Department of Biology, University of North Carolina at Charlotte, Charlotte, North Carolina 28223
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ABSTRACT |
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The present study was undertaken to investigate hepatic microcirculatory response following partial portal vein ligation (PPVL) in rats. Portal pressure was markedly increased 2-6 wk after PPVL, but no significant reduction in sinusoidal perfusion and hepatocellular injury were detected. However, marked neovascularization was observed in PPVL rats using intravital microscopy and scanning electron microscopy (SEM). Extremely high red blood cell velocity (2,000-4,900 µm/s) was seen in these vessels. Injection of fluorescein sodium via the carotid artery revealed that the neovessels originated from the hepatic arterial vasculature. This was further confirmed by clamping the common hepatic artery and phenylephrine injection from the carotid artery. These vessels maintained sufficient flow after massive sinusoidal shutdown elicited by the portal infusion of endothelin receptor B agonist IRL-1620. SEM also showed extensive neovascularization at the hilum. Additionally, clamping the portal vein decreased sinusoidal perfusion only by 9.5% in PPVL, whereas a 71.2% decrease was observed in sham. These results strongly suggest that the liver maintains its microcirculatory flow by vascular remodeling from the hepatic arterial vasculature following PPVL.
liver microcirculation; intravital microscopy; portal hypertension; hepatic vascular casts
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INTRODUCTION |
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PARTIAL PORTAL VEIN LIGATION (PPVL) has been used as an animal model to induce prehepatic portal hypertension, which is characterized by increased cardiac output, reduced systemic vascular resistance, increased plasma volume, and decreased vascular responsiveness to vasopressors (27). Although much attention has been drawn to the pathophysiological changes in the preportal splanchnic circulation and systemic circulation (2, 3, 7, 22), little information is available on how the liver responds to the sudden and sustained decrease in portal blood flow following PPVL.
The liver receives blood supplies from both the portal vein and the hepatic artery, and these circulations are regulated independently. Portal venous flow is regulated by the resistance vessels in the splanchnic viscera upstream and is determined by the net outflow of the splanchnic organs. The liver does not control portal blood flow. A change in blood perfusion to the liver is brought about by regulation of hepatic arterial blood flow via the hepatic arterial buffer response (HABR) (14, 15). HABR, first reported by Lautt et al. (13) as the primary intrinsic regulator of the hepatic artery, increases hepatic arterial blood flow in response to decreased portal flow. This compensatory mechanism of hepatic blood flow has been proposed to be mediated by adenosine washout. If portal blood flow is severely reduced, less adenosine is washed away into the portal blood and the accumulated adenosine leads to hepatic arterial dilation and increased hepatic arterial flow. Although HABR has been generally accepted as a regulatory mechanism of the total hepatic blood flow with an acute change in portal blood flow, information on how the liver regulates its blood flow under conditions in which portal blood flow is chronically reduced, such as occurs with PPVL, is still lacking. It is conceivable that the liver responds to diminished portal blood flow following PPVL with chronic functional and/or structural changes. Tsoporis et al. (26) reported the structural changes in mesenteric arterioles after chronic treatment with a vasodilator. Another study performed by Sumanovski et al. (24) showed increased angiogenesis in the abdominal cavity in a rat PPVL model. However, little information is available on the effects of PPVL on the microcirculatory hemodynamics and morphological adaptations in the liver. Therefore, the present study was conducted to investigate the chronic response of hepatic microcirculation following PPVL.
Using intravital fluorescence microscopy and scanning electron microscopy (SEM), we discovered that the liver adapted to permanently diminished portal blood flow after PPVL by initiating neovascularization from the hepatic arterial system. The neovessels bear characteristics of arterioles and function similarly to the arterial vasculature. We therefore propose that neovascularization of the hepatic artery may represent a novel mechanism for a long-term regulation of hepatic microcirculatory flow under conditions in which portal blood flow is chronically deprived.
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MATERIALS AND METHODS |
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Chemicals
IRL-1620, an endothelin receptor B agonist, was purchased from American Peptide. Batson's No. 17 anatomic corrosion compound was purchased from Polysciences (Warrington, PA). All other chemicals were purchased from Sigma (St. Louis, MO).PPVL
Experiments were performed using male Sprague-Dawley rats (Charles River Labs) weighing 250-350 g at the time of operation. All procedures were performed in accordance with National Institutes of Health guidelines under a protocol approved by the Institutional Animal Care and Use Committee of the University of North Carolina at Charlotte. Prehepatic portal hypertension was induced by PPVL as described by Vorbioff et al. (27). Briefly, rats were anesthetized with metaphane (Schering Plough). After an upper midline abdominal incision, the portal vein proximal to the confluence of the right and left branches was exposed and carefully separated from the hepatic artery. A 20-gauge needle was placed beside the portal vein, and a tight ligature was performed around the needle and portal vein with 4-0 silk. Subsequent removal of the needle resulted in constant calibrated stenosis of the portal vein. After washing the abdominal cavity with physiological saline, the abdomen was closed and the animals were allowed to recover for 2 or 6 wk. Sham animals were operated using the same procedure except for the ligation of the portal vein.Lactic Dehydrogenase and Alanine Aminotransferase Assays
To determine whether PPVL induces liver injury, blood samples were obtained from the arterial line of PPVL and sham rats for measurement of serum lactic dehydrogenase (LDH) and alanine aminotransferase (ALT) levels. Measurements were made spectrophotometrically using diagnostic kits from Sigma.Fluorescent Cell Labeling
FITC-labeled red blood cells (RBCs) were prepared as originally described by Zimmerhackl et al. (30) in a modified technique. Briefly, RBCs from 10 ml of heparinized blood of a donor animal were washed three times in Alsever's buffer [in g/l: 20.5 glucose, 8.0 citric acid trisodium salt, 0.55 citric acid, and 3.766 NaCl, pH 6.2] and once in bicine-saline buffer [in g/l: 3.264 bicine, 0.399 NaOH, and 7.288 NaCl, pH 8.3]. For each washing step, 3 min of centrifugation at 2,500 rpm was used. Subsequently, FITC, at a dose of 4 mg/ml RBC suspension, was dissolved in 0.1 ml N,N-dimethylformamide and added into 1:1 diluted RBCs in the bicine-saline buffer. After incubation for 3 h at room temperature on a rocking plate, cells were washed once in bicine-saline buffer and 3 times in physiological saline by centrifugation. After resuspension in physiological saline with citrate-phosphate-dextrose (0.14 ml/ml RBC suspension) to a hematocrit of ~50%, RBCs were refrigerated until use within 7 days.Intravital Microscopy
Surgical preparation. Rats were fasted overnight but were allowed free access to water. Every procedure was performed on a heating pad to maintain body temperature at 36-37°C. Following induction of anesthesia (pentobarbital sodium, 50 mg/kg body wt ip), a tracheotomy was performed to allow spontaneous breathing. After cannulation of the right carotid artery, a midline laparotomy was performed. The bowel was wrapped with wet gauze and shifted to the left side to expose the portal and splenic veins. After careful dissection of the connective tissue around the portal vein and the splenic vein, a double-lumen catheter (outer catheter PE-90, inner catheter PE-10) was inserted from the splenic vein into the portal vein. The tip of the catheter was positioned at the proximal site of PPVL. This cannulation allowed for the simultaneous pressure measurement and infusion. The bowel was repositioned, and the lower half of the incision was approximated. Each animal was transferred to the stage of an Olympus IX70 inverted microscope and turned on its left side. The left lobe of the liver was gently exteriorized and positioned onto a window, which was covered with a Corning no. 1 micro-cover glass so that the working distance between the objective lens and the specimen could be minimized. To prevent evaporation, the surface of the liver was covered with Saran plastic wrap (Dow Chemical).
In vivo microscopy. The liver surface was epi-illuminated with a 100-W mercury lamp with 460-500 nm excitation and 515-560 nm emission band-pass filters. The images were projected onto a DAGE-MTI SIT 66 camera and processed with an ARGUS-10 image processor (Hamamatsu Photonics). The processed images were viewed on a high-resolution video monitor and recorded with a S-VHS VCR (Panasonic AG 7300). Quantitative analysis of the images was performed offline during video playback using digitized frame-by-frame analysis with Image-Pro (Media Cybernetics, Silver Spring, MD). A total magnification (specimen to monitor) of ×760 was used for the experiment. Mean arterial pressure (MAP), heart rate, and portal pressure (PP) were recorded simultaneously using Digi-Med 200 pressure analyzer system (Micro-Med, Louisville, KY) during the entire procedure. Measurements of sinusoidal diameters and evaluation of sinusoid density (defined as number of perfused sinusoids per 150 µm) were made directly from video playback as previously described (4).
Detection of neovascularization.
The neovessels were first identified using epi-illumination with
460- to 500-nm excitation/515- to 560-nm emission band-pass filters.
Neovessels exhibit characteristics that easily distinguish them from
sinusoids, i.e., larger diameters and clearly defined vascular
boundaries (Fig. 1). The blood flow in
those vessels was extremely fast. To determine whether those vessels
originated from the hepatic artery or the portal vein, 1 ml of
fluorescein sodium (0.2 mg in saline) was injected from the carotid
artery while the sequential images of appearance of the fluorescent dye in those vessels were recorded. In addition, the origin of the neovessels was confirmed by clamping the common hepatic artery that was
isolated during the surgical preparation while the blood flow rate in
those vessels was monitored. In separate experiments, 0.2 ml of
FITC-labeled RBCs (hematocrit = 50%) was injected intravenously for measurement of the RBC velocity in both the neovessels and sinusoids.
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Response of the neovessels to vasoconstrictors.
To compare the responses of the neovessels and sinusoids to
vasoconstrictors, the -adrenergic receptor agonist phenylephrine (PE) and endothelin receptor B agonist IRL-1620 were used. A bolus of
PE (4 × 10
7 mol) was injected via the carotid
artery (n = 3) or portal vein while the response of the
neovessels was monitored. IRL-1620 was infused through the portal vein
at a rate of 5 pmol · min
1 · 100 g
1 for 20 min, and responses of the sinusoids and
neovessels were continuously monitored for 1 h (sham,
n = 7; PPVL, n = 8).
Effect of portal vein clamping.
To determine the relative significance of the blood supply to the
hepatic microcirculation from the hepatic arterial system following
PPVL or sham operation, acute portal vein clamping was performed to
occlude the portal flow while the blood perfusion in sinusoids was
monitored using intravital microscopy. Before portal clamping, 0.2 ml
of FITC-labeled RBCs was injected from the carotid artery for
measurement of the RBC velocity (VRBC). Sinusoidal diameters (Ds) and
VRBC were measured during offline analysis.
Perfusion index (PI), which reflects the blood perfusion in the hepatic
microcirculation, was calculated using the following equation
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Hepatic Vascular Casting
Hepatic vascular casting was carried out using a modified method of Lim et al. (16). Under pentobarbital anesthesia, the portal vein and thoracic aorta were cannulated with a PE-160 catheter. A cannula for the thoracic aorta was inserted caudally to position the catheter tip at the radix of the celiac artery. Tight ligation was performed on abdominal aorta just proximal to the branching point of the renal artery. The inferior vena cava was also ligated at the level proximal to the right renal vein. After dissecting the right atrium as a drainage route of perfusate, the liver was perfused with sterilized saline containing 10,000 U/l heparin for 10 min to wash out the blood. Pressure of the arterial line was monitored by Digi-Med 200 pressure analyzer system (Micro-Med, Louisville, KY). During the perfusion, Batson's No. 17 anatomic corrosion compound (Polysciences) was formulated for use as the casting medium. The ingredients of the mixture consisted of 10.0 ml of monomer base, 2.0 ml of catalyst, and 2 drops of promoter. The casting medium of different colors was injected from portal vein (blue) and thoracic aorta (red) manually by using a 10-ml disposable polyethylene syringe. The pressure of the arterial line was kept under 180 mmHg to prevent the formation of arterial aneurysm. Infusion was terminated when the casting medium appeared on the surface of the liver. The liver was covered with Saran plastic wrap and left in the abdominal cavity overnight to allow complete polymerization. After polymerization, the liver was excised carefully and soaked in potassium hydroxide (6 M) to macerate the tissue. The solution was changed at least once a day. More than 2 days were required to dissolve the tissue surrounding the cast entirely. Additional maceration was carried out in a 60°C incubator when the room temperature digestion of the tissue was incomplete. The casts were then dehydrated in ethanol and dried.SEM. Dried casts of the liver were mounted on aluminum stubs and sputter-coated with gold (96%)-palladium (4%). Samples were viewed and photographed by Cambridge scanning electron microscope (Cambridge, MA) at an accelerating voltage of 15 kV.
Statistical Analysis
Statistical significance was tested using one-way ANOVA, with individual means detected by Student-Newman-Keuls test. A P value <0.05 was considered significant. All results are presented as means ± SE. ![]() |
RESULTS |
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Hemodynamic and Enzymatic Data
The hemodynamic data of PPVL and sham-operated rats are shown in Table 1. PP was markedly increased in PPVL rats compared with sham-operated rats. MAP of the PPVL group shows a trend of decrease but did not reach statistical difference from the sham group. The sinusoidal diameters and number of perfused sinusoids per 150-µm length in each microscopic field (sinusoidal density) showed no significant differences between PPVL rats and sham controls. No deficits in microcirculatory perfusion and necrosis were observed in PPVL rats when compared with sham rats. To determine the level of hepatocellular injury, plasma LDH and ALT levels were measured 2 wk after PPVL or sham operation (Fig. 2). No significant differences were found between PPVL and sham (LDH: sham, 54.0 ± 7.0; PPVL, 57.4 ± 10.0 IU/l; ALT: sham, 20.0 ± 6.0; PPVL, 31.4 ± 10.0 IU/l), indicating that the procedure of PPVL did not produce significant hepatocellular injury.
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Detection of Neovascularization
Using in vivo intravital fluorescence microscopy, we were able to observe extensive neovascularization in livers that were subjected to PPVL for 2 or 6 wk, but no neovessels were found in the liver of sham-operated animals (Fig. 1). The neovessels were usually observed near the edge of the left liver lobe. Because of the difficulty of maneuvering the liver for microscopic viewing, no attempt was made to search for the neovessels in other lobes. The neovascularization was observed in 13 out of 37 PPVL rats (35.1%), with no difference in the frequency of observation between 2 and 6 wk of PPVL.These neovessels showed features that clearly distinguished them from
sinusoids. The size was larger than sinusoids, ranging from 8 to 60 µm in diameter, and they conducted flow at extremely high rates (RBC
velocity 2,000-4,900 µm/s vs. the normal sinusoid rates of
200-500 µm/s). The average RBC velocity was >2,000 µm/s even
in the smaller neovessels (<20 µm in diameter) and was higher than
that of terminal hepatic venules with similar size (Fig. 3). The vessels displayed a smooth border
in their vascular walls and often branched into smaller vessels, most
of which drained into sinusoids (Fig. 1). In some places, the vessels
drained directly into the terminal hepatic venules. To determine the
origin of these neovessels, a bolus of fluorescein sodium was injected
via the carotid artery. The fluorescent dye appeared first in the neovessels within 3 s following the injection and then became visible in sinusoids seconds later (Fig.
4). These results strongly suggest that
the neovessels originate from the hepatic arterial system. This was
further confirmed by clamping the common hepatic artery, which
substantially decreased blood flow in the neovessels. Releasing of the
clamp immediately restored the flow rate in the neovessels.
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Response of Neovessels to Vasoconstrictors
To compare the responses of the neovessels and sinusoids to vasoactive substances, vasoconstrictors,
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In separate experiments, IRL-1620, an endothelin receptor B agonist,
was infused through the portal vein in a concentration of 5 pmol · min1 · 100 g body
wt
1 for 20 min. IRL-1620 did not significantly change PP
in the PPVL group, but it induced marked increase of PP in the sham
group (Fig. 6A). Neither the
MAP nor the average sinusoidal diameter showed significant difference
in either group following infusion of IRL-1620 (Fig. 6, B
and C). IRL-1620, previously shown to induce an increase in
PP and heterogeneous responses of sinusoidal flow in normal rats
(1), induced massive sinusoidal flow shutdown in some
areas of the PPVL livers, which was manifested with the decreased
number of perfused sinusoids (Fig. 6D). However, blood flow
in the neovessels remained unaltered. No change in vascular diameter
was detected in these vessels either. In the areas where the sinusoidal
shutdown of blood flow occurred, the neovessels were seen to supply
blood flow to the neighboring acini, which was revealed by the clear
staining of injected fluorescein sodium (Fig.
7). This observation strongly suggests
that the neovessels provide an important route for blood flow
compensation under conditions in which the portal blood flow is
deprived.
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Effect of Portal Vein Clamping
To evaluate the relative contribution of hepatic artery blood flow in feeding the sinusoids following PPVL, the portal vein was temporarily clamped while the sinusoidal flow was monitored. PI decreased only by 9.5% after clamping the portal vein in the PPVL group, whereas the sham group showed a decrease by 71.2%, suggesting an important compensatory role for the hepatic artery system following PPVL (Fig. 8).
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SEM of Hepatic Vascular Casts
To confirm that this vascular remodeling is not limited to the surface of the liver, SEM of corrosion casts of hepatic vessels was performed. Extensive growth of tortuous vessels was observed around the portal vein at the level of the hepatic hilum following PPVL (Fig. 9C). A greater number of hepatic arterial vessels were found surrounding the intrahepatic portal veins with numerous branches twisted, clustered, and disorganized in the PPVL group (Fig. 9D). In contrast, the hepatic arteries in sham showed fewer, relatively straight, and orderly vessels next to the portal vein (Fig. 9, A and B). These tortuous and clustered vessels of the hepatic artery branches were seen to a different degree in all three corrosion casts of the PPVL group.
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DISCUSSION |
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The liver receives its blood supply from both the portal vein and the hepatic artery. Although the portal venous flow comprises ~75% of the total blood flow volume to the liver, the liver does not control the volume of portal blood flow. Intrahepatic regulation in total hepatic blood flow is thus mediated by regulation of hepatic arterial blood flow via the HABR (14). HABR increases hepatic artery blood flow in response to acutely decreased portal venous flow. Kawasaki et al. (11) reported a significant increase of hepatic arterial flow (436% increase compared with sham) in the acute PPVL model. This increase in hepatic arterial flow could well be induced by HABR. However, studies have been limited to the acute response of hepatic arterial flow after the change of portal venous flow, and no previous reports have shown the chronic effects of portal venous flow reduction on the hepatic arterial system. In particular, the functional and morphological changes in the hepatic vasculature, especially at microvascular levels, following PPVL are unknown. In the present study, we report for the first time the observation of neovascularization from the hepatic arterial system that takes place to accommodate the permanent reduction of the portal venous flow following PPVL in rats. We therefore propose that this vascular remodeling serves as an important mechanism in regulating total hepatic blood flow under conditions in which the portal venous blood flow is chronically compromised.
In this model of PPVL-induced portal hypertension, we did not detect significant changes in sinusoidal perfusion when compared with the sham-controlled animals. The sinusoidal density, sinusoidal diameter, RBC velocity in the sinusoids, and calculated sinusoidal volumetric flow and PI showed no significant difference between sham-operated and PPVL rats 2 wk (data not shown) or 6 wk after PPVL. No differences in the enzyme levels of LDH and ALT were observed between the two groups 2 wk after the operation. These data raise the essential question: what mechanisms exist to compensate for the blood flow loss due to the PPVL procedure, ultimately maintaining the integrity of sinusoidal perfusion and preventing the hepatocyte injury? The answer lies, at least partially, in neovascularization initiating from the hepatic arterial system.
Using in vivo intravital fluorescence microscopy, we were able to observe extensive neovascularization in the livers that were subjected to PPVL for 2 or 6 wk. There was no significant difference in frequency of the neovessels observed following PPVL at either 2 or 6 wk. The neovessels were easily identifiable in 35.1% of PPVL rats that were subjected to the procedure (Fig. 1). Because of the difficulty of maneuvering the liver for microscopic viewing, only a small area of the left lobe was viewed in each rat. In the PPVL rats in which no neovessels were seen in the limited viewing area, no attempt was made to search for the neovessels in other areas or other lobes. However, it was clear that the phenomenon of neovascularization we observed was specific for the PPVL rats because no neovessels were identified in the sham-operated rats unless severe adhesions occurred on the surface of the observed liver lobe. As seen in Fig. 1, the neovessels were clearly distinguished from sinusoids, with smooth vascular walls. The size of the vessels ranged from 8 to 60 µm in diameter. The blood flow velocity was extremely fast, with RBC velocity >2,000 µm/s even in the smaller vessels (Fig. 3). The morphological and functional properties of those neovessels clearly demonstrated characteristics of arterioles. Injection of fluorescein sodium via the carotid artery showed the appearance of the fluorescent dye first in the neovessels (within 3 s) and then in sinusoids ~8-10 s later. These results provide strong evidence that these neovessels originate from the hepatic arterial system and not from the portal vein because the fast appearance of the dye in these neovessels following intra-arterial injection indicates a direct traveling route from the aorta to the neovessels through the hepatic artery. On the other hand, the delayed appearance of the dye in the sinusoids can be explained by the extra traveling distance of the splanchnic circulation and the portal vein. This was further confirmed by clamping the common hepatic artery, which substantially decreased blood flow in those vessels. Releasing the clamp immediately restored the blood flow rate in the neovessels.
Since the limitation of intravital microscopy in observing the hepatic microcirculation only allows us to view the peripheral region of the liver, we performed additional experiments in which we examined the hepatic vascular corrosion casts under SEM. Substantial neovascularization from the hepatic arterial vessels observed on the hepatic vascular casts by SEM (Fig. 8) confirmed that the vascular remodeling also occurred around the smaller portal vein (diameter ~300 µm) as well as at the hilum of the liver in the PPVL group. These results suggest that the occurrence of vascular remodeling is not limited to the surface of the liver.
To determine the functional importance of these neovessels, PE and
IRL-1620 were administered through the carotid artery and the portal
vein, respectively. PE, an -adrenergic receptor agonist, which has
been shown previously to induce vasoconstriction in the arterial
vasculature and portal vein but not in sinusoids (1),
resulted in transient but substantial constriction and decrease in
blood flow in the neovessels. In contrast, the diameter of the
neovessels was not changed following PE injection via the portal vein
even though the sinusoidal flow was substantially decreased. These
results demonstrated the functional similarity of the neovessels to
arterioles. It is conceivable that those neovessels play an important
role in regulating the hepatic blood flow. It was observed that those
vessels make connections with more than one acinus and their branches
directly drained into sinusoids and the terminal hepatic venules in
some cases. Their functional importance was exemplified by the
experiment using IRL-1620, an endothelin receptor B agonist. We have
previously shown that the compound produces heterogeneous hemodynamic
responses in the hepatic microcirculation depending on activation of
receptor B subtypes (1). In the present study, we showed
that IRL-1620 induced a significant decrease in sinusoidal flow in both
sham and PPVL rats without changing the average sinusoidal diameter (Fig. 6). Interestingly, the sinusoidal flow was substantially diminished in some acini with infusion of IRL-1620 via the portal vein,
whereas the blood flow in the neovessels was well maintained and
essentially fed the flow-deprived acini. These observations suggest
that the neovessels contribute to the regulation of the hepatic
microvascular perfusion, and neovascularization following PPVL may
represent a novel mechanism of regulation of the hepatic blood flow
under conditions in which the portal venous flow is chronically
deprived. The difference seen in the number of perfused sinusoids
between sham and PPVL after IRL-1620 can most likely be attributed to
the fact that the neovessels only supply the sinusoids they directly
feed, whereas the areas without the supply by the neovessels suffer a
greater loss of flow perfusion.
The notion that the hepatic artery system compensates and becomes an important blood supply to the liver after permanently depriving the portal venous flow has been previously reported by McCuskey et al. (18). This is further supported by the present study, in which the sinusoidal perfusion was compared between sham and PPVL groups while portal flow was temporarily stopped by clamping the portal vein. The sinusoidal perfusion decreased only by 9.5% in PPVL rats, whereas it decreased by 71.2% in sham when the portal flow was occluded (Fig. 8). These results clearly demonstrate the importance of the hepatic blood flow as the dominant supply to the liver following PPVL. It is likely that the hepatic arterial system becomes the primary blood supplier for the sinusoids following PPVL by remodeling the existing vasculature and/or generating neovessels.
Although the existence of the hepatic artery-originated vessels following PPVL was clearly demonstrated in the present study, whether these vessels were newly synthesized via angiogenesis or simply the result of hypertrophy and expansion of the existing arterial vessels is still of debate. Previously, McCuskey et al. (18) reported that numerous arteriosinus twigs (branches of hepatic arterioles terminating in some of the sinusoids) were seen in rats with portacaval anastomosis, and they were observed to be greatly enlarged and contain higher rates of blood flow than the sinusoids. The neovessels seen in the present study following PPVL seem to be different from the arteriosinus twigs. They were not generally seen across the surface of the liver and tended to cluster in isolated areas. Moreover, we did not observe similar vessels in the sham-operated rats. Most recently, Ekataksin (6) showed an isolated artery that ramified and anastomosed extensively to form a capsular plexus in the liver of humans and other mammals. It could be argued that the vessels we observed in this study using intravital microscopy are vessels of the capsular plexus but are not neovessels generated through angiogenesis. The evidence available, however, would argue against that possibility. First of all, the capsular plexus fed by the isolated artery was only observed in mammals such as pig, ox, horse, and human and was not found in rats and mice according to the study of Ekataksin (6). Secondly, we not only observed the vessels in isolated areas of the liver surface in PPVL rats with intravital fluorescence microscopy but also observed the extensive network of the hepatic arterial vessels intrahepatically, particularly at the hilum, using SEM on the hepatic vascular cast. Furthermore, we have never seen the capsular arterial vessels in livers of normal and sham-operated rats. Therefore, the results of the current study favor the notion that the vessels we observed after PPVL are newly synthesized as a result of angiogenesis.
Neovascularization or angiogenesis is essential in a variety of conditions such as inflammation, hypoxia, increased shear stress, and wound healing (5, 8, 9, 19, 21, 25). In the liver, the development of solid tumors, chronic viral infection by hepatitis C virus, and cirrhosis have been proposed as inducing factors for angiogenesis (12, 17, 19, 20, 28). Neovascularization observed in the current PPVL model is likely to be induced by hypoxia resulting from diminished portal venous flow due to PPVL. Another possibility for neovascularization is the increased shear stress, which can be induced by the sudden increase of hepatic arterial flow due to HABR following PPVL (9, 10). Using a rat PPVL model, Sumanovski et al. (24) reported increased angiogenesis in the abdominal cavity using quantitative angiogenesis assay in vivo. However, to our knowledge, our present report is the first time that neovascularization in the liver after PPVL was reported. This discovery may be of functional significance in several areas. First, understanding the mechanisms that control the angiogenesis derived from the hepatic arterial system following the chronic restriction of portal venous flow may help us understand the fundamental mechanisms of how the liver regulates its total blood flow under the chronic disease conditions that affect the portal venous flow. Second, the finding will help us learn more about prehepatic portal hypertension resulting from extrahepatic portal vein obstruction, a relatively rare disease occurring primarily in children. The information on neovascularization after PPVL will certainly allow us to gain more insight into pathological changes in the liver and how the liver responds to those changes under disease conditions. Finally, angiogenesis plays an essential role in the growth of tumors. Formation and development of the hepatic tumor requires extensive angiogenesis from the hepatic artery. The results reported here suggest that PPVL can be used as a simple useful animal model to conduct mechanistic studies of hepatic artery-derived angiogenesis associated with hepatic tumors.
In summary, we investigated the microhemodynamic and structural changes of microvessels in the rat liver following PPVL. Using intravital microscopy, we showed extensive neovascularization of the hepatic arterial system in the rat liver subjected to PPVL. The neovessels supplied blood flow directly to sinusoids or terminal hepatic venules. This phenomenon may represent a very important regulatory mechanism that maintains total hepatic blood flow under conditions in which the portal flow is chronically compromised. In addition, our results suggest that the rat model of PPVL can serve as a useful animal model to study hepatic artery-derived angiogenesis in hepatic tumors.
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ACKNOWLEDGEMENTS |
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We thank Sandra F. Zane and John Hudok for their expert technical assistance in the SEM experiments.
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FOOTNOTES |
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This study was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-38201) and faculty grants from the University of North Carolina at Charlotte.
Address for reprint requests and other correspondence: J. X. Zhang, Biology Dept., Univ. of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223 (E-mail: jxzhang{at}emailuncc.edu).
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. Section 1734 solely to indicate this fact.
Received 20 April 2000; accepted in final form 4 August 2000.
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