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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) pathways are crucial to angiogenesis, a process that contributes significantly to the pathogenesis of portal hypertension. This study determined the effects of inhibition of VEGF and/or PDGF signaling on hyperdynamic splanchnic circulation and portosystemic collateralization in rats with completely established portal hypertension, thus mimicking the situation in patients. Portal vein–ligated rats were treated with rapamycin (VEGF signaling inhibitor), Gleevec (PDGF signaling inhibitor), or both simultaneously when portal hypertension was already fully developed. Hemodynamic studies were performed by transit-time flowmetry. The extent of portosystemic collaterals was measured by radioactive microspheres. The expression of angiogenesis mediators was determined by Western blotting and immunohistochemistry. Combined inhibition of VEGF and PDGF signaling significantly reduced splanchnic neovascularization (i.e., CD31 and VEGFR-2 expression) and pericyte coverage of neovessels (that is, α-smooth muscle actin and PDGFR-β expression) and translated into hemodynamic effects as marked as a 40% decrease in portal pressure, a 30% decrease in superior mesenteric artery blood flow, and a 63% increase in superior mesenteric artery resistance, yielding a significant reversal of the hemodynamic changes provoked by portal hypertension in rats. Portosystemic collateralization was reduced as well. Conclusions: Our results provide new insights into how angiogenesis regulates portal hypertension by demonstrating that the maintenance of increased portal pressure, hyperkinetic circulation, splanchnic neovascularization, and portosystemic collateralization is regulated by VEGF and PDGF in portal hypertensive rats. Importantly, these findings also suggest that an extended antiangiogenic strategy (that is, targeting VEGF/endothelium and PDGF/pericytes) may be a novel approach to the treatment of portal hypertension. (HEPATOLOGY 2007.)

Portal hypertension (PH) is the most important complication of chronic liver diseases and is a leading cause of mortality and liver transplantation worldwide.1 A characteristic feature of the PH syndrome is development of hyperdynamic splanchnic circulation, with an increase in blood flow in splanchnic organs draining into the portal vein and a subsequent increase in portal venous inflow.2 Such increased portal venous inflow is a significant factor in maintaining and worsening portal pressure elevation. We have recently demonstrated that an increase in the splanchnic vascular bed size mediated by a vascular endothelial growth factor (VEGF)–dependent angiogenic process significantly contributes to increased overall blood flow in splanchnic tissues of PH animals.3–6 In addition, this VEGF-dependent angiogenesis also plays a crucial role in the formation of portal-systemic collateral vessels, which include the gastroesophageal varices.3–6 Collectively, these prior studies highlighted the importance of angiogenesis in the pathogenesis of PH and suggested that antiangiogenic treatment might be a promising therapeutic strategy to prevent the progression of the PH syndrome.

In clinical practice, however, an antiangiogenic drug would have to be given to patients presenting with PH at rather advanced stages.7 Thus, it was imperative to confirm our previous prophylactic studies by therapeutic models that most closely mirror advanced human PH and to determine whether antiangiogenic agents could not only prevent but also get the circulatory abnormalities associated with PH to revert once they are fully developed. Therefore, we evaluated long-term antiangiogenic treatment in an animal model of established portal hypertension induced by partial portal vein ligation.

Recent studies indicate that in the process of neovascularization, VEGF plays the predominant role in the formation of new blood vessels by activating proliferation of endothelial cells and the subsequent formation of an endothelial tubule, whereas maturation of newly formed vessels is mainly modulated by proangiogenic platelet-derived growth factor (PDGF), which regulates the investiture of the endothelial tubule with mural cell and pericyte populations, thereby stabilizing the vascular architecture of the nascent vessel.8–12 Thus, it can be hypothesized that established PH could be associated with overexpression of PDGF in splanchnic tissues and that reversal of splanchnic circulatory changes associated with PH might be greater after the combined blockade of VEGF and PDGF (i.e., after simultaneous targeting of endothelial cells and pericytes) than after either alone.

The purpose of the study was to test this hypothesis. We used rapamycin and Gleevec as strategies to inhibit the VEGF and PDGF signaling pathways, respectively.13–18 We chose these drugs because, in addition to being well-known inhibitors of the VEGF and PDGF pathways, they are already broadly used in the treatment of several human malignancies and have been proven to be well tolerated in these indications.13–18

Our study is the first to our knowledge to describe the successful therapeutic use of the combination of rapamycin and Gleevec in an animal model of PH. We demonstrate that treatment with rapamycin plus Gleevec reversed the hemodynamic and vascular changes provoked by PH in the partial portal vein ligated rat model. Taking into account the limitations of experimental studies, we believe our findings will stimulate consideration of this novel therapeutic approach in patients suffering from advanced PH, in which VEGF and PDGF signaling pathways may be up-regulated, as suggested by the findings in the currently investigated PH animals.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Materials.

Rapamycin (RAPA) was purchased from Wyeth Europe (Berkshire, UK). Gleevec was from Novartis (West Sussex, UK). Polyclonal antibodies against rat CD31 (sc-8306 and sc-1506), VEGF (sc-507), VEGF receptor–2 (VEGFR-2; sc-315), PDGF-BB (sc-7878), PDGF receptor-β (PDGFR-β sc-1627), and GAPDH (sc-32233) were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti–α-smooth muscle actin (α-SMA; A-2547) and anti-α-tubulin (T-9026) were from Sigma (St. Louis, MO). Peroxidase-conjugated secondary antibody was from Stressgen (Sidney, British Columbia, Canada). 51Cr-labeled microspheres were from Perkin-Elmer (Boston, MA). A protein assay kit and nitrocellulose membranes were from Bio-Rad (Hercules, CA). All other reagents and chemicals were from Sigma.

Animals and Treatments.

Portal hypertension (PH) was induced in male Sprague-Dawley rats (300–350 g body weight) by partial portal vein ligation (PPVL) as previously described.19 Briefly, under anesthesia (80 mg/kg ketamine plus 12 mg/kg xylacin, intramuscularly), calibrated constriction of the portal vein was performed using a single ligature of 3-0 silk tied around the portal vein and a 20-gauge blunt-tipped needle. The needle was then removed, leaving a calibrated constriction of the portal vein. In sham-operated (SO) rats, the portal vein was isolated and similarly manipulated but not ligated.

In a first experimental protocol, rats were treated with the VEGF signaling inhibitor RAPA (2 mg kg−1 day−1 intraperitoneally; PPVL: n = 12, SO: n = 4),13, 14 the PDGF signaling inhibitor Gleevec (20 mg kg−1 day−1 intraperitoneally; PPVL: n = 6, SO: n = 5),15–18 a combination of RAPA plus Gleevec (PPVL: n = 4, SO: n = 4), or vehicle (700 μL of NaCl 0.9%; PPVL: n = 13, SO: n = 6) for 5 days starting immediately after PPVL or SO.

In a second protocol, rats were treated with RAPA (PPVL: n = 9, SO: n = 5), Gleevec (PPVL: n = 9, SO: n = 5), RAPA plus Gleevec (PPVL: n = 8, SO: n = 5), or vehicle (PPVL: n = 12, SO: n = 4) over a 2-week period, commencing 1 week after PPVL (or SO in controls), when portal hypertension was fully established.3, 4

Doses of RAPA and Gleevec were selected on the basis of earlier work showing that these doses efficiently inhibit VEGF and PDGF signaling, respectively.13–18 All procedures were approved by the Laboratory Animal Care and Use Committee of the University of Barcelona and were conducted in accordance with the “Guide for the Care and Use of Laboratory Animals” (NIH publication 86-23, revised 1985).

Hemodynamic Studies.

Under anesthesia, PE-50 catheters were introduced into a femoral artery and the portal vein and connected to highly sensitive pressure transducers to measure arterial pressure (MAP, mm Hg) and portal pressure (PP, mm Hg), respectively. Then, a nonconstrictive perivascular transit-time ultrasonic flow probe (Transonic Systems, New York, NY) was placed around the superior mesenteric artery and connected to a flowmeter to measure superior mesenteric artery blood flow (SMABF, mL min−1 100 g−1). Superior mesenteric artery resistance (SMAR, mmHg mL−1 min−1 100 g−1) was calculated as: (MAP − PP)/SMABF.20

Determination of Extent of Portal-Systemic Collateral Vessel Formation.

The extent of portal-systemic collateralization was quantified by injection of 51Cr-labeled microspheres (diameter 15 ± 3 μm; specific activity 52.68 mCi/g) into the spleen.3–6 Rats were then sacrificed, and radioactivity in liver and lungs was determined in a γ-scintillation counter. This enabled quantification of the degree of collateral formation on a scale from 0% to 100% by this equation: collateralization (%) = (lung radioactivity/[lung radioactivity + liver radioactivity]) × 100.

Western Blot Analysis.

Tissue samples were homogenized in ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.4), 0.1 mM EGTA, 0.1 mM EDTA, 2 mM leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1% Nonidet P40, 0.1% sodium dodecyl sulfate, and 0.1% deoxycholate. Samples were then centrifuged for 30 minutes at 10,000g. The supernatant was collected, and the protein concentration was measured using a colorimetric assay. Proteins (100 and 200 μg) were separated by SDS-PAGE electrophoresis and subsequently transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk in incubation buffer and incubated with antibodies against rat CD31, VEGF, VEGFR-2, PDGF, PDGFR-β, and α-SMA (1:500 dilution). Bound antibody was detected with peroxidase-linked secondary antibody and a chemiluminescence detection system. Loading accuracy was evaluated by membrane rehybridization with monoclonal antibodies against α-tubulin or GAPDH.3–6

Immunohistochemistry.

Immunohistochemical detection of CD31 and α-SMA was carried out on sections from paraffin-embedded mesenteric tissues fixed in 10% neutral-buffered formalin solution. Endogenous peroxidase activity was blocked using methanol containing 0.3% H2O2. Antigen retrieval for CD31 detection was performed using citrate buffer. Nonspecific antibody binding was prevented by incubation with 10% normal horse serum. Sections were incubated with primary antibodies against CD31 (diluted 1:200), and α-SMA (diluted 1:500), and then the bound antibodies were visualized using diaminobenzidine (DAB) as the chromogen. For the negative control, phosphate-buffered saline was used instead of the primary antibody.

Measurement of Vascular Areas.

Tissue samples were fixed in 10% neutral-buffered formalin and embedded in paraffin. Tissues were sectioned (thickness of 2 μm), and slides were stained with hematoxylin and eosin (H&E) or immunostained for CD31 and α-SMA. Quantitative analysis of angiogenesis was performed with the assistance of ImageJ version 1.37 software (NIH, Bethesda, MD) by scanning the entire tissue section and counting the areas occupied by vascular structures. We obtained a value from each tissue section (1 section per rat) and then averaged the results from individual sections in each experimental group (3–5 sections per group). Group values reflect the average readings from all sections in the group. The results of neovascularization measurements are expressed as the mean area (± SEM; expressed in square micrometers) occupied by vascular structures in a tissue field measuring 1 mm2.

Determination of Rapamycin Concentration in Whole Blood.

Blood rapamycin was measured by high-performance liquid chromatography (HPLC) coupled with tandem ultraviolet detection (HPLC-UV) as previously described.21 In brief, to each whole-blood sample was added sodium acetate buffer (0.1M, pH 4.7), demethoxyrapamycin (2.5 μg/mL), and 1-chlorobutane. After sample centrifugation, the organic extract was evaporated at 45°C under nitrogen gas. The dried extracts were reconstituted with methanol/H2O, and the supernatants were analyzed with a reverse-phase C18 column at 50°C under a flow rate of 1.1 mL/min for 30 minutes in the HPLC system. Rapamycin was detected by ultraviolet absorption at 278 nm. The calibration range was 2.5–100 ng/mL.

Statistical Analysis.

Values are expressed as means ± SEMs and compared using the unpaired Student t test and ANOVA analysis. Statistical significance was established at P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Increased VEGF and PDGF Expression in PH Rats.

Given the importance of growth factors VEGF and PDGF in vascular formation and maturation, we hypothesized that their expression could be up-regulated in PH and that simultaneous inhibition of both the VEGF and the PDGF pathways might be expected to show greater antiangiogenic activity than inhibition of only 1 of these signaling pathways. To demonstrate this, we first measured VEGF and PDGF protein expression in intestines from SO rats and also 1, 5, 10 and 20 days after induction of PH. PDGF expression was minimal in SO rats (Fig. 1). After PPVL, however, expression of PDGF increased progressively, with the highest expression observed on day 10 (Fig. 1). VEGF expression also increased as the PH syndrome progressed, but peak VEGF expression (on day 5) preceded the appearance of the PDGF peak (Fig. 1).

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Figure 1. Expression of VEGF and PDGF on days 1, 5, 10, and 20 after PPVL and also in SO rats. Representative blots are shown on top, and quantification of expression normalized to α-tubulin and expressed as arbitrary units is shown on bottom: (a) P < 0.05 versus SO rats.

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Effects of Angiogenesis Blockade When PH Was Actively Developing.

Next, we determined the effects of VEGF and/or PDGF inhibition when PH was actively developing in rats. For that purpose, treatment with RAPA, Gleevec, a combination of RAPA plus Gleevec, or vehicle was started immediately after PPVL or SO and terminated 5 days later.

RAPA effectively prevented increased VEGF expression (Fig. 2A) and the formation of new blood vessels in the splanchnic territory of PPVL rats, as reflected by significant inhibition in the intestinal expression of vascular endothelial cell markers CD31 and VEGFR-2 (Fig. 2A).11 These findings were further identified on mesenteric sections stained with H&E and immunostained for CD31 (Fig. 2B). Quantitative analysis confirmed a significantly (P < 0.05) greater vascular area in PPVL-vehicle rats than in SO-vehicle rats and a significant decrease in vascular area after RAPA treatment in PPVL rats but not in SO rats (Fig. 2C). We also found that expression of the vascular smooth muscle cell marker α-SMA was not significantly modified by the RAPA treatment in the mesentery from PPVL or SO rats (Fig. 2D), indicating that, at least in the experimental settings used in this study, RAPA had no significant antiproliferative effects on smooth muscle. These Western blot results show that the amount of α-SMA per cell was not modified by RAPA, although the net amount of α-SMA in the mesentery tissue was decreased by RAPA as a consequence of the overall reduction in vascularization caused by this treatment, as we demonstrated by immunohistochemistry (Fig. 2B).

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Figure 2. Effects of RAPA on angiogenesis mediators when PH was actively developing. (A) Expression of CD31, VEGF, and VEGFR-2 in the intestines of PPVL rats treated with RAPA or vehicle. Representative Western blots are shown on the left, and quantification of protein expression normalized to α-tubulin and expressed as percentages of controls (PPVL–vehicle) is shown on the right. (B) Representative histological images of mesentery tissues stained with H&E or immunostained for CD31 and α-SMA from PPVL and SO rats treated with RAPA or vehicle. (Original magnification ×40 [×100 magnification in CD31 staining of PPVL–vehicle].) (C) Quantitative analysis of neovascularization in the mesentery from RAPA- or vehicle-treated PPVL and SO rats. (a) P < 0.05 versus PPVL–vehicle. (D) Expression of α-SMA in the mesentery of PPVL and SO rats treated with RAPA or vehicle. Representative Western blot is shown on the left, and quantification of protein expression normalized to GAPDH and expressed as arbitrary units is shown on the right.

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The RAPA-induced inhibition of splanchnic neovascularization was paralleled by significant prevention of the hyperdynamic splanchnic circulation of PPVL rats, as indicated by a significant 24% decrease in SMABF and a 66% increase in SMAR compared with that in vehicle-treated PPVL rats (Fig. 3), reaching values not significantly different from those observed in RAPA-treated SO rats (Fig. 3). Despite the decrease in SMABF, PP was not reduced by RAPA in PPVL animals. Instead, it increased compared with that in vehicle-treated PPVL rats (P = 0.19; Fig. 3). That was most likely a result of the marked, 67% reduction in portal-systemic collateral vessel formation observed in PPVL rats after VEGF signaling inhibition (Fig. 3). Notably, the extent of portal-systemic collaterals in PPVL rats after RAPA treatment was not statistically different from that in RAPA-treated SO rats (P = 0.12; Fig. 3). In addition, RAPA increased MAP, both in PPVL rats (108.68 ± 3.70 mm Hg in vehicle vs. 131.18 ± 3.33 mm Hg in RAPA; < 0.001) and in SO animals (111.02 ± 4.53 mm Hg in vehicle vs. 140.73 ± 2.97 mm Hg in RAPA; P < 0.01), whereas it did not significantly change heart rate compared with the vehicle (PPVL rats: 348.58 ± 9.77 vs. 356.77 ± 10.73 beats/min, respectively; P = 0.6; SO rats: 326.00 ± 15.11 vs. 324.17 ± 14.18 beats/min, respectively; P = 0.9). Values of PP, SMABF, and SMAR and the extent of collateralization were similar when comparing SO rats treated with RAPA or with vehicle (Fig. 3). Whole-blood RAPA concentration in RAPA-treated rats was 46.63 ± 3.5 ng/mL.

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Figure 3. Effects of RAPA on splanchnic hemodynamic parameters and extent of portal-systemic collateral vessels when PH was actively developing. (a) P < 0.05 versus SO–vehicle; (b) P < 0.05 versus SO–RAPA; (c) P < 0.05 versus PPVL–vehicle.

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In addition, Gleevec had no significant effects on any of the hemodynamic parameters studied (Table 1). Moreover, the combination of RAPA+Gleevec had hemodynamic effects that were not significantly different from those observed after treatment with RAPA alone (Table 1). In PPVL rats, the decrease in the extent of portal-systemic collaterals observed after RAPA+Gleevec (78% decrease) was more pronounced than that observed after RAPA alone (67% decrease), but the difference was not statistically significant (14.77 ± 6.48% vs. 21.64 ± 7.30%, respectively; P = 0.6).

Table 1. Hemodynamic Effects of Treatment with Gleevec or Combination of RAPA+Gleevec for 5 Days Starting Immediately after Induction of Portal Hypertension or Sham Operation
 PP (mm Hg)SMAR (mm Hg mL−1 min−1 100 g−1)MAP (mm Hg)HR (beats/min)
  • *

    P < 0.05 versus PPVL-vehicle.

  • P < 0.05 versus PPVL-Gleevec.

  • P < 0.05 versus PPVL-RAPA+Gleevec.

PPVL-Vehicle (n = 13)14.9 ± 1.014.4 ± 0.8108.7 ± 3.7356.8 ± 10.7
PPVL-Gleevec (n = 6)14.0 ± 1.314.2 ± 1.1116.7 ± 7.5378.0 ± 13.5
PPVL-RAPA+Gleevec (n = 4)13.0 ± 0.719.5 ± 1.8*144.2 ± 4.8*363.0 ± 9.2
SO-vehicle (n = 6)6.8 ± 0.5*20.7 ± 1.7*111.0 ± 4.5324.2 ± 14.2
SO-Gleevec (n = 5)5.3 ± 0.318.3 ± 2.797.4 ± 6.4344.0 ± 15.4
SO-RAPA+Gleevec (n = 4)7.2 ± 0.525.0 ± 4.5123.0 ± 12.2320.5 ± 21.1

Collectively taken, these data indicate that blockade of VEGF signaling by RAPA markedly prevents the development of hyperdynamic splanchnic circulation, splanchnic neovascularization, and portal-systemic collateral formation when administered at the time of portal hypertension initiation.

Effects of Angiogenesis Blockade When PH Was Completely Established.

We then addressed the effects of inhibiting VEGF signaling, PDGF signaling, or signaling by both pathways simultaneously on rats that had an already established PH syndrome. For this purpose, 7 days after induction of PH (or after SO), rats were given RAPA, Gleevec, a combination of RAPA and Gleevec, or vehicle over a 2-week period.

Therapy with RAPA alone in PPVL rats was effective in reducing the intestinal expression of VEGF and translated into substantial inhibition of splanchnic neovascularization, as indicated by significant down-regulation of intestinal CD31 and VEGFR-2 expression compared with that in vehicle-treated PPVL rats (Fig. 4A). RAPA did not significantly affect expression of PDGF, PDGFR-β, and α-SMA in PPVL rats (Fig. 4A). To examine this further, we identified vascular structures in mesentery sections by H&E staining and by immunohistochemical staining for CD31 and α-SMA (Fig. 4B). Quantitative analysis of neovascularization revealed that vehicle-treated PPVL mesenteries were more angiogenic than were those of SO-vehicle and that RAPA treatment markedly and significantly reduced this neovascularization (Fig. 4C).

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Figure 4. Effects of RAPA, Gleevec, or RAPA+Gleevec on angiogenesis mediators when PH was completely established. (A) Expression of CD31, VEGF, VEGFR-2, PDGF, PDGFR-β, and α-SMA in the intestines of PPVL rats after treatment with RAPA, Gleevec, RAPA+Gleevec, or vehicle. Representative blots are shown on the left, and quantification of expression normalized to α-tubulin and expressed as percentages of controls (PPVL vehicle) is shown on the right: (a) P < 0.05 versus PPVL–vehicle; (b) P < 0.05 versus PPVL–RAPA. (B) Representative mesentery photographs and representative histological images of mesentery sections stained with H&E or immunostained for CD31 and α-SMA from PPVL and SO rats treated with RAPA or vehicle. Original magnification, ×40. (C) Quantitative analysis of neovascularization in the mesentery from PPVL and SO rats treated with RAPA or vehicle. (a) P < 0.05 versus PPVL–vehicle.

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In PPVL rats, hyperdynamic splanchnic circulation was also significantly reduced after RAPA treatment, with a 17% decrease in SMABF and a 38% increase in SMAR (Fig. 5). Of note, PP was significantly decreased by RAPA (by 17%, P < 0.01; Fig. 5), whereas MAP increased compared with vehicle (117.93 ± 2.32 vs. 104.12 ± 3.62 mm Hg, respectively; P < 0.05). RAPA did not significantly modify heart rate (351.83 ± 24.14 beats/min in vehicle vs. 316.67 ± 17.43 beats/min in RAPA; P = 0.3). In contrast to the inhibitory effect of RAPA on splanchnic neovascularization, PP, and mesenteric blood flow, the degree of portal-systemic collateralization was not significantly different when comparing RAPA-treated and vehicle-treated rats (Fig. 5). In SO rats, RAPA did not significantly affect any of the hemodynamic parameters studied (Fig. 5). Whole-blood RAPA concentration (steady-state) in RAPA-treated rats was 16.3 ± 1.5 ng/mL, which was in the 10–60 ng/mL range required to get maximum effectiveness with limited toxicity.21

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Figure 5. Effects of RAPA, Gleevec, or RAPA+Gleevec on splanchnic hemodynamic parameters and the extent of portal-systemic collaterals when PH was completely established. (a) P < 0.05 versus SO–vehicle; (b) P < 0.05 versus SO–RAPA; (c) P < 0.05 versus SO–Gleevec; (d) P < 0.05 versus SO–RAPA+Gleevec; (e) P < 0.05 versus PPVL–vehicle; (f) P < 0.01 versus PPVL–RAPA.

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Treatment with Gleevec alone significantly diminished the intestinal expression of PDGF as well as that of pericyte-specific markers α-SMA and PDGFR-β in PPVL rats compared with vehicle but did not significantly modify splanchnic neovascularity (i.e., CD31 and VEGFR-2 expression), SMABF, SMAR, PP, and the extent of portal-systemic collaterals (Figs. 4A and 5). MAP was significantly higher in Gleevec-treated PPVL rats than in those receiving vehicle (118.57 ± 4.96 vs. 104.12 ± 3.62 mm Hg, respectively; P < 0.05), whereas heart rate was similar in both groups (359.57 ± 8.16 vs. 351.83 ± 24.14 beats/min, respectively; P = 0.8). In SO rats, Gleevec had no significant hemodynamic effects (Fig. 5).

Interestingly, treatment of PPVL rats with a combination of RAPA plus Gleevec effectively inhibited expression of VEGF and PDGF protein and also caused a marked and significant reduction in the extent of splanchnic neovascularization (i.e., in expression of endothelial cell markers CD31 and VEGFR-2) and in the perivascular cell coverage of neovessels (i.e., in expression of pericyte markers α-SMA and PDGFR-β), shown in Fig. 4A. In addition, this therapy translated into hemodynamic effects as marked as a 40% decrease in PP, a 30% decrease in SMABF, and a 63% increase in SMAR (Fig. 5), whereas MAP increased compared with vehicle (127.05 ± 3.37 vs. 104.12 ± 3.62 mm Hg, respectively; P < 0.01), and heart rate was unchanged (366.00 ± 6.66 vs. 351.83 ± 24.14 beats/min, respectively; not significant). Importantly, RAPA+Gleevec yielded significantly greater effects than the addition of the effects of either drug alone. Of note, the RAPA+Gleevec treatment was also accompanied by a modest but significant decrease in the degree of portal-systemic collateral vessels (9% decrease, P < 0.05; Fig. 5). In SO rats, treatment with RAPA+Gleevec had no significant hemodynamic effects (Fig. 5).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

The present study demonstrates that the development of hyperdynamic splanchnic circulation and splanchnic neovasculature and the formation of portal-systemic collateral vessels in PH rats are in part VEGF-dependent angiogenic processes that can be significantly prevented by administration of the VEGF pathway inhibitor RAPA starting at the time of PH initiation. This is very much in line with our recent findings in which other angiogenesis inhibitors with different modes of action were used in experimental models of PH to interfere with the VEGF signaling pathway.3–6 Altogether, these results provide additional support for the idea that angiogenesis plays a key role in the pathogenesis of PH.

However, the principal novel findings of this study were derived from experiments in which antiangiogenesis treatments were begun when PH was already fully developed. The idea was to determine whether antiangiogenic agents could not only prevent the circulatory abnormalities associated with PH but also get them to revert once these were completely established. This is closer to the clinical situation of the PH patient, who is usually diagnosed and eventually treated when PH is already advanced and has caused clinical manifestations. Because VEGF and PDGF are both crucial to the angiogenic process, contributing to the formation, stabilization, and maturation of newly formed vessels,8–12 it seems reasonable to hypothesize that established PH should be associated with overexpression of not only VEGF but also PDGF and that greater benefit in reducing circulatory disorders associated with PH could be obtained by simultaneous targeting of the VEGF and PDGF pathways rather than by targeting either alone. Indeed, this study has demonstrated for the first time that in addition to VEGF, PH development is associated with progressive overexpression of PDGF, which reached its peak later in the course of PH than did VEGF overexpression. In addition, in rats with fully established PH, a 2-week course of treatment with RAPA induced a significant decrease in VEGF expression, which was associated with substantial attenuation of the increased splanchnic neovascularity (i.e., attenuation of overexpression of endothelial cell markers CD31 and VEGFR-2 and decrease in vascular area) and a marked reduction in PP and splanchnic hyperemia. These effects were specific for the neovasculature of PH rats because they were not observed in SO control animals. We therefore concluded that VEGF signaling is required not only for development but also for maintenance of the PH syndrome and that inhibition of the VEGF pathway results in significant attenuation of increased PP and of hyperdynamic splanchnic circulation in rats with advanced PH. However, despite the significant decreases in PP, SMABF, and splanchnic neovascularization, the extent of collateralization was unchanged. This finding is discussed later.

Treatment with the PDGF inhibitor Gleevec alone had no major effects other than reduced PDGF expression and decreased perivascular cell coverage of splanchnic neovessels, as indicated by the significantly decreased expression of pericyte-specific markers α-SMA and PDGFR-β. However, combined treatment with RAPA and Gleevec resulted in, on top of the expected significantly decreased expression of VEGF, VEGFR-2, CD31, PDGF, PDGFR-β, and α-SMA, virtually complete reversal of the increases in PP and SMABF, which were reduced by 40% and 30%, respectively. Notably, the magnitude of the effects of the combination treatment was greater than the addition of the effects of either drug alone, suggesting a synergistic regulatory interaction between the VEGF and the PDGF signaling pathways in mediating maintenance of the vascular and circulatory abnormalities observed in PH rats. These findings also have important clinical implications; in the absence of perivascular cells (i.e., after PDGF signaling inhibition), the endothelium is more vulnerable to antiangiogenic therapies targeting endothelial cells, such as a VEGF signaling blockade.22

As already noted, although VEGF inhibition suppressed the development of new portal-systemic collateral vessels, selective VEGF signaling blockade did not have a significant effect on the extent of already established collateral vessels. The simultaneous inhibition of VEGF plus PDGF signaling, despite much greater effects on PP and SMABF, caused a significant but mild reduction in the extent of portal-systemic collaterals. Why did the antiangiogenic therapy prevent the formation of collaterals when these vessels began to develop but not when collateralization was extensive? A possible explanation is that a measurable decrease in collateralization may take longer than the 2-week course of treatment evaluated in this study. It should be noted in this regard that patients with cirrhosis receiving an orthotopic liver transplant maintain increased portocollateral blood flow for months.23 Actually, the extent of portal-systemic collaterals decreased slightly (9% decrease) but significantly (P < 0.05) after the combined inhibition of VEGF plus PDGF signaling. On the other hand, it is also possible that there are intrinsic differences in the sensitivity of vessels to the lack of growth factors or differences in the extent of vessel maturation.8–10, 24, 25 In this regard, it is important to note that collaterals are frequently large-size vessels, suggesting a high degree of vessel maturation.26 Therefore, it could be that the dependence on VEGF and PDGF for collateral vessel growth and maintenance changes over time and that on their maturation, portal-systemic collaterals dependence from VEGF and PDGF is less accentuated. Nevertheless, the marked decrease in PP and splanchnic hyperemia in response to the combined VEGF and PDGF signaling inhibition suggests that even if collaterals remain, the possible risk that they could cause clinical complications could be substantially reduced by this treatment.26–28 Importantly, the use of RAPA and Gleevec as inhibitors of the VEGF and PDGF signaling pathways, respectively, has the additional advantages of being safe and effective in the treatment of several human malignancies, which has already been broadly documented.13–18

In conclusion, the results of this study provide new and important biological insights into the role of growth factors and angiogenesis in PH by suggesting the existence of a regulatory interaction between the VEGF and PDGF pathways to maintain hyperdynamic splanchnic circulation, splanchnic neovascularization, and increased portal pressure in PH rats and further underline their potential importance in therapeutic strategies for patient treatment based on the combined blockade of the VEGF and PDGF signaling pathways.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  • 1
    Bosch J, Pizcueta P, Feu F, Fernandez M, Garcia-Pagan JC. Pathophysiology of portal hypertension. Gastroenterol Clin North Am 1992; 21: 113.
  • 2
    Bosch J, Garcia-Pagan JC. The splanchnic circulation in cirrhosis. In: GinesP, ArroyoV, RodesJ, SchrierRW, eds. Ascites and Renal Dysfunction in Liver Disease. Pathogenesis, Diagnosis, and Treatment. Malden, MA: Blackwell Publishing; 2005: 125136.
  • 3
    Fernandez M, Vizzutti F, Garcia-Pagan JC, Rodes J, Bosch J. Anti-VEGF receptor-2 monoclonal antibody prevents portal-systemic collateral vessel formation in portal hypertensive mice. Gastroenterology 2004; 126: 886894.
  • 4
    Fernandez M, Mejias M, Angermayr B, Garcia-Pagan JC, Rodes J, Bosch J. Inhibition of VEGF receptor-2 decreases the development of hyperdynamic splanchnic circulation and portal-systemic collateral vessels in portal hypertensive rats. J Hepatol 2005; 43: 98103.
  • 5
    Angermayr B, Mejias M, Gracia-Sancho J, Garcia-Pagan JC, Bosch J, Fernandez M. Heme oxygenase attenuates oxidative stress and inflammation, and increases VEGF expression in portal hypertensive rats. J Hepatol 2006; 44: 10331039.
  • 6
    Angermayr B, Fernandez M, Mejias M, Gracia-Sancho J, Garcia-Pagan JC, Bosch J. NAD(P)H oxidase modulates angiogenesis and the development of portosystemic collaterals and splanchnic hyperemia in portal hypertensive rats. Gut 2007; 56: 560564.
  • 7
    Groszmann RJ, Garcia-Tsao G, Bosch J, Grace ND, Burroughs AK, Planas R, Escorsell A, et al. Beta-blockers to prevent gastroesophageal varices in patients with cirrhosis. N Engl J Med 2005; 353: 22542261.
  • 8
    Folkman J, D'Amore PA. Blood vessel formation: what is its molecular basis? Cell 1996; 87: 11531155.
  • 9
    Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med 2000; 6: 389395.
  • 10
    Jain RK. Molecular regulation of vessel maturation. Nat Med 2003; 9: 685693.
  • 11
    Ferrara N, Gerber H-P, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003; 9: 669676.
  • 12
    Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev 1999; 79: 12831316.
  • 13
    Guba M, von Breitenbuch P, Steinbauer M, Koehl G, Flegel S, Hornung M, et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med 2002; 8: 128135.
  • 14
    Yatscoff RW, Wang P, Chan K, Hicks D, Zimmerman J. Rapamycin: distribution, pharmacokinetics, and therapeutic range investigations. Ther Drug Monit 1995; 17: 666671.
  • 15
    Cohen MH, Williams G, Johnson JR, Duan J, Gobburu J, Rahman A, et al. Approval summary for imatinib mesylate capsules in the treatment of chronic myelogenous leukemia. Clin Cancer Res 2002; 8: 935942.
  • 16
    Schermuly RT, Dony E, Ghofrani HA, Pullamsetti S, Savai R, Roth M, et al. Reversal of experimental pulmonary hypertension by PDGF inhibition. J Clin Invest 2005; 115: 28112821.
  • 17
    Capdeville R, Buchdunger E, Zimmermann J, Matter A. Glivec (STI571, imatinib), a rationally developed, targeted anticancer drug. Nat Rev Drug Discov 2002; 1: 493502.
  • 18
    Pietras K, Sjöblom T, Rubin K, Heldin CH, Ostman A. PDGF receptors as cancer drug targets. Cancer Cell 2003; 3: 439443.
  • 19
    Fernandez M, Garcia-Pagan JC, Casadevall M, Bernadich C, Piera C, Whittle BJ, et al. Evidence against a role for inducible nitric oxide synthase in the hyperdynamic circulation of portal hypertensive rats. Gastroenterology 1995; 108: 14871495.
  • 20
    Fernandez M, Garcia-Pagan JC, Casadevall M, Mourelle MI, Pique JM, Bosch J, Rodes J. Acute and chronic cyclooxygenase blockade in portal hypertensive rats: Influence on nitric oxide biosynthesis. Gastroenterology 1996; 110: 15291535.
  • 21
    Jimenez O, Campistol JM, Vidal E, Faura A, Oppenheimer F, Corbella J, Brunet M. Defining the initial doses of sirolimus and tacrolimus in the period immediately after renal transplantation. Transplant Proc 2003; 35: 16861688.
  • 22
    Bergers G, Song S, Meyer-Morse N, Bergsland E, Hanahan D. Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors. J Clin Invest 2003; 111: 12871295.
  • 23
    Navasa M, Forns X, Sanchez V, Andreu H, Marcos V, Borras JM, Rimola A, et al. Quality of life, major medical complications and hospital service utilization in patients with primary biliary cirrosis after liver transplantation. J Hepatol 1996; 25: 129134.
  • 24
    Eberhard A, Kahlert S, Goede V, Hemmerlein B, Plate KH, Augustin HG. Heterogeneity of angiogenesis and blood vessel maturation in human tumors: implications for antiangiogenic tumor therapies. Cancer Res 2000; 60: 13881393.
  • 25
    Gee MS, Procopio WN, Makonnen S, Feldman MD, Yeilding NM, Lee WM. Tumor vessel development and maturation impose limits on the effectiveness of anti-vascular therapy. Am J Pathol 2003; 162: 183193.
  • 26
    Escorsell A, Bosch J. Pathophysiology of variceal bleeding. In: GroszmannRJ, BoschJ, eds. Portal Hypertension in the 21st Century. Dordrecht, The Netherlands: Kluwer Academic Publishers; 2004: 155166.
  • 27
    Abraldes JG, Tarantino I, Turnes J, Garcia-Pagan JC, Rodes J, Bosch J. Hemodynamic response to pharmacological treatment of portal hypertension and long-term prognosis of cirrhosis. HEPATOLOGY 2003; 37: 902908.
  • 28
    D'Amico G, Garcia-Pagan JC, Luca A, Bosch J. Hepatic vein pressure gradient reduction and prevention of variceal bleeding in cirrhosis: a systematic review. Gastroenterology 2006; 131: 16111624.