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Placental growth factor levels neither reflect severity of portal hypertension nor portal-hypertensive gastropathy in patients with advanced chronic liver disease
1 BeSi and AS contributed equally to this manuscript.
Benedikt Simbrunner
Footnotes
1 BeSi and AS contributed equally to this manuscript.
Affiliations
Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, AustriaVienna Hepatic Hemodynamic Laboratory, Medical University of Vienna, Vienna, AustriaChristian-Doppler Laboratory for Portal Hypertension and Liver Fibrosis, Medical University of Vienna, Vienna, Austria
1 BeSi and AS contributed equally to this manuscript.
Alexander Stadlmann
Footnotes
1 BeSi and AS contributed equally to this manuscript.
Affiliations
Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, AustriaVienna Hepatic Hemodynamic Laboratory, Medical University of Vienna, Vienna, AustriaHospital Hietzing, Vienna, Austria
Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, AustriaVienna Hepatic Hemodynamic Laboratory, Medical University of Vienna, Vienna, AustriaChristian-Doppler Laboratory for Portal Hypertension and Liver Fibrosis, Medical University of Vienna, Vienna, Austria
Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, AustriaVienna Hepatic Hemodynamic Laboratory, Medical University of Vienna, Vienna, Austria
Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, AustriaVienna Hepatic Hemodynamic Laboratory, Medical University of Vienna, Vienna, Austria
Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, AustriaVienna Hepatic Hemodynamic Laboratory, Medical University of Vienna, Vienna, Austria
Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, AustriaVienna Hepatic Hemodynamic Laboratory, Medical University of Vienna, Vienna, Austria
Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, AustriaVienna Hepatic Hemodynamic Laboratory, Medical University of Vienna, Vienna, AustriaChristian-Doppler Laboratory for Portal Hypertension and Liver Fibrosis, Medical University of Vienna, Vienna, Austria
Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, AustriaVienna Hepatic Hemodynamic Laboratory, Medical University of Vienna, Vienna, AustriaChristian-Doppler Laboratory for Portal Hypertension and Liver Fibrosis, Medical University of Vienna, Vienna, Austria
Experimental data indicates that placental growth factor (PLGF) is involved in the pathophysiology of portal hypertension (PH) due to advanced chronic liver disease (ACLD). We investigated serum levels of PLGF and its “scavenger”, the receptor soluble fms-like tyrosine kinase-1 (sFLT1, or sVEGFR1), in ACLD patients with different severity of PH and portal-hypertensive gastropathy (PHG).
Methods
PLGF and sVEGFR1 were measured in ACLD patients with hepatic venous pressure gradient (HVPG) ≥6 mmHg (n = 241) and endoscopic evaluation of PHG (n = 216). Patients with pre-/posthepatic PH, TIPS, liver transplantation and hepatocellular carcinoma were excluded.
Results
Thirty-two (13%) patients had HVPG 6–9 mmHg, 128 (53%) 10–19 mmHg and 81 (34%) ≥20 mmHg; 141 (59%) had decompensated ACLD (dACLD). PLGF (median 17.2 vs. 20.8 vs. 22.4 pg/mL; p = 0.002), sVEGFR1 (median 96.0 vs. 104.8 vs. 119.3 pg/mL; p < 0.001) levels increased across HVPG strata, while PLGF/sVEGFR1 ratios remained similar (0.19 vs. 0.20 vs. 0.18 pg/mL; p = 0.140). The correlation between PLGF and HVPG was weak (Rho = 0.190,95%CI 0.06–0.31; p = 0.003), and the PLGF/sVEGFR1 ratio did not correlate with HVPG (p = 0.331). The area-under-the-receiver operating characteristics (AUROC) for PLGF to detect clinically significant PH (CSPH;i.e. HVPG ≥ 10 mmHg) yielded only 0.688 (0.60–0.78; p < 0.001).
When compared to ACLD patients without PHG, PLGF levels (20 without vs. 21.4 with mild vs. 17.1 pg/mL with severe PHG, respectively; p = 0.005) and PLGF/sVEGFR1 ratios (0.20 vs. 0.19 vs. 0.17; p = 0.076) did not increase with mild and severe PHG.
Conclusion
While PLGF levels tended to increase with severity of PH, the PLGF/sVEGFR1 ratio remained stable across HVPG strata. Neither PLGF nor the PLGF/sVEGFR1 ratio had diagnostic value for prediction of CSPH. The severity of PHG was also not associated with stepwise increases in PLGF levels or PLGF/sVEGFR1 ratio.
Lay summary: The placental growth factor (PLGF) supports growth and proliferation of blood vessels in disease. Animal studies have suggested that PLGF is relevant for the progression of portal hypertension (PH), which is an important feature of advanced chronic liver disease (also known as cirrhosis). However, in our well-characterized cohort of cirrhotic patients with PH, the association with PLGF levels with severity of PH was limited and there was no correlation with the severity of PH-related changes of gastric mucosa.
1. Introduction
Portal hypertension (PH) drives the development of complications in advanced chronic liver disease (ACLD). Clinically significant portal hypertension (CSPH) is defined by a hepatic venous pressure gradient (HVPG) ≥10 mmHg and associated with a substantially increased risks of hepatic decompensation [
]. Mechanistically, PH is initiated by liver fibrosis and sinusoidal endothelial dysfunction that both increase intrahepatic resistance. Abnormal angiogenesis drives PH by impacting on sinusoidal remodeling and development of portosystemic collateralization [
Placental growth factor (PLGF) belongs to the vascular endothelial growth factor (VEGF) subfamily and is involved in pathological angiogenesis as present in cancer and inflammatory diseases [
Placental growth factor and its receptor, vascular endothelial growth factor receptor-1: novel targets for stimulation of ischemic tissue revascularization and inhibition of angiogenic and inflammatory disorders.
]. PLGF exerts its angiogenic effects via VEGF receptor-1 (VEGFR-1, i.e. fms-like tyrosine kinase-1 [FLT1]), resulting in proliferation of endothelial cells as well as growth and maturation of blood vessels [
An experimental study in mice indicated an important role of PLGF in PH-related angiogenesis: PLGF was upregulated in the mesenteric tissue after partial portal vein ligation (PPVL) and associated with pathologic angiogenesis and portal hypertension, which was alleviated upon PLGF gene knockout or after administration of anti-PLGF antibodies [
]. Importantly, a relative deficiency of the soluble VEGFR-1 (sVEGFR-1, i.e. sFLT1) - which sequesters PLGF and thus, exerts antagonistic effects - was reported in this study [
]. A subsequent publication from the same group demonstrated upregulation of PLGF and an amelioration of liver fibrosis, portal hypertension, and mesenteric artery blood flow after PLGF blockade in mice with carbon tetrachloride (CCl4)-induced cirrhosis [
]. PLGF expression in liver biopsies and PLGF serum levels were significantly elevated in patients with cirrhosis as compared to control subjects and PLGF serum levels also showed a direct correlation with HVPG in a small cohort of patients with cirrhosis [
Up-regulation of proproliferative genes and the ligand/receptor pair placental growth factor and vascular endothelial growth factor receptor 1 in hepatitis C cirrhosis.
] with chronic hepatitis C, as compared to controls without chronic liver disease. Finally, Gelman et al. recently confirmed the weak but statistically significant positive correlation between HVPG and PLGF serum levels in patients with cirrhosis due to chronic hepatitis C or alcohol-related liver disease (ALD) [
While experimental data indicates an important impact of PLGF-driven angiogenesis on PH, clinical studies on the relevance of PLGF for PH in patients with ACLD are scarce and did not account for serum levels of sFLT1, which has been combined with PLGF serum levels for preeclampsia risk stratification [
] as the only current clinical application for PLGF measurements. Moreover, although VEGF-driven angiogenesis has been implicated in the pathogenesis of portal hypertensive gastropathy (PHG) [
], PLGF has yet to be evaluated in this context. To this end, we prospectively recruited ACLD patients undergoing measurement of HVPG for concomitant assessment of PLGF and sVEGFR1 serum levels. In addition, we also investigated PLGF levels and PLGF/sVEGFR1 ratios in a group of ACLD patients undergoing endoscopic assessment of portal hypertensive gastropathy (PHG).
2. Patients and methods
2.1 Study design
241 patients with advanced chronic liver disease (ACLD; defined by hepatic venous pressure gradient ≥6 mmHg) undergoing hepatic vein catheterization at the Vienna Hepatic Hemodynamic Lab of the Medical University of Vienna were consecutively included in the prospective VICIS study (NCT03267615). Patients with non-cirrhotic PH, pre- or post-hepatic PH, hepatocellular carcinoma (HCC), transjugular intrahepatic portosystemic shunt (TIPS), or liver transplantation were excluded. Furthermore, patients under treatment with non-selective betablockers (NSBB) at the time of HVPG measurement were excluded. Blood samples for the assessment of PLGF and sVEGFR1 serum levels were obtained via the catheter introducer sheath placed in the internal jugular vein after HVPG measurement. Compensated ACLD (cACLD) was defined as the absence of hepatic decompensation events prior to HVPG measurement, i.e. ascites, variceal bleeding, or hepatic encephalopathy [
HVPG measurements were performed in fasting condition by trained physicians of the Vienna Hepatic Hemodynamic Lab following a standard operating procedure [
]. Briefly, after puncture of the right internal jugular vein under local anesthesia and ultrasound guidance, a catheter introducer sheath (8.5 F, Arrow International, Reading, PA, USA) was inserted using the Seldinger technique. The liver vein was cannulated by an angled balloon occlusion catheter (Medical University of Vienna/Medizintechnik Pejcl, Austria). Adequate placement and wedge position were verified by X-ray after injection of contrast agent while the balloon was inflated. At least three measurements of free and wedged hepatic vein pressure were performed to assess HVPG.
2.3 Endoscopic procedures and grading of portal hypertensive gastropathy
Portal-hypertensive gastropathy was diagnosed and classified according to consensus guidelines [
] as no PHG, mild PHG, and severe PHG. PLGF serum levels obtained within 3 months prior to or after endoscopic procedures were considered for analysis, provided that inclusion and exclusion criteria for this study were maintained within this time span. However, NSBB intake at the time of endoscopy was not considered an exclusion criterium for this particular analysis.
2.4 Analysis of placental growth factor and soluble vascular endothelial growth factor receptor-1 serum levels
PLGF and sVEGFR1 were measured using the Cobas PLGF and sFLT-1 fully automated electrochemiluminescence immunoassays (ECLIA) on Cobas e 602 analyzers (Roche, Mannheim, Germany) at the Department of Laboratory Medicine of the Medical University of Vienna according to the manufacturer's instructions and complying to ISO 9001 and ISO 15,189 quality standards. Laboratory staff performing the measurements was blinded to clinical, hemodynamic, and endoscopic characteristics of patients.
2.5 Statistics
Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, La Jolla, California, USA) and IBM SPSS Statistics 26 (IBM, Armonk, New York, USA). Continuous variables are reported as mean ±standard error of the mean (SEM) or median and interquartile range (IQR), and categorical variables are presented as numbers (n) and proportions (%) of patients. Comparisons of continuous variables were performed using Student's t-test or Mann-Whitney U test for two groups, and one-way analysis of variance or Kruskal-Wallis test for three or more groups, as applicable. Post-hoc analysis was performed using Dunn's multiple comparison test. Categorical variables were compared with Chi-squared or Fisher‘s exact test, as applicable. Correlations between parameters were assessed by calculation of either Spearman or Pearson correlation coefficients dependent on the distribution of the respective parameters. The diagnostic accuracy for the detection of CSPH was evaluated by area under the receiver operating characteristic curve (AUROC) analysis. In all analyses, a two-sided p-value < 0.05 was defined to denote statistical significance.
2.6 Ethics
This study was conducted in accordance with the most recent version of the Helsinki declaration and approved by the local ethics committee of the Medical University of Vienna (EK1262/2017). All patients gave written informed consent to liver vein catheterizations, endoscopy, and for enrolment into the VICIS study (NCT03267615).
3. Results
3.1 Patient characteristics
The study population comprised 241 patients with a median age of 57.4 (49.5–64.2) years and predominantly male gender (n = 157, 65%). The most common etiologies of ACLD were alcohol-related liver disease (ALD; n = 102, 42%) and viral hepatitis (n = 47, 20%; Supplementary Table-S1). Prevalence of CSPH was 87%, with a median HVPG of 18 (12–21) mmHg in the overall cohort. HVPG was 6–9 mmHg in 32 (13%) patients, 10–19 mmHg in 128 (53%), and ≥20 mmHg in 81 (34%) patients. Most patients were classified as Child-Turcotte-Pugh (CTP) stage A (n = 134, 56%), while 87 (36%) were CTP stage B, and 20 (8%) CTP stage C. Median model for end-stage liver disease including sodium (MELD-Na) score yielded 12 (9–16) points. One hundred (41%) patients had no history of hepatic decompensation at the time of HVPG measurement (i.e., cACLD). Variceal status was unknown in 6 (2%) patients, while 96 (40%) had large, 62 (26%) had small, and 77 (32%) had no varices. One-hundred and fourteen (47%) patients either had ascites or received diuretics at the time of HVPG measurement, while 49 (20%) had mild or medically controlled hepatic encephalopathy (HE).
3.2 Placental growth factor and severity of portal hypertension
PLGF and sVEGFR1 serum levels were assessed in patients stratified by severity of portal hypertension (i.e. HVPG 6–9 mmHg, 10–19 mmHg, and ≥20 mmHg; Fig. 1, Table 1). While median PLGF levels significantly increased in patients with subclinical PH as compared to patients with CSPH, no significant difference between HVPG strata 10–19 mmHg and ≥ 20 mmHg was observed, with a median PLGF of 17.2 [13.6–20.2] vs. 20.8 [17.0–27.0] vs. 22.4 [16.6–29.1] pg/mL (p = 0.002; post-hoc analysis: 6–9/10–19 mmHg p = 0.005, 6–9/≥20 mmHg p = 0.003, 10–19/≥20 mmHg p > 0.999). sVEGFR1 levels displayed a similar pattern, however, with statistically significant stepwise increases across all HVPG strata: median sVEGFR1 yielded 96.0 [81.4–108.0] vs. 104.8 [90.8–127.3] vs. 119.3 [99.7–140.0] pg/mL (p < 0.001; post-hoc analysis: 6–9/10–19 mmHg p = 0.024, 6–9/≥20 mmHg p < 0.001, 10–19/≥20 mmHg p = 0.005). Of note, the lower boundaries of PLGF and sVEGFR1 serum levels almost entirely overlapped between PH strata (Fig. 1AB). Interestingly, the PLGF/sVEGFR1 ratio was similar between patients with different severity of PH (0.19 [0.14–0.22] vs. 0.20 [0.17–0.25] vs. 0.18 [0.14–0.24] pg/mL; p = 0.140; Fig. 1C).
Fig. 1Placental growth factor (PLGF), soluble vascular endothelial growth factor receptor-1 (sVEGFR1) serum levels and PLGF/sVEGFR1 ratio in patients stratified by (A-C) hepatic venous pressure gradient (HVPG) and (D-F) Child-Turcotte-Pugh (CTP) stage. Abbreviations: (PLGF) placental growth factor, (sVEGFR1) soluble vascular endothelial growth factor receptor-1, (HVPG) hepatic venous pressure gradient, (CTP) Child-Turcotte-Pugh, (ns) not significant, (*) p < 0.05, (**) p < 0.01, (***) p < 0.001.
3.3 Placental growth factor, hepatic (dys-)function and comparison of PLGF terciles
Furthermore, we assessed PLGF and sVEGFR1 serum levels in patients stratified by CTP stage (Fig. 1D-F). We observed a statistically significant increase of PLGF levels between CTP A and B patients, with a median PLGF of 19.0 [16.1–25.5] vs. 21.2 [17.9–29.1] vs. 23.7 [15.9–32.8] pg/mL (p = 0.021; post-hoc analysis: CTP A/B p = 0.040, CTP A/C p = 0.225, CTP B/C p > 0.999). The lack of statistical significance between PLGF levels of CTP A and CTP C patients might, however, be attributed to the small sample size of the latter group. Similarly, PLGF levels were significantly higher in patients with decompensated ACLD, however, displaying only small numerical differences and extensive overlap of upper and lower boundaries (median PLGF 19.4 [16.1–25.2] vs. 21.1 [16.9–29.1] for cACLD and dACLD, respectively; p = 0.047; Supplementary figure-S1). Again, we observed a more distinct difference among sVEGFR1 serum levels (median 101.1 [86.6–112.5] vs. 118.7 [97.3–134.7], respectively; p < 0.001), while PLGF/sVEGFR1 ratio was similar (p = 0.223) between compensated and decompensated patients.
Finally, patients were stratified by PLGF terciles to identify conditions associated with high PLGF levels (Supplementary table-S2). Gender distribution displayed no significant differences regarding PH severity or PLGF levels. Analogous to the analyses depicted above, PH and disease severity showed a significant but small increase across terciles. Interestingly, platelet counts (PLT) displayed an increase between low and high PLGF terciles (91 [57–130] G/L in T1 vs. 103 [75–143] G/L in T2 vs. 114 [76–160] G/L in T3, p = 0.009).
Moreover, PLGF/sVEGFR1 ratio also incremented with higher PLGF terciles (median ratio 0.15 [0.12–0.18] in T1 vs. 0.19 [0.17–0.23] in T2 vs. 0.26 [0.21–0.31] in T3; p < 0.001; Supplementary figure-S2), indicating that high PLGF levels might not always relate to proportional increase of its antagonist-like soluble receptor. Nevertheless, HVPG and PLT counts were not significantly different between PLGF/sVEGFR1 ratio terciles: 19 (13–26) vs. 17.5 (12–20) vs. 18 (14–21) mmHg (p = 0.209) for HVPG and 98 (66–144) vs. 98 (70–136) vs. 114 (76–159) G/L (p = 0.140) for PLT counts (Supplementary Fig.-S3).
3.4 Placental growth factor weakly correlates with portal hypertension
Importantly, PLGF and sVEGFR1 levels exhibited distinct interrelation upon correlation analysis (Rho = 0.455, 95%CI 0.34–0.55; p < 0.001; Supplementary figure-S4). Next, we assessed whether PLGF and PLGF/sVEGFR1 showed a clinically meaningful correlation with portal hypertension (Fig. 2). However, PLGF displayed a statistically significant but only very weak association with HVPG (Rho = 0.190, 95%CI 0.06–0.31; p = 0.003), whereas sVEGFR1 displayed a slightly stronger direct correlation (Rho = 0.378, 95%CI 0.26–0.49; p < 0.001). Notably, the PLGF/sVEGFR1 ratio did not correlate with HVPG (p = 0.331).
Fig. 2(A-B) Correlation between placental growth factor (PLGF) and PLGF/soluble vascular endothelial growth factor receptor-1(sVEGFR1) ratio and hepatic venous pressure gradient (HVPG). (C-D) Area-under-the-receiver operating characteristics (AUROC) for prediction of clinically significant portal hypertension (CSPH) by PLGF and PLGF/sVEGFR1 ratio. Abbreviations: (PLGF) placental growth factor, (sVEGFR1) soluble vascular endothelial growth factor receptor-1, (HVPG) hepatic venous pressure gradient, (CSPH) clinically significant portal hypertension.
Furthermore, we performed AUROC analyses to assess whether PLGF or PLGF/sVEGFR1 ratio displayed any discriminative ability for prediction of CSPH (Fig. 2). However, AUROC analyses only yielded 0.688 (95%CI 0.60–0.78, p < 0.001) for PLGF and 0.556 (95%CI 0.45–0.66, p = 0.309) for the PLGF/sVEGFR1 ratio.
3.5 Placental growth factor and portal-hypertensive gastropathy
Finally, we aimed to investigate whether PLGF was associated with PHG, as previous experimental studies reported an association between PLGF and intestinal angiogenesis, which has also been implicated in the pathogenesis of PHG. PLGF and sVEGFR1 serum levels were assessed in 216 patients undergoing endoscopy for variceal screening. Only 56 (26%) patients had no endoscopic signs of PHG, of whom 134 (62%) had mild and 26 (12%) had severe PHG (Fig. 3). PLGF levels were similar between patients with no and mild PHG, and even lower in patients with severe PHG: 20 (14.9–25.7) vs. 21.4 (17.3–28.0) vs. 17.1 (14.4–20.3) pg/mL, respectively (p = 0.005; post-hoc analysis: no/mild PHG p = 0.244, no/severe PHG p = 0.349, mild/severe PHG p = 0.007). Furthermore, no differences of sVEGFR1 levels (p = 0.179) and PLGF/sVEGFR1 ratio (p = 0.209) were observed between the PHG strata.
Fig. 3Placental growth factor (PLGF), soluble vascular endothelial growth factor receptor-1 (sVEGFR1) serum levels and PLGF/sVEGFR1 ratio stratified by severity of portal-hypertensive gastropathy (PHG). Grading of portal-hypertensive gastropathy: (0) no, (1) mild, (2) severe. Abbreviations: (PLGF) placental growth factor, (sVEGFR1) soluble vascular endothelial growth factor receptor-1, (PHG) portal hypertensive gastropathy, (ns) not significant, (*) p < 0.05, (**) p < 0.01, (***) p < 0.001.
Conversely, PHG was associated with increased HVPG: median HVPG was 15 (10–19) mmHg in patients without, 18 (14–21) mmHg in patients with mild, and 17.5 (14–19) mmHg in patients with severe PHG (p = 0.013; post-hoc analysis: A/B p = 0.010, A/C p = 0.588, B/C p > 0.999; Supplementary figure-S5). Median time interval between liver vein catheterization and gastroscopy was 0.6 (0.0–1.7) months.
4. Discussion
In our large prospective series of 241 ACLD patients, PLGF levels only weakly correlated with HVPG and mildly increased with severity of PH. Neither PLGF nor the PLGF/sVEGFR1 ratio displayed diagnostic value for identifying ACLD patients with CSPH. In addition, PLGF levels and the PLGF/sVEGFR1 ratio did not correlate with the presence and severity of PHG in 216 patients undergoing upper GI endoscopy.
Hepatic and splanchnic angiogenesis play important roles in the remodeling of the hepatic and splanchnic vasculature that contributes to the progression of liver disease and PH [
Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.
]. Importantly, alternative splicing of messenger RNA encoding VEGFR-1 generates sVEGFR1, which lacks a transmembrane-spanning amino acid sequence and, thus, is released into circulation and acts as an antagonist for VEGF and PLGF [
]. The serum levels of circulating PLGF and sVEGFR1 and their ratio reflect an angiogenic or antiangiogenic net effect specific for distinct physiological or pathophysiological processes. For example, the ratio of sVEGFR1 and PLGF serum levels has emerged as an accurate predictor of preeclampsia in pregnant women and, notably, was of superior prognostic value than the respective individual parameters [
] suggested an important role of PLGF-induced angiogenesis towards the development of PH. Interestingly, the longitudinal expression profile in mesenteric tissue of the PPVL model displayed an immediate increase of PLGF and VEGF after PPVL, whereas sVEGFR1 and soluble VEGFR-2 where similar to controls, which resulted in the consequent abundance of PLGF and VEGF, respectively [
]. While considerable differences between these experimental animal models and patients with ACLD must be acknowledged, first human data reported PLGF upregulation in patients with chronic hepatitis C (HCV): Huang et al. displayed upregulation of PLGF expression in explant livers of patients with HCV-related cirrhosis undergoing orthotopic liver transplantation as compared to non-diseased liver tissue [
Up-regulation of proproliferative genes and the ligand/receptor pair placental growth factor and vascular endothelial growth factor receptor 1 in hepatitis C cirrhosis.
In our study, we noted an increase of median PLGF serum levels between patients with subclinical PH and CSPH, whereas patients with HVPG ≥ 20 mmHg displayed no significant further increment of PLGF. Similar trends were observed in patients stratified by CTP stage. This was paralleled by a gradual increase of sVEGFR1 serum levels across PH and CTP strata in our study cohort. Consequently, PLGF/sVEGFR1 ratio displayed no differences between PH strata, whereas CTP-C patients even exhibited a decrease in PLGF/sVEGFR1 ratio. These results indicate that concordant upregulation of sVEGFR1 dampens angiogenic effects of PLGF in ACLD patients with increasing severity of PH.
This interpretation is supported by several observations: First of all, PLGF and sVEGFR1 serum levels were directly correlated, i.e., increases in PLGF were paralleled by increases in sVEGFR1. Interestingly, upregulation of sVEGFR1 tended to be more pronounced than that of PLGF across HVPG and CTP strata. Next, we observed a weak correlation between PLGF and HVPG, and no meaningful association between PLGF/sVEGFR1 ratio and PH. Van Steenkiste et al. reported significant correlation (r = 0.386) between HVPG and PLGF serum levels in a small number of patients with alcoholic hepatitis [
]. Similarly, another study has reported slightly better correlation between PLGF serum levels and HVPG (r = 0.338 as compared to r = 0.190 in our study) [
]. However, this study by Gelman et al. included non-ACLD patients with HVPG below 6 mmHg, which likely accounts for the observed difference, as a number of studies have displayed significant PLGF increments from controls/non-ACLD patients and ACLD patients [
. Still, we found a significant increase of PLGF serum levels between patients with subclinical PH and patients with CSPH. Thus, we calculated AUROC analyses to assess whether PLGF was suitable to detect CSPH. However, PLGF yielded a poor AUROC of 0.688 in our study cohort, and therefore, has no value for the non-invasive diagnosis of CSPH. These results are in line with previously reported data on the lack of diagnostic accuracy of PLGF to detect CSPH (AUROC 0.67) [
Interestingly, platelet counts gradually increased across PLGF terciles, although the severity of portal hypertension (as assessed by HVPG) increased, which seems counterintuitive. Intriguingly, prior studies have reported stimulation of hematopoiesis and, more specifically, megakaryocyte (i.e., the precursor of platelets) maturation by VEGF and PLGF via interaction with membrane-bound VEGFR-1 [
]. While these studies are mostly experimental and did not focus on liver diseases, it seems plausible to consider these mechanisms for the observations made in our study, even though platelets counts were similar across PLGF/sVEGFR1 ratio terciles. In any case, this observation could be relevant for studies investigating non-invasive tests that include platelet count [
Changes in hepatic venous pressure gradient predict hepatic decompensation in patients who achieved sustained virologic response to interferon-free therapy.
, as a potential PLGF-induced effect on megakaryopoiesis may distort the accuracy of platelet counts to reflect PH.
In regard to mesenteric angiogenesis, experimental data suggest that splanchnic vasodilatation and neovascularization are associated with increased PLGF expression [
]. Similarly, Geerts et al. demonstrated an increment of mesenteric angiogenesis in rats with either cirrhotic PH or pre-hepatic PH, which was associated with increased VEGF expression [
]. Moreover, capillary angiogenesis and/or congestion were frequently observed in upper gastrointestinal biopsies from patients with chronic liver disease and PH and have been implicated in the pathogenesis of PHG [
]. Thus, we additionally explored whether PLGF levels are associated with the presence and severity of PHG as potential paradigm clinical setting for such pathomechanisms but found no meaningful association between PHG and PLGF levels. Conversely, median HVPG increased in patients with PHG, which is in accordance with prior findings [
]. The limited sample size of patients with severe PHG may, however, limit the statistical power to detect significant differences in this particular subgroup.
Our study provides novel data on the relationship between PLGF and sVEGFR1 serum levels as well as the correlation between sVEGFR1 and PH and their relationship with PHG in a large cohort of prospectively recruited patients who were thoroughly characterized by HVPG measurements. However, we acknowledge that this study has several limitations: First, VEGF serum levels were not available, which prevents us from investigating whether VEGF-driven angiogenesis rather than PLGF associates with PH and PHG severity. Second, PLGF serum levels within 3 months prior or after endoscopy were considered for assessing the association between PLGF and PHG. Nevertheless, PLGF was assessed on the day of endoscopy in the majority (54%) of patients. Third, the assessment of systemic PLGF levels might not essentially reflect the activation of intrahepatic and splanchnic PLGF-driven angiogenesis but could also be reflective of the situation in other vascular beds, such as in pulmonary angiogenesis in hepatopulmonary syndrome [
In summary, our study demonstrates that there is no clinically meaningful association between PLGF or sVEGFR1 serum levels or their ratio and PH/PHG. Thus, PLGF serum levels do not represent a valuable biomarker for CSPH or PHG severity in patients with ACLD. The limited impact of PLGF in humans with ACLD may be explained by simultaneous upregulation of sVEGFR1, which acts as an antagonist for PLGF. The diagnostic value of PLGF for other complications of cirrhosis (e.g., hepatopulmonary sydrome), as well as its prognostic value for hepatic decompensation, risk of developing hepatocellular carcinoma, and mortality should be evaluated in future studies.
Author's contribution
Study design (BeSi, AS, MM, TR). Extraction of data (BeSi, AS, EE). Statistical analysis (BeSi, MM, TR). Drafting of the manuscript (BeSi, AS, TR). Critical revision for important intellectual content (all authors).
Ethics approval statement
This study was conducted in accordance with the most recent version of the Helsinki declaration and approved by the local ethics committee of the Medical University of Vienna (EK1262/2017). All patients gave written informed consent to liver vein catheterizations, endoscopy and for enrolment into the VICIS study (NCT03267615).
Trial registration number
NCT03267615
Funding
None.
Declaration of Competing Interest
BeSi received travel support from AbbVie and Gilead.
PS received speaking honoraria from Bristol-Myers Squibb and Boehringer-Ingelheim, consulting fees from PharmaIN, and travel support from Falk and Phenex Pharmaceuticals.
DB received travel support from AbbVie and Gilead.
TB received speaker honoraria from BMS, travel support from Abbvie, BMS, and Gilead; travel grant, financial award/grant from Medis.
BeSc received travel support from Abbvie and Gilead.
MP is an investigator for Bayer, BMS, Lilly, and Roche; he received speaker honoraria from Bayer, BMS, Eisai, Lilly, and MSD; he is a consultant for Bayer, BMS, Ipsen, Eisai, Lilly, MSD, and Roche; he received travel support from Bayer and BMS.
MT received speaker fees from BMS, Falk Foundation, Gilead, Intercept and MSD; advisory board fees from Albireo, BiomX, Boehringer Ingelheim, Falk Pharma GmbH, Genfit, Gilead, Intercept, MSD, Novartis, Phenex and Regulus. He further received travel grants from Abbvie, Falk, Gilead and Intercept and unrestricted research grants from Albireo, Cymabay, Falk, Gilead, Intercept, MSD and Takeda.
MM served as a speaker and/or consultant and/or advisory board member for AbbVie, Bristol-Myers Squibb, Collective Acumen, Gilead, and W. L. Gore & Associates and received travel support from AbbVie, Bristol-Myers Squibb, and Gilead.
TR received grant support from Abbvie, Boehringer-Ingelheim, Gilead, MSD, Philips Healthcare, Gore; speaking honoraria from Abbvie, Gilead, Gore, Intercept, Roche, MSD; consulting/advisory board fee from Abbvie, Bayer, Boehringer-Ingelheim, Gilead, MSD, Siemens; and travel support from Boehringer-Ingelheim, Gilead and Roche.
AFS, AS, EE, KW, AB, TS, KL and RP declare no conflict of interest.
Placental growth factor and its receptor, vascular endothelial growth factor receptor-1: novel targets for stimulation of ischemic tissue revascularization and inhibition of angiogenic and inflammatory disorders.
Up-regulation of proproliferative genes and the ligand/receptor pair placental growth factor and vascular endothelial growth factor receptor 1 in hepatitis C cirrhosis.
Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.
Changes in hepatic venous pressure gradient predict hepatic decompensation in patients who achieved sustained virologic response to interferon-free therapy.
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