinkling was not artifactually due to effects of the gas flow because cultures exposed to comparable flow rates of normoxic gas for 10 min showed no change in tension. However, when these same cultures were subsequently switched to a hypoxic atmosphere, a marked increase in wrinklng occurred (data not shown). The changes in tension brought about by hypoxia could sometimes be readily reversed simply by returning the cells to a normoxic environment (relaxation of hypoxic contraction upon return to normoxia was seen in six of 13 cultures examined). Figure 2a shows a rather densely populated culture surface in which some resting tone is apparent. Increased distortion of the growth surface was noted within 2 min of beginning the flow of hypoxic gas into the culture flask. The wrinkling of the surface progressively increased with time and was quite prominent by 10 min of hypoxic gas flow (Figure 2b). Replacing the hypoxic gas mixture with a
normoxic one (5% COz/95% air) brought about a return to the resting level of tension seen earlier in these cells (Figure 2c versus Figure 2a). Also apparent in this sequence is a small island of cells (upper right-hand corner) that shrinks in size with hypoxia and then relaxes with a return to normoxia. (No wrinkles are seen here because these cells are off the polymerized portion to the silicone, which ends at the edge of the confluent area below.) Exposing cells to a repeated hypoxic challenge was found to increase the intensity of the contractile response in six of 13 cultures tested. Figure 3a shows the baseline level of tension in a sparse, normoxic culture that is modestly increased by an initial hypoxic challenge (Figure 3b). Little change occurred during a subsequent normoxic period (Figure 3c). However, a second hypoxic challenge caused a marked increase in contraction of these same cells (Figure 3d). Stimulation of MLC Phosphorylation by Hypoxia Phosphorylation of the 20,000-D light chains on the contractile protein myosin was analyzed by a two-dimensional gel electrophoresis technique that readily resolved MLCs from other cellular proteins. MLCs from normoxic cultures (Figure 4a) were predominantly in the unphosphorylated form (pI = 5.1), with only about 20% of the light chains phosphorylated. Exposure to hypoxia during the brief experimental period resulted in a marked increase in the proportion of the MLCs in the phosphoryated form (Figure 4b). Quantification of these hypoxia-induced changes in the phosphorylation ofMLCs was obtained from densitometric scans of the stained two-dimensional gels. Evaluation of VSM cultures exposed to a Paz of 145 mmHg during a 6-min study
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Figure 3. Effect of repeated hypoxic challenges on PA VSM cells. Cells were photographed before (a) and after (b) exposure to hypoxic gas flows for 25 min. Normoxic gas was then infused for 11 min (c) followed by a second hypoxic challenge for 17 min (d). Arrowheads indicate areas where changes in tension were most prominent. Bar = 20 JLm.
period revealed that 22 % of the MLCs were in the phosphorylated form under these conditions (Table 1). This is comparable to the level of phosphorylation of MLCs seen in resting cultures of PAVSM cells under standard growth conditions in our laboratory (21). Exposure to hypoxia during the experimental period brought about significant increases (45%) in the amount of phosphorylated MLCs.
32p Incorporation into MLCs Autoradiographs of two-dimensional gels illustrated the baseline level of 32p incorporation into the MLCs of cultures first preincubated with the label and then exposed to normoxia during the 5-min experimental period (data not shown). A marked increase in 32p incorporation into MLCs was observed when cultures were exposed to hypoxia during the 5-min experimental period prior to harvesting and this was readily apparent as darker MLC bands on autoradiographs. Quantitative analysis of these changes revealed that hypoxia caused the amount of 32p incorporated into the MLCs to more than double (Table 2). Immunofluorescence Staining for von Willebrand Factor The hypoxia-induced contraction studies described herein
were conducted on cultures of VSM cells isolated from the medial layer of pulmonary arteries. The absence of endothelial cell contamination of these VSM preparations was confirmed using an immunohistochemical technique (25) to detect the presence of the endothelial-derived protein von Willebrand factor. Endothelial cells in culture continue to synthesize and store von Willebrand factor, whereas neither VSM cells nor other vessel wall cells produce this protein. A primary rabbit antibody to human von Willebrand factor was first reacted with the cells, followed by incubation with rhodamine-conjugated anti-rabbit IgG antibody. Endothelial cells show the typical punctate staining appearance as expected (Figure 5a). Conversely, endothelial cells exposed only to the conjugated anti-IgG antibody (and not the antivon Willebrand antibody) lack the specific staining pattern (Figure 5c). The smooth muscle cell preparation (Figure 5e) treated with both antibodies (like the cells in Figure 5a) revealed only nonspecific background fluorescence just as the endothelium exposed only to the second antibody (Figure 5c). Thus, the absence of any cells with the specific binding pattern of endothelium indicated that the VSM cell studies were conducted in cultures free from the influence of endothelium.
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TABLE 2
Incorporation of 32P into myosin light chains Condition
32p Incorporated*
Normoxia Hypoxia
1,729 3,821
± 607
± 693
* Numbers represent mean counts per minute per milligram of cellular protein ± SD for n = 3. The difference between conditions was statistically significant at the P = 0.02 level using Student's t test.
Figure 4. Hypoxia-induced increases in MLC phosphorylation. Confluent cultures of PA VSM cells were exposed to normoxic (a) or hypoxic (b) atmospheres during a 6-min experimental period prior to harvesting. Homogenates of the cultures were subjected to two-dimensional electrophoresis. Isoelectric focusing (pH 4.0 to 6.5) was followed by electrophoresis in 12 % polyacrylamide gels in the second dimension. Arrows indicate the bands corresponding to the MLC protein in the unphosphorylated (D) and phosphorylated (P) forms.
Discussion It is apparent in observations from several investigators that the strength of the hypoxic pressor response is modified and modulated by the complex interaction of numerous variables
that affect pulmonary vascular tone. In order to understand these interactions at the cellular and ultimately at the molecular level, it has been advantageous to reduce the complexity of the systems being analyzed. As a result, there has been a steady progression in refinement in experimental systems aimed at simplifying the preparation to the essential components (28-30) while still maintaining the basic aspects of the mechanism being studied. The present studies represent the beginnings of an attempt to extend this progression to the cellular level. A previously described model system of cultured smooth muscle cells that demonstrated appropriate responses to a variety of vasoactive agents in long-term culture was employed here (21). Results demonstrate that isolated PAVSM cells contract when challenged with hypoxic stimuli and do so in the absence of any other cell types. Hypoxic contraction was documented through two distinct lines of evidence. First, hypoxia-induced increases in tension forces generated by the VSM cells resulted in wrinkling and distortion of a flexible growth surface, which was followed by photomicroscopy. The results of these studies were corroborated and complemented by measurements of increases in MLC phosphorylation, a biochemical marker for activation of VSM cell contraction (31). The cross-linked polydimethyl siloxane growth surface provides a very sensitive indicator of cellular traction forces and is capable of detecting small changes in tension generated by the cultured cells (26). It was employed here to demonstrate that hypoxia stimulates increases in smooth muscle cell tone and that these changes are sometimes reversible with a return to normoxic atmospheres. Further, the hypoxic contractile response was shown to be maintained through several passages in culture without any apparent diminution during prolonged culture periods (analogous to the preservation of responsiveness to pharmacologic agonists of contraction in long-term culture reported previously for this system [21]). The difficulty with quantifying the response using this technique led us to choose measurements of MLC phosphorylation as a means to corroborate these findings and to provide a more quantitative assessment of the changes.
TABLE 1
Hypoxia-induced increases in MLC phosphorylation
Normoxia Hypoxia-l Hypoxia-2
Oxygen in Gas Phase
P02 of Culture Medium
(%)
(mmHg)
20
145 59 25
6
o
* Statistically significant at P < 0.02
Percentage of MLC Phosphorylated (± SD [nD
22 32 32
compared with nonnoxic condition in Student's t test.
± 5 (6) ± 6 (5)* ± 5 (5)*
Percent Increase versus Normoxic Control
o 45 45
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Figure 5. Immunohistochemical staining for von Willebrand factor in PA VSM and endothelial cell cultures. Cultures of PA VSM and endothelial cells were isolated independently and maintained as described in MATERIALS AND METHODS. Endothelial clones treated first with rabbit IgG antibodies to von Willebrand factor and then rhodamine-labeled goat anti-rabbit IgG show a punctate staining appearance (a). Light (b) and fluorescent (c) photographs of endothelial cells treated only with the second antibody show background, nonspecific staining, which is not related to von Willebrand factor. A VSM culture representative of those used in the contraction studies that was treated with both antibodies is shown under light (d) and fluorescent illumination (e). Bar = 50 JLm.
Conversion of the light chains of myosin from an unphosphorylated to a phosphorylated form facilitates the interaction of actin with myosin, which leads to VSM contraction. It has been shown to correlate well with initial force development for several smooth muscle preparations (31). Brief exposure of PA VSM to moderate hypoxia (Po, = 59 mmHg) is sufficient to activate this conversion and leads to significant increases over baseline levels in the proportion of
the MLCs that are in the phosphorylated form. Decreasing O2 in the culture media from 59 to 25 mmHg did not cause significant increases in phosphorylaton. It is not known if further decreases in Po, of the media bathing the cells would cause higher levels ofMLC phosphorylation. Oxygen partial pressure in the medium was lowered by flowing hypoxic gas through the space above the thin layer of media bathing the cells in covered culture dishes. Po, measured in the
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liquid phase dropped quickly during the first 60 to 75 s after hypoxic gas flow was begun, but then plateaued and stayed relatively constant for the remainder of the 5- or 6-min hypoxic periods. Oxygen levels in the culture media overlying the cells consistently remained higher than those of the gas phase and only decreased to an average Po2 of 25 mmHg when gas containing 95 % N 2/5 % CO 2 was employed. Thus, some modification of the hypoxic chamber will be needed in order to assess the responses of isolated VSM to more severe levels of hypoxia. The two-dimensional electrophoresis gels of MLCs revealed about 22 % of the light chains to be in the phosphorylated form under normoxic conditions. This is comparable to the levels reported earlier for unstimulated PA VSM cells (21) The relatively high levels of basal phosphorylation in cultured cells seen here and by others (32) compared with that of arterial muscle strips (33) is likely a reflection of basal tone in VSM cells in culture where they are attached to a growth surface. Consistent with this concept are the findings by Anderson and associates (32), who noted that cultures of mesenteric artery VSM cells had significant amounts of basal phosphorylation of MLCs that they were able to markedly reduce by treating cultures with dibutyryl cyclic AMP or chlorpromazine, agents that can cause vasodilation. The 32p incorporation studies and the presence of wrinkles under some unstimulated cells on the polymerized polydimethyl siloxane are also consistent with the presence of basal tone. In the former case, there is some 32p incorporated into the light chains of the control cultures not stimulated to contract with hypoxia (Table 2). Cross-bridge cycling between actin and myosin in cells with basal tone is a likely explanation for much of the 32p incorporation. Under conditions of constant stress, there continues to be a steady turnover of actin-myosin cross-bridges and MLCs undergo associated phosphorylation/dephosphorylation reactions (34). 32p taken up into the VSM cells during the 4-h pre-incubation used for these experiments could readily have exchanged with the unlabeled pool of intracellular phosphate and become attached to MLCs during turnover of the phosphorylated species of the protein. The response to hypoxia in individual cells is somewhat variable. .Photomicrographs of cells on the polymerized polydimethyl siloxane demonstrate that not all cells contract when challenged. Typically, the best responses were seen in areas of the cultures where there was already some baseline distortion of the growth surface prior to testing with hypoxia. The reason for this is uncertain at present, but it suggests that the response may be affected by the basal tone of the muscle. It has been shown with PA vessel loops and isolated lung preparations that hypoxic contractions are potentiated by prestimulating the tissue with various vasoconstrictors (16, 28,35). Rodman and colleagues (16) have suggested that a "threshold synergy" (36) between hypoxia and other vasoactive agents may be responsible for the potentiation of hypoxic contraction. This concept was based on observations by Stupecky and coworkers (36), who suggested that addition of threshold concentrations of an agonist converted resting muscle to an activated state that resulted in greater than additive contraction upon addition of a second agonist. Our observations with the flexible growth surface, while not designed to test this, are consistent with the proposal that
hypoxia acts as a more potent stimuli for VSM contraction in cells with elevated basal tone. The concept of an "oxygen sensor" has been discussed in recent reviews of HPV and a variety of different locations in the lungs have been proposed for it (2, 3, 37). However, the anatomic site and mechanism by which hypoxia is sensed and coupled to VSM cell excitation and contraction remain to be identified. Studies by Marshall and Marshall (38) with isolated rat lungs in which alveolar and mixed venous oxygen levels were varied independently suggested that the oxygen sensor behaved as ifit were located within the medial portion of the vessel wall. A significant finding of the present work is that VSM cells isolated from the PA medial layer are able to sense hypoxia and initiate a contractile response to it in the absence of other cell types. Thus, PA VSM cells are not dependent upon nonmuscle cells to initiate a hypoxia-induced contraction, although a role for other cells in modulating basal tone and modifying the hypoxic responses seems likely. The contribution of endothelium to the HPV response has been an area of ongoing controversy. An obligatory role for them has been reported in some cases (13) but not in others (14, 15). Recently, it has been shown (16, 39) that the response to hypoxia of PA vessel loops denuded of endothelium was diminished, but not abolished, while antagonists of endothelial-derived relaxing factor (EDRF) were shown to augment the HPV response in isolated rat lung (40-42). Some of the confusion undoubtedly lies in the ability of endothelium to synthesize several factors that can cause VSM relaxation or constriction. For example, endothelia exposed to hypoxia continue to release prostacyclin, a potent vasodilator (43), as well as endothelium-derived constricting factors (6). The various conditions that lead to release of these endothelial-derived products are not all understood and therefore may not always be appropriately controlled for in the different experimental preparations used. Results presented here show hypoxia-induced contractions of isolated PA VSM cells in cultures devoid of endothelium, findings more consistent with a modulatory rather than obligatory role for endothelium. A relaxation toward baseline levels of tension in cells first contracted with hypoxia and then returned to normoxia was noted (Figure 2). However, this response time was slower than that previously reported for cultured cells treated with the smooth muscle relaxant isoproterenol (21), as well as that for vessel loops and isolated lungs (which contained intact endothelium) following termination of a hypoxic challenge. It has been suggested that the pulmonary circulation behaves like the systemic circulation by generating vasodilatory prostaglandins in response to vasoconstrictive stimuli and these act to attenuate the constriction (44). Endothelia are known to be the source of multiple vasodilatory factors, including prostacyclin and EDRF. Further, hypoxia promotes increased synthesis of prostacyclin from animal lungs (45, 46) and prostacyc1in continues to be produced by cultured bovine jlA endothelial cells during hypoxic stimulation (43). Inhibitors of these compounds have been shown to augment the HPV response in some in vitro (28, 40) and in vivo (47) preparations. It is not clear at this time what role if any the absence of endothelium may play in the somewhat slower return toward basal tone seen in our cultures.
Murray, Chen, Marshall et al.: Hypoxic Contraction of Cultured Vascular Smooth Muscle Cells
In summary, the present work has demonstrated hypoxic contraction of isolated pulmonary artery smooth muscle cell cultures that occurs in the absence of nonmuscle cells. The applicability of these findings to the in vivo phenomenon of hypoxic pulmonary vasoconstriction remains to be established. However, if analogous, they suggest that the presence of endothelium and other lung cells in vivo serve a modulator rather than mediator role in this important physiologic response. Ackno:vledg"!ents: It is a pleasure to acknowledge Robert Rogers for his valuable technical assistance. We thank Drs. Albert K. Harris, Mike A. Clarke, Thomas M. Butler, Robert S. Moreland, Stephen F. Gorfien, and Pamela S. Howard for their assistance and advice and Dr. Phyllis M. Sampson for the use of her laser densitometer. A preliminary report of this work was presented at the 1988 meeting of the American Society of Anesthesiologists, San Francisco, California. This work was supported in part by grants from the Pennsylvania Thoracic Society (TRM) , the American Society of Anesthesiologists (TRM) , and Grants 5T32GM-07612 (TRM), GM-29629 (BEM), and HL-34005 (EJM) from the National Institutes of Health.
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