Division of Digestive Diseases and CURE: Digestive Diseases Research Center, University of California School of Medicine, Los Angeles, California 90095
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ABSTRACT |
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Although it has been hypothesized that contraction of hepatic stellate cells (HSC) regulates blood flow by modulating sinusoidal resistance, neither HSC contraction nor relaxation has been directly quantitated. To test this hypothesis, a force transducer was employed to directly measure the magnitude and rate of contraction and relaxation by primary rat HSC (4.7 × 105 ± 0.2 × 105 cells) cultured within a collagen gel. Serial exposures to 10% fetal bovine serum stimulated 81 ± 14 and 82 ± 10 dyn of contractile force, respectively. Subsequent stimulation with 2 nM endothelin-1 (ET-1) resulted in the development of 185 ± 25 dyn of force. Contractions began within 10 s of ET-1 stimulation, and the half time of maximal force development was <5 min. Removal of agonist resulted in complete or nearly complete relaxation within 45 min. These results suggest that the magnitude and rate of HSC contraction and relaxation are capable of modulating blood flow via sinusoidal constriction.
liver; portal pressure; endothelin-1; non-muscle cells; blood flow
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INTRODUCTION |
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HEPATIC STELLATE CELLS (HSC) have been postulated to play a role in the control of blood flow through the liver based on their anatomic location and contractile characteristics (15, 21). HSC, occupying the space of Disse, extend up to four cellular processes that run along one or more sinusoids (1). Branching perpendicularly from the primary processes are secondary processes that encircle the sinusoid in a cylindrical manner (1). On the basis of these observations, HSC have long been theorized to buttress the sinusoid and perhaps even modulate the sinusoidal caliber. Over the past several years, HSC in culture have been shown to contract in response to various agonists, including the vasoactive peptide, endothelin-1 (ET-1), which is found in elevated concentrations in cirrhotic rats and humans (8, 18). In addition, investigations of isolated perfused rat livers showed that ET-1 increased portal pressure and decreased the sinusoidal radius in regions colocalized with HSC (31). Finally, recent preliminary studies demonstrated that ET-1 antagonists reduce portal pressure and increase hepatic blood flow in laparotomized rats (28). These results led to the hypothesis that HSC contraction and relaxation around the sinusoid may control hepatic blood flow by regulating sinusoidal resistance.
Heretofore, contractile force generation by HSC has not been directly measured; consequently, it has not previously been possible to test whether HSC contraction is forceful or rapid enough to modulate sinusoidal blood flow. Although prior studies of isolated HSC have shown that a variety of agonists can stimulate HSC contraction (9, 17, 22), none of these investigations directly quantitated force development by HSC. Similarly, studies in isolated perfused liver have indicated that ET-1 mediates sinusoidal caliber (in association with HSC), portal pressure, and hepatic blood flow (31), but these studies did not directly evaluate whether HSC were independently capable of sinusoidal constriction. The aim of this study was to directly quantitate contractile force development by HSC, in order to test the hypothesis that HSC contraction and relaxation around the sinusoid control hepatic blood flow by regulating sinusoidal resistance. The data presented here suggest that the magnitude and rate of HSC contraction and relaxation are sufficient to modulate sinusoidal blood flow.
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MATERIALS AND METHODS |
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Materials. Cell culture products, including fetal bovine serum (FBS; lot 300060742) and DMEM with 20 mM HEPES, were obtained from Irvine Scientific (Santa Ana, CA). DMEM (10×) was bought from Sigma (St. Louis, MO). ET-1 was purchased from Calbiochem (La Jolla, CA). Rat tail collagen I was bought from Upstate Biotechnology (Lake Placid, NY). The JB-4 embedding kit was from Polysciences. All other salts were obtained from Fisher (Tustin, CA) and Sigma.
Cell culture. Primary rat HSC, obtained from H. Tsukamoto (Non-Parenchymal Liver Cell Subcore Facility of University of Southern California Research Center for Liver Disease), were isolated by gradient centrifugation (5, 27). HSC were cultured in DMEM supplemented with 10% FBS, 200 mM L-glutamine, 50,000 units of penicillin G, 50,000 units of streptomycin, 1% nonessential amino acids, and 0.5 mM sodium pyruvate at 37°C in 10% CO2. Tests to detect the presence of mycoplasma were not performed. Cells were grown on Primaria-treated plates (Falcon), and medium was exchanged every 2 days.
Quantitation of HSC contractile force development. HSC were removed from Primaria-treated plates at day 7 (i.e., after they had acquired the activated phenotype; Ref. 30) by exposure to trypsin (1:4,000 dilution of 5 g trypsin-2 g EDTA/L-DMEM, 2 min at 37°C); 4.7 × 105 ± 0.2 × 105 HSC were mixed with an ice-cold solution composed of rat tail collagen I, 10× DMEM, NaOH, and FBS to obtain final concentrations of 2 mg/ml of collagen, 1× DMEM, and 12.5% FBS at pH 7.4 (11, 12). The number of HSC per gel varied due to the yield of HSC after isolation. The collagen-HSC solution was poured into rectangular molds (1 cm3 volume) containing, at opposite ends, a section of Velcro (i.e., the loop end) attached to a glass rod. Metal spacers were used to keep a distance of 10 mm between the glass rods. The collagen-HSC solution was warmed to 37°C for 1 h, causing polymerization of the collagen into a solid but elastic gel containing HSC (~10 mm × 10 mm × 2 mm). Culture medium was added onto the gels and replaced every 2 days.
After 4-5 days, the glass rod at one end of a gel containing HSC was immobilized, whereas the glass rod at the free end was attached to an isometric force transducer (Harvard Apparatus). The gel was stretched to its original length (i.e., 10 mm) and submerged in an organ bath containing serum-free DMEM with HEPES. The gels containing HSC were equilibrated for 1 h to ensure a stable baseline tension. Agonists were added directly to the bath, and contractions were terminated after 15 min by replacement of agonist-containing medium with agonist-free medium. Contraction experiments were carried out at 37°C.Data acquisition and analysis. Changes in isometric tension were acquired on a Pentium-based computer (Dell) with an analog-to-digital converter (DAQ-500, National Instruments) using commercially available software (Virtual Bench Logger, National Instruments). Analysis and fits were performed using a commercial software package (Axum 5.0, MathSoft). One sample was discarded with an r2 < 0.5 because of vibrations that caused an inordinate amount of noise within the measurements.
Determination of HSC in cross section.
At the end of contraction experiments, collagen gels containing HSC
were fixed in 10% Formalin and stored at 4°C until embedding was
performed (JB-4 embedding kit protocol). Collagen gels were dehydrated
with a graded ethanol treatment (30-100%), and the glass
rod-Velcro attachments were removed. Gels were placed in a 50:50
mixture of 95% ethanol and JB-4 plastic solution overnight. Gels were
then placed in 100% JB-4 plastic solution, and the solution was
changed twice. After infiltration, gels were embedded in the JB-4
plastic, and the plastic was allowed to solidify. Cross sections (2.5 µm) of gels were cut in the plane perpendicular to the force vector
measured by the force transducer. HSC within the cross sections were
stained with toludine blue. Cross sections were visualized
microscopically (Olympus), and images were acquired (Sensys cooled
charge-coupled device camera, Photometrics). HSC were defined as
toludine blue-stained objects >10 µm × 10 µm to distinguish
cell bodies from primary and secondary processes (Fig. 1). HSC were counted manually.
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Determination of HSC orientation within the gel. In addition to cross sections of the HSC within the collagen gels, several lengthwise sections were also cut, stained, and visualized in the same manner as above.
Statistical analysis. Data are presented as means ± SE, and groups were compared by Student's paired t-test. Significance level was defined as P < 0.05.
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RESULTS |
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To directly characterize their contractile behavior, HSC were suspended
within a rectangular collagen gel and attached to an isometric force
transducer, permitting measurement of contractile force development by
the cells. This method allowed quantitation of contraction and
relaxation elicited by exposure to and removal of agonists,
respectively. Reproducible cycles of contraction and relaxation were
evoked by sequentially stimulating the same sample of HSC with FBS
(Fig. 2). Subsequent stimulation with ET-1 also stimulated force development (Fig. 2). HSC contraction curves were
well fit by a monoexponential function described by the equation a + b(kt)
(where k is the rate constant and
t is time) (Fig.
3A). The
predicted maximal amplitude and the kinetics of contraction were
determined from the fit curve because contractile force did not come to
a steady state during the duration of the experiment. Exposing collagen gels containing dead cells to agonists did not induce contraction.
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Initial stimulation with 10% FBS resulted in 81 ± 14 dyn (n = 7) of developed force by the HSC, and this was reproduced by a second exposure to 10% FBS (82 ± 10 dyn, n = 7). Subsequently, 2 nM ET-1 stimulated 185 ± 25 dyn of contractile force (n = 7), which was significantly different from that stimulated by FBS (P < 0.001). In all cases, changes in tension were observed within 10 s of agonist addition. Half time of maximal contractile force development (t1/2) was 187 ± 18 s (n = 7) and 137 ± 16 s (n = 7) for the initial and second FBS-stimulated contractions (not significantly different) and 282 ± 30 s (n = 7) for the ET-1-stimulated contraction. This ET-1-stimulated t1/2 was different (P < 0.01) from the second FBS contraction but was not different from the first FBS-stimulated t1/2.
HSC relaxation occurred when agonist was removed from the bath (Fig 2). This relaxation curve could, in some experiments with FBS, be fit by the monoexponential equation a + b(kt) (Fig. 3B). HSC relaxation was complete within 45 min of FBS removal after both the initial (94 ± 28% relaxation at 45 min; n = 6) and second (135 ± 15% relaxation at 45 min; n = 6) FBS-stimulated contractions. Removal of ET-1 following the ET-1-induced contraction resulted in 74 ± 7% (n = 6) relaxation at 45 min. The time course of relaxation following removal of ET-1 could not be fit by a monoexponential equation. The amount of relaxation following the second FBS stimulation was different from the amount of relaxation following the first FBS stimulation and the ET-1-stimulated contraction (P < 0.05). There was no difference between the relaxation following the first FBS stimulation and the relaxation following the ET-1 stimulation.
To determine if HSC contractile force was robust enough to permit sinusoidal constriction, it was necessary to determine the quantity of HSC in cross section. Microscopic analyses of gels containing HSC showed that there were 806 ± 137 cells per cross section (n = 4; Fig. 1B). Primary processes extended from the cell bodies in a manner similar to that observed in situ (Fig. 1A) (1, 29). To investigate the orientation of HSC within the collagen gels, cross sections and lengthwise sections were visualized. HSC within the gels were randomly oriented in all three dimensions (data not shown).
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DISCUSSION |
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The goal of this study was to test the hypothesis that HSC contraction and relaxation around the sinusoid may control hepatic blood flow by regulating sinusoidal resistance. Therefore, a novel method was employed to directly quantitate HSC contractile force development. The results of these experiments indicate that the magnitude and rate of HSC contraction and relaxation are sufficient to influence sinusoidal blood flow.
In contrast to the techniques utilized in prior studies of HSC
contraction, the method employed in this study allowed direct and
quantitative measurement of contractile force development and
relaxation by HSC. Previously, HSC contraction has been assayed using a
variety of surrogate measurements. Microscopic analyses of cell
shrinkage have been employed to demonstrate contraction by cultured HSC
(17). Such measurements, however, could be influenced by noncontractile
processes, including alterations in cellular adhesion or volume,
three-dimensional shape changes, and focusing artifacts. HSC
contraction has also been detected by culturing HSC on top of collagen
lattices and measuring shrinkage of collagen lattice area (22). This
method allows neither measurement of relaxation nor rapid changes in
contractile force development and may not allow differentiation between
active contraction and passive changes in tension (such as that
potentially generated by cellular spreading). Wrinkle formation by HSC
cultured on top of an elastic rubber substrate has also been used to
examine contraction (9). This method, similar to the previous two
methods, does not permit direct quantitation of force development and
relaxation. Finally, high-power intravital microscopy of isolated rat
livers has been used to demonstrate that ET-1 stimulated decreases in sinusoidal diameter and increases in sinusoidal resistance in proximity
to HSC (31). This method, however, does not allow actual measurement of
the contractile force generated by HSC, nor does it completely rule out
the possibility that other cells (e.g., Kupffer or endothelial cells,
see Table 1) or noncontractile events
(e.g., cellular swelling) may mediate the observed sinusoidal constriction. Although all of these techniques have been instrumental in investigating HSC contraction, none of them has allowed direct or
quantitative force measurements; consequently, the question of whether
HSC are independently capable of modulating sinusoidal resistance has
remained unanswered. The method employed in this study has enabled us
to directly investigate the force and speed with which a population of
HSC contract and subsequently relax.
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These direct measurements of the contractile force generated by a population of HSC have allowed us to estimate the contractile force generated by a single HSC. ET-1 (2 nM) elicited the development of 185 ± 25 dyn of measured contractile force by HSC. Fundamental principles of mechanics and empiric data from prior studies of contractile force development by striated muscle (2, 7, 19, 23) indicate that the force produced by a tissue in a given direction is determined by the number of cells in parallel (i.e., in cross section). In contrast, the number of cells in series (i.e., in longitudinal section) determines the velocity of contractile force development. There were 806 ± 137 HSC in each cross section of the collagen gels containing HSC. With the assumption that all of the HSC in a gel contracted simultaneously and with equal force, the average force produced by a single HSC in the direction of the force transducer is equal to the contractile force generated by all of the HSC in that direction (i.e., ~185 dyn) divided by the number of HSC in cross section (i.e., ~800 cells), that is, ~0.23 dyn per HSC in the direction of the force transducer. Note that contractile force was measured only in the direction of the force transducer. However, HSC within the collagen gels were randomly oriented in three dimensions and consequently were capable of producing contractile force in all three dimensions. Therefore, the average total force that could be generated by a single HSC was ~0.69 dyn, that is, three times the force measured in the direction of the transducer.
Electron microscopic studies of HSC, in situ, indicate that HSC extend
up to four primary processes that may run along the length of more than
one sinusoid (1, 26). Sinusoids are encircled by secondary processes
that branch perpendicularly from the primary processes. The primary
processes are ~20 µm in length, and the secondary processes wrap
around the circumference of the sinusoids. If the cell body of a
typical HSC lies along one sinusoid, extending two processes along that
sinusoid and two processes along an adjacent sinusoid (29), then the
HSC would potentially encircle a total length of 80 µm (i.e., the
combined length of the four processes). The external radius of a
sinusoid is 7-12 µm [i.e., internal radius of ~6 µm
(31) and endothelial thickness of 1-6 µm (20)]. On average
the circumference of a sinusoid would be 60 µm [i.e., (2)(9.5 µm)]. The contractile force developed by an HSC is
consequently exerted across a surface area around the sinusoids of ~ 4,800 µm2 (i.e., the length
along the sinusoid, 80 µm, multiplied by the average circumference,
60 µm). On the basis of the average total contractile force produced
by a single HSC of ~0.69 dyn, a typical HSC would be predicted to
generate ~14,000 dyn/cm2 (i.e.,
contractile force per HSC, ~0.69 dyn, divided by surface area around
the sinusoid, 4.8 × 10
5
cm2) of pressure around the sinusoid.
This estimate for the pressure that an HSC is capable of producing was based on a number of assumptions. In each case, conservative assumptions were made to avoid overestimating this value. For instance, it is doubtful that HSC within the gel contract simultaneously. If HSC contract asynchronously, then the estimate of the force of HSC contraction represented an underestimate because in that case the measured force would actually have been produced by a smaller number of cells. Furthermore, average data (e.g., average force generated by an HSC, average HSC length, average sinusoidal diameter, average sinusoidal pressures) were used in the calculations. Use of average data obscures the likelihood that at some locations within the liver HSC that can generate greater than average pressure may encircle sinusoids of less than average blood pressure. Certainly, it would be significant even if HSC regulate sinusoidal resistance in only certain regions of the liver. Finally, these calculations did not take into account the intrinsic resisting force of the collagen gels (i.e., the force necessary to deform the collagen gel and permit detection of HSC force generation by the transducer). Because it was not possible to accurately measure the gel intrinsic resisting force, the estimate of the pressure generated by a single HSC was underestimated.
For an HSC to constrict a sinusoid and modulate resistance to hepatic blood flow, an HSC must be capable of generating sufficient contractile force to overcome the sinusoidal blood pressure. Blood pressure within the sinusoid resides between the terminal hepatic venule pressure and the terminal portal venule pressure. In normal rats, hepatic venule pressure was ~2,700 dyn/cm2 (~2 mmHg), and portal venule pressure was ~6,700 dyn/cm2 (~5 mmHg) (24, 25). In cirrhotic rats, hepatic venule pressure was ~3,200 dyn/cm2 (~2.4 mmHg) and portal venule pressure was ~10,500 dyn/cm2 (~8 mmHg) (24, 25). In a study of pressure gradients along the rabbit hepatic vasculature, it was observed that sinusoidal pressure lay 70% of the way between the hepatic venule and portal venule pressures (14). With extrapolation of this data, sinusoidal blood pressure was estimated to be ~5,500 dyn/cm2 for normal rats and ~8,200 dyn/cm2 for cirrhotic rats. Therefore, the predicted pressure produced by a single HSC, ~14,000 dyn/cm2, was greater than the sinusoidal pressures in normal or cirrhotic rats, indicating that HSC can constrict the sinusoids and thereby raise sinusoidal resistance.
For HSC to regulate sinusoidal resistance in a physiologically significant manner, it is not only necessary that these cells generate sufficient contractile force but that force development be reversible and rapid enough. In other words, physiological regulation of sinusoidal resistance would be impossible if HSC were unable to relax or if the time course of contraction or relaxation were too long. In this study, contractile force development began within 10 s of ET-1 stimulation and the t1/2 was <5 min. Moreover, these kinetic data underestimate the rate of force development, since the diffusional distance across the gel was longer than the diffusional distance across the space of Disse for FBS or ET-1. These data are consistent with the time course of changes in sinusoidal radius in response to ET-1 (31) and suggest that HSC contraction is both robust enough and rapid enough to regulate sinusoidal resistance. Also, in this study, HSC exhibited complete or nearly complete relaxation within 45 min of the removal of stimulus. These results are, again, likely to underestimate the rate of relaxation because agonist washout from the gel was probably many times slower than that from the space of Disse. In addition, in situ, HSC relaxation would also be facilitated by relaxing factors, including nitric oxide (10, 15, 21). This study demonstrates for the first time that the magnitude and kinetics of contraction and relaxation are consistent with the hypothesis that HSC regulate sinusoidal resistance in a physiologically important manner.
Although this study permitted the first direct quantitation of HSC contraction and relaxation, there are certain limitations to these results. Contractile force development by a single HSC needed to be estimated from measurements of the contractile force generated by a population of HSC within a collagen gel. Current technology, however, does not permit the measurement of force development by a single HSC. Indeed, the data reported here represent the first direct and quantitative measurements of HSC contraction and relaxation. Primary cultures of HSC may not behave the same as HSC in the liver. Again there are, at present, no methods for directly measuring HSC contraction and relaxation in situ. HSC in primary culture are currently recognized as the model system most similar to HSC in situ, in contrast to the passaged HSC, which have been used in many of the prior studies of HSC contraction (17). Moreover, this concern may be minimized by growing HSC within a three-dimensional collagen gel, which may more closely mimic the milieu within the liver compared with HSC cultured on top of substrates. Finally, HSC, in situ, would never be acted on by a single stepped exposure to an agonist. The method used in this study, however, may permit investigation of HSC contraction and relaxation in response to multiple agonists and antagonists. Despite the limitations of this study, the data compare favorably with results from experiments on isolated perfused liver. For example, in this study 2 nM ET-1 stimulated a rapid (t1/2 <5 min) contraction that was sustained until the agonist was removed, whereas intravital microscopic studies on isolated liver showed that 1 nM ET-1 stimulated a rapid (~3 min) and sustained decrease in the sinusoidal diameter (31). Furthermore, these data are consistent with the hemodynamic response of the whole liver, in which 1 nM ET-1 stimulated increases in total vascular resistance and decreases in hepatic blood flow that reached a peak at ~3 min and were sustained for at least 30 min (31).
These results suggesting that the contractile force produced by HSC is capable of modulating sinusoidal blood flow are compatible with studies indicating that the contractile forces generated by other non-muscle cell types play important physiological roles (Table 1). For example, a variety of different kinds of fibroblasts have been demonstrated to produce magnitudes of contractile force similar to that of HSC (Refs. 3, 12, 13, 16, and L. Kernochan, B. N. Tran, and H. F. Yee unpublished data). These fibroblasts have been recognized to play important roles in the wound-healing response of the skin and gut. Phagocytes have been demonstrated to generate significant contractile force as they carry out their important function of clearing foreign particles and microorganisms (4). Furthermore, the contractile forces generated by endothelial cells have been measured and were predicted to be capable of regulating venous blood flow (6, 13). This study of endothelial cells in particular draws attention to the possibility that contraction of sinusoidal endothelial cells may also contribute to sinusoidal blood flow regulation. Thus HSC contractile force development and its role in the regulation of sinusoidal blood flow may be a general phenomena applicable to many other non-muscle cell types involved in specific biological processes.
The aim of this study was to directly quantitate HSC contraction and relaxation to investigate their contribution to sinusoidal blood flow control. ET-1 or FBS stimulated robust and rapid development of contractile force by HSC, and removal of agonist resulted in a complete or nearly complete relaxation within 45 min. These observations provide important new evidence supporting the hypothesis that HSC contraction and relaxation around the sinusoid may control hepatic blood flow by regulating sinusoidal resistance.
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ACKNOWLEDGEMENTS |
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We thank Dr. Michael Kolodney for technical assistance and helpful comments, Drs. Albert Kim, Kenneth Roos, and Vivek Dixit for valuable discussion, and Dr. John Inadomi for a thoughtful critique of the manuscript. Rat HSC were provided by the Non-Parenchymal Liver Cell Subcore Facility of University of Southern California Research Center for Liver Disease (P30-DK-48522).
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FOOTNOTES |
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This work was supported by awards to H. F. Yee from the National Institute of Diabetes and Digestive and Kidney Diseases (K08-DK-02450) and the Glaxo-Wellcome Institute for Digestive Health and a gift from the United Liver Association.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. F. Yee, Jr., 675 Charles E. Young Dr., South, 1519 MacDonald Research Labs, UCLA School of Medicine, Los Angeles, CA 90095 (E-mail: hyee{at}med1.medsch.ucla.edu).
Received 21 January 1999; accepted in final form 30 March 1999.
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