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. 2023 Jun 17;5(10):100816. doi: 10.1016/j.jhepr.2023.100816

Recombinant VEGF-C (Cys156Ser) improves mesenteric lymphatic drainage and gut immune surveillance in experimental cirrhosis

Pinky Juneja 1,†, Syed Nazrin Ruhina Rahman 2,†, Deepika Jakhar 1, Akash Kumar Mourya 1, Dinesh M Tripathi 1, Impreet Kaur 1, Vaibhav Tiwari 1, Sumati Rohilla 1, Abhishek Gupta 1, Preety Rawal 1, Sukriti Baweja 1, Archana Rastogi 3, VGM Naidu 4, Shiv K Sarin 5, Subham Banerjee 2,∗, Savneet Kaur 1,∗
PMCID: PMC10472308  PMID: 37663117

Abstract

Background & Aims

Lymphatic vessels (LVs) are crucial for maintaining abdominal fluid homoeostasis and immunity. In cirrhosis, mesenteric LVs (mLVs) are dilated and dysfunctional. Given the established role of vascular endothelial growth factor-C (VEGF-C) in improving LVs, we hypothesised that VEGF-C treatment could ameliorate the functions of mLVs in cirrhosis.

Methods

In this study, we developed a nanoformulation comprising LV-specific growth factor, recombinant human VEGF-C (Cys156Ser) protein (E-VEGF-C) and delivered it orally in different models of rat cirrhosis to target mLVs. Cirrhotic rats were given nanoformulation without VEGF-C served as vehicles. Drainage of mLVs was analysed using tracer dye. Portal and systemic physiological assessments and computed tomography were performed to measure portal pressures and ascites. Gene expression and permeability of primary mesenteric lymphatic endothelial cells (LyECs) was studied. Immune cells in mesenteric lymph nodes (MLNs) were quantified by flow cytometry. Endogenous and exogenous gut bacterial translocation to MLNs was examined.

Results

In cirrhotic rats, mLVs were dilated and leaky with impaired drainage. Treatment with E-VEGF-C induced proliferation of mLVs, reduced their diameter, and improved functional drainage. Ascites and portal pressures were significantly reduced in E-VEGF-C rats compared with vehicle rats. In MLNs of E-VEGF-C animals, CD8+CD134+ T cells were increased, whereas CD25+ regulatory T cells were decreased. Both endogenous and exogenous bacterial translocation were limited to MLNs in E-VEGF-C rats with reduced levels of endotoxins in ascites and blood in comparison with those in vehicle rats. E-VEGF-C treatment upregulated the expression of vascular endothelial-cadherin in LyECs and functionally improved the permeability of these cells.

Conclusions

E-VEGF-C treatment ameliorates mesenteric lymph drainage and portal pressure and strengthens cytotoxic T-cell immunity in MLNs in experimental cirrhosis. It may thus serve as a promising therapy to manage ascites and reduce pathogenic gut bacterial translocation in cirrhosis.

Impact and Implications

A human recombinant pro-lymphangiogenic growth factor, VEGF-C, was encapsulated in nanolipocarriers (E-VEGF-C) and orally delivered in different models of rat liver cirrhosis to facilitate its gut lymphatic vessel uptake. E-VEGF-C administration significantly increased mesenteric lymphatic vessel proliferation and improved lymph drainage, attenuating abdominal ascites and portal pressures in the animal models. E-VEGF-C treatment limited bacterial translocation to MLNs only with reduced gut bacterial load and ascitic endotoxins. E-VEGF-C therapy thus holds the potential to manage ascites and portal pressure and reduce gut bacterial translocation in patients with cirrhosis.

Keywords: Liver cirrhosis, Lymphangiogenesis, Mesenteric lymphatic vessels, Portal hypertension, Targeted nano-delivery

Graphical abstract

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Highlights

  • •

    Mesenteric lymphatic vessels are dilated and dysfunctional in experimental liver cirrhosis.

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    Treatment with recombinant VEGF-C improves mesenteric lymphatic vessel drainage and prevents leakage in experimental cirrhosis.

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    VEGF-C ameliorates ascites and portal pressure in cirrhotic and non-cirrhotic portal hypertensive rats.

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    VEGF-C increases the trafficking of immune cells and antigen to mesenteric lymph nodes in cirrhotic rats.

Introduction

Chronic liver disease can progress from mild fibrosis to cirrhosis, which may further progress to end-stage liver disease with the onset of decompensation, such as ascites, hepatic encephalopathy, and variceal bleeding.[1], [2], [3] In addition to other factors, deranged gut–liver axis plays a significant role in the progression of liver disease.4,5

Lymphatic vessels (LVs) in the gut bypass the portal circulation and directly connect the gut to systemic circulation via the thoracic duct.6 Mesenteric LVs (mLVs) prevent oedema and abdominal ascites by facilitating removal of interstitial fluid and returning it back to blood circulation.7 Gut lymphangiogenesis compensates for lymphatic insufficiency in patients with inflammatory bowel disease (IBD).8,9 During cirrhosis, the production of abdominal lymph increases 30-fold, and there is a positive correlation between increased lymph flow and portal pressures (PPs).10,11 In carbon tetrachloride (CCl4)-induced cirrhosis models and patients with cirrhosis, previous studies reported that gut LVs show an impaired phenotype and reduced contractility.[12], [13]

Vascular endothelial growth factor-C (VEGF-C), a key pro-lymphangiogenic factor, activates lymphangiogenesis by binding to tyrosine kinase receptor vascular endothelial growth factor receptor-3 (VEGFR-3).14 VEGF-C is known to be effective in improving lymphatic drainage and ameliorating inflammation diseases such as IBD, rheumatoid arthritis, skin inflammation, and hepatic encephalopathy.[15], [16], [17], [18]

We hypothesised a therapeutic role of VEGF-C for restoring gut lymphatic drainage and immune response in cirrhosis. VEGF-C, a hydrophilic molecule, is highly unstable and has a positive charge at physiological pH.19,20 To minimise its side effects of systemic delivery and increase its gut bioavailability,21 we fabricated an engineered VEGF-C protein using recombinant human VEGF-C protein (rhVEGF-C) (Cys156Ser), which specifically binds to VEGFR-3 homodimers, present on lymphatic endothelial cells (LyECs) lining the LVs. VEGF-C protein was encapsulated within nanoscale reverse micelle (RM)-based lipocarriers and delivered via oral route to ensure its uptake in mLVs in vivo.

Materials and methods

Preparation of E-VEGF-C

To prepare RMs, high-pressure homogenisation/microfluidisation techniques were adapted as described by Bai et al.,22 followed by slight modification. rhVEGF-C (Cys156Ser, R&D Systems, 752-VC-025) was incorporated in prepared RMs to obtain nanosized RMs containing VEGF-C protein termed as engineered VEGF-C (E-VEGF-C) (Fig. S1). E-VEGF-C was characterised, and its uptake was studied in mesenteric LyECs in vitro, as given in Supplementary information.

Study groups and treatment

All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23 revised 1985). The Animal Ethics Committee of ILBS, New Delhi, approved the study according to standard guidelines (Ethics Protocol No.: IAEC/ILBS/18/01). Rats were housed in a room at 22 ± 3 °C for a 12-h light–dark cycle and were given food and water ad libitum. Studies were performed on 8-week-old male Sprague Dawley rats weighing 250–300 g. Three cirrhotic models of portal hypertension and one non-cirrhotic portal hypertension model were prepared. Cirrhosis was induced by (1) i.p. injection of 1.0 ml/kg body weight of CCl4:olive oil in a 1:1 ratio, two times a week for 12–14 weeks until ascites formation; (2) i.p. injection of 250 mg/kg body weight of thioacetamide (TAA), two times a week for 12 weeks; and (3) a bile duct ligation (BDL) model developed by ligating the common bile duct as previously described.23 Non-cirrhotic animal models of portal hypertension were developed using partial portal vein ligation (PPVL), as previously described.23 To inhibit LVs, a selective VEGFR-3 inhibitor, SAR131675, was orally administered in TAA cirrhotic rats. For in vivo studies, animals were randomised to study the efficacy of E-VEGF-C. In cirrhotic models of portal hypertension, the following groups were prepared: healthy control, RMs without VEGF-C treated vehicle (CCl4-V, TAA-V, and BDL-V), and E-VEGF-C treated (E-VEGF-C, TAA + E-VEGF-C, and BDL + E-VEGF-C). After haemodynamics, these rats were sacrificed 48 h after the last dose of the vehicle or E-VEGF-C. In the non-cirrhotic model of portal hypertension, PPVL rats were prepared and randomised into two groups: (1) vehicle (PPVL-V) and (2) treated (PPVL + E-VEGF-C). The preparation of animal models and treatments are described in detail in Supplementary methods.

Statistical analysis

Continuous variables are expressed either as mean ± SD for normal distribution or as median values for skewed distribution. Continuous variables were compared between the two groups using an unpaired two-tailed Student’s t test or Mann–Whitney U test. Variables greater than 2 were compared using one-way ANOVA followed by a post hoc Tukey’s test. Bar diagrams with various data points, dot plots, and box whisker plots were plotted using GraphPad Prism (version 8.0.1; GraphPad Software, San Diego, CA, USA), and statistical analysis was performed using GraphPad Prism. Statistical significance was set at p <0.05.

Results

mLVs are dilated in experimental liver cirrhosis

To first evaluate mLVs in liver cirrhosis, we performed immunohistochemical staining of mesentery sections in control rats and CCl4 experimental models of liver cirrhosis with antibodies recognising podoplanin (Pdpn), a widely accepted marker of lymphatic vasculature. Control rats showed sporadic, thin LVs near blood vessels, whereas cirrhotic rats’ mesentery contained numerous, readily detectable, and dilated LVs compared to control rats (Fig. 1A). Quantitative analysis revealed that the total number of Pdpn + LVs per field in CCl4 cirrhotic rats was significantly higher with increased diameter than that in control rats (Fig. 1B and C; p <0.05 each).

Fig. 1.

Fig. 1

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mLV density increased in liver cirrhosis.

(A) IHC with Pdpn antibody was performed in mesentery tissue of control and CCl4 model of liver cirrhosis. Scale bar: 500 μm. (B) Number of the pdpn + mLVs was quantified using ImageJ. p = 0.0240. (C) Diameter of mLVs in control and CCl4 rats was measured using ImageJ’s diameter plugin. n = 5 each. p = 0.0119. (D) mRNA levels for Prox1, LyVE1, VEGF-C, CCL21, eNOS, and COX2 were quantified in mesentery tissue. n = 5 each. p = 0.05–0.01. The dotted line represents control. (E) Immunofluorescence staining for VEGFR-3, and DAPI was performed in mesentery tissue of control and CCl4 rats. Scale bar: 186.2 μm. (F) VEGFR-3 expression was quantified using ImageJ. n = 5 each. p = 0.0147. Data expressed as mean ± SD. Two-tailed, unpaired t test.∗p < =0.05. IHC, immunohistochemistry; LV, lymphatic vessel; MFI, mean fluorescence intensity; mLV, mesenteric LV; Pdpn, podoplanin; VEGF-C, vascular endothelial growth factor-C; VEGFR-3, vascular endothelial growth factor receptor-3.

The mRNA levels of LV markers Prox1 and LyVE1 were also enhanced in the mesentery of CCl4 rats compared with control rats (Fig. 1D; p <0.05 each). In addition, the mRNA levels of CCL21, COX2, and eNOS were also upregulated, reflecting an inflamed gut (p <0.05 each). VEGF-C expression was enhanced, suggesting inflammation-induced lymphangiogenesis (p <0.05). The VEGF-C/VEGFR-3 axis is pronounced in intestinal lymphangiogenesis and inflammation-induced lymphangiogenesis.17 Therefore, we checked the expression of VEGFR-3 by immunofluorescence staining in mesentery sections. The protein expression of VEGFR-3 was significantly increased in CCl4 cirrhotic rats compared with that seen in controls (Fig. 1E and F; p <0.01).

Development of nanoengineered VEGF-C for targeted delivery to mLVs

Dilation and lymph transport failure of the mLVs in experimental cirrhosis with ascites have been previously reported12; therefore, we hypothesised that enhancing the number of new functional mLVs by treatment with VEGF-C may improve lymphatic drainage in cirrhotic rats. To this end, we developed an engineered VEGF-C (E-VEGF-C) nanoformulation to enhance its uptake by gut LVs after oral delivery (Fig. S1). rhVEGF-C (Cys156Ser), which specifically binds to VEGFR-3 homodimer present on LyECs, was encapsulated inside lipocarriers prepared with distearoyl-rac-glycerol-PEG2K (Fig. 2A). In dynamic light scattering analysis, the z-average/mean particle size of E-VEGF-C was found to be 134.8 ± 0.47 nm with a polydispersity index value of 0.126 ± 0.01 (Fig. 2B). The zeta potential and pH value of the E-VEGF-C were −21.9 ± 1.24 mV and 6.369 ± 0.004, respectively (Fig. 2C). A stability study of up to 1 month indicated no drastic change in the physicochemical properties of E-VEGF-C nanoformulation (Table S1). The surface morphology and size of E-VEGF-C were studied using field emission scanning electron microscopy, and field emission transmission electron microscopy indicated that particles were spherical, smooth in appearance, and uniformly distributed (Fig. 2D and E). Atomic force microscopy micrographs provided two-dimensional and three-dimensional images of E-VEGF-C surface morphology and showed that E-VEGF-C was compact, smooth, and spherical (Fig. 2F). The average diameter of individual particles was 16 nm with an encapsulation efficiency of 93.28 ± 1.85%. The release profile of E-VEGF-C showed an initial burst release of 31.95 ± 1.52% VEGF-C observed at 2 h post mixing, followed by an increase in VEGF-C release of 84.66 ± 1.82% at 4 h, which then gradually decreased at other post-mixing time points (Fig. 2G). After the in vitro release study, R2 values obtained from different kinetic models listed in Table S2 suggested that the VEGF-C release from RMs showed zero-order kinetic and, therefore, is independent of concentration. To investigate the internalisation of E-VEGF-C in vitro, we isolated LyECs from rat mesentery and mesenteric lymph nodes (MLNs) using FACS (Fig. S2A and B) and incubated them with coumarin-6-labelled E-VEGF-C. Fluorescence microscopy revealed the efficient internalisation of E-VEGF-C by LyECs at 4 h (Fig. 2H and I). Next, to ensure the specificity of E-VEGF-C delivery in vivo, tissue biodistribution studies of coumarin-tagged E-VEGF-C were performed 2 h after oral administration using spectrofluorimetry and fluorescence microscopy (Fig. 2J). A weak fluorescence signal was observed in all tissues of control and CCl4 rats (Fig. S2C), with an intense signal observed in the mesentery of both groups (Fig. 2K and L). CCl4 rats also showed increased human VEGF-C levels in mesenteric and duodenal tissues compared with those in control rats (Fig. 2M; p <0.05). In the serum of CCl4 animals, levels of VEGF-C exhibited a biphasic peak; the first peak appeared at about 10 min, and the second peak appeared at approximately 5 h (Fig. 2N). There were no adverse effects or mortality after E-VEGF-C treatment in either control or CCl4 rats.

Fig. 2.

Fig. 2

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Formation, characterisation, uptake, and biodistribution of E-VEGF-C.

(A) Schematic representation of VEGF-C-engineered stealth nanolipocarriers (E-VEGF-C). Histograms showing (B) mean particle size and polydispersity index, and (C) zeta potential of E-VEGF-C. (D) FE-SEM of E-VEGF-C. Surface morphology and size of E-VEGF-C were visualised using FE-SEM at 4.70KX and 50.67KX magnification. (E) FE-TEM analysis of E-VEGF-C. The morphology and size of E-VEGF-C were examined using FE-TEM, indicating spherical shape. (F) Atomic force microscopy micrographs – two-dimensional and three-dimensional images of E-VEGF-C surface morphology. (G) In vitro release profile of E-VEGF-C in PBS at pH 7.4. (H) Primary LyECs were cultured and incubated with coumarin-6-labelled E-VEGF-C and observed at different time points. Scale bar: 100 μm. (I) Line graph of coumarin-positive LyECs depicting percentage of E-VEGF-C uptake at different time points. (J) Schema of in vivo biodistribution studies. n = 6 each. Tissues were collected after 2 h and measured in a fluorimeter. (K) Representative fluorescence images of the control and CCl4-V rats’ mesentery section showed localisation of coumarin-tagged E-VEGF-C in the gut. Scale bar: 200 μm. (L) Quantitative fluorescence analysis in different tissues of control and CCl4 rats using a fluorimeter. n = 6 for each group. (M) ELISA of human VEGF-C (pg/mg of total protein) in different tissues of control and CCl4 rats. n = 3 each. p = 0.01–0.001. (N) Levels of human VEGF-C (pg/ml) in control and CCl4-V rat plasma samples at indicated time points were measured using ELISA. n = 3 each. Data expressed as mean ± SD. Data expressed as mean ± SD. Two-tailed, unpaired t test. ∗p <0.05; ∗∗p <0.01. CCl4, carbon tetrachloride; E-VEGF-C, engineered VEGF-C; FE-SEM, field emission scanning electron microscopy; FE-TEM, field emission transmission electron microscopy; LyEC, lymphatic endothelial cell; VEGF-C, vascular endothelial growth factor-C.

mLV proliferation by E-VEGF-C

Next, we studied the therapeutic effects of E-VEGF-C in different animal models of liver cirrhosis. Rats were injected i.p. with CCl4 and TAA for 14 and 12 weeks, respectively. All CCl4 rats had ascites at 14 weeks, whereas ascites were absent in cirrhotic TAA rats. E-VEGF-C was administered orally, 600 μg/kg, on alternate days for up to the next 2 weeks in CCl4 and TAA rats (Fig. 3A). SAR, a VEGFR-3 selective inhibitor, was orally administered to ablate LVs in cirrhotic TAA rats from 8 to 14 weeks. Rats were sacrificed 48 h after the last dose of E-VEGF-C or SAR. Immunohistochemical analysis of mesentery showed increased numbers of Pdpn + mLVs (p >0.05) with a significantly reduced diameter (p <0.05) in E-VEGF-C-treated rats compared with CCl4-V rats (Fig. 3B–D). In the mesentery of TAA-V and TAA + SAR rats, we observed dilated mLVs. Treatment with E-VEGF-C increased the number of mLVs and reduced their dilation in TAA rats (p <0.05; Fig. 3E–G). There was a significant increase in rhVEGF-C and VEGFR-3 protein in the mesentery of E-VEGF-C-treated rats vs. CCl4-V rats (p <0.01 each; Fig. 3H–J). Along with the mesentery, marked LV proliferation was observed in MLNs and the duodenum of E-VEGF-C rats compared with CCl4-V and control rats (p <0.05 each; Fig. 3K–N). Mesenteric tissues also revealed reduced inflammation in E-VEGF-C rats compared with CCl4-V rats. There was a reduction in the expression of inflammatory markers eNOS, iNOS, IL-6, and interferon-gamma and increased transforming growth factor-beta in mesentery tissues of E-VEGF-C rats vs. CCl4-V rats (Fig. S3A and B). Because VEGF-C also participates in angiogenesis, we evaluated whether E-VEGF-C affected angiogenesis in the mesentery. We observed no significant difference in the number of CD31+ blood vessels between CCl4-V and E-VEGF-C-treated rats (p >0.05; Fig. S3C and D).

Fig. 3.

Fig. 3

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Effect of E-VEGF-C treatment on mLVs in a cirrhotic rat model of PHT.

(A) Schema of in vivo studies. CCl4 and TAA cirrhotic rats were treated with E-VEGF-C 600 μg/kg on alternate days for 2 weeks. n = 8 for each group. TAA rats treated with SAR, 30 mg/kg, twice a week for 7 weeks. n = 6. (B) IHC staining of mesentery sections of CCl4-V and E-VEGF-C rats. Scale bar: 200 μm. Arrow indicated at Pdpn + LVs. (C) Number of mLVs quantified using ImageJ. p >0.05. (D) Diameter of Pdpn + mLVs was measured using ImageJ. p = 0.0073. (E) IHC staining of mesentery sections of TAA-V, TAA + SAR, and TAA + E-VEGF-C rats. Scale bar: 200 μm. Arrow indicated at LyVE1 + LVs. (F) Number of LyVE1+ mLVs quantified using ImageJ. p <0.0001. (G) Diameter of LyVE1+ mLVs was measured using ImageJ. p <0.0001. (H) Expression of human VEGF-C and VEGFR-3 protein in the mesentery was measured using Western blotting in the control, CCl4-V, and E-VEGF-C groups. (I and J) Quantitative analysis of VEGF-C (p = 0.0076) and VEGFR-3 (p = 0.0263) protein is represented in the bar graph. The dotted line represents control. n = 4 each. (K) IHC of MLN tissue in control, CCl4-V, and E-VEGF-C-treated rats. Scale bar: 500 μm. (L) Stained area mean intensity was quantified as IHC scores using ImageJ. p = 0.0197 for control vs. CCl4-V and p = 0.0346 for CCl4-V vs. E-VEGF-C. (M) IHC staining of duodenum sections of control, CCl4-V, and E-VEGF-C-treated rats. Scale bar: 200 μm. (N) Stained area mean intensity was quantified as IHC scores using ImageJ. p = 0.0002 for control vs. CCl4-V and p = 0.0017 for CCl4-V vs. E-VEGF-C. Data expressed as mean ± SD. One-way ANOVA with Tukey’s post hoc test was performed. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001. CCl4, carbon tetrachloride; E-VEGF-C, engineered VEGF-C; IHC, immunohistochemistry; LV, lymphatic vessel; MLN, mesenteric lymph node; mLV, mesenteric LV; Pdpn, podoplanin; ns, not significant; PHT, portal hypertension; TAA, thioacetamide; VEGF-C, vascular endothelial growth factor-C; VEGFR-3, vascular endothelial growth factor receptor-3.

Improved drainage of mLVs by E-VEGF-C

To investigate the effect of E-VEGF-C on LV patterning, whole-mount immunostaining of the mesentery was performed using Pdpn antibody. In control rats, thin LVs of diameter around 50 μm were observed, which were increased to 100–150 μm in CCl4-V rats. In E-VEGF-C-treated CCl4 rats, sprouting of new LVs from the existing one was marked with reduced diameter ranging from 60 to 100 μm compared with that in CCl4-V rats (Fig. 4A and B). In addition, branching points of mLVs close to the intestine were increased in E-VEGF-C rats compared with CCl4-V rats (Fig. 4C and D). To assess the functionality of LVs, we gavaged BODIPY FL-C16 in all study groups and analysed drainage and leakage of mLVs after 2 h (Fig. 4E–L and Fig. S4A and B). CCl4-V, TAA-V, and TAA + SAR rats displayed increased BODIPY fluorescence inside mLVs with an increased diameter compared with that in control rats, representing incomplete drainage (p <0.05 each). By contrast, E-VEGF-C-treated CCl4 and TAA rats had significantly reduced fluorescence inside the mLVs with decreased diameter (p <0.05 each). As dilated LVs would cause leakage of the lymph in tissue spaces, we assessed leakage from mLVs by estimating fluorescence outside the mLVs. We observed increased fluorescence outside the LVs in both CCl4 and TAA vehicle rats compared with controls and reduced fluorescence in both E-VEGF-C-treated rats (p <0.05 each).

Fig. 4.

Fig. 4

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Effect of E-VEGF-C on proliferation and drainage of mLVs.

(A) Whole-mount immunostaining of mesentery for visualisation of mLVs in the control, CCl4-V, and E-VEGF-C study groups. The upper two panels represent collecting mLVs, and the lower two represent lymphatic capillaries. Scale bar: 309.4 μm. (B) Diameter of collecting mLVs was measured using the ImageJ diameter plugin. n = 3 or 4 rats in each group. p <0.0001 for control vs. CCl4-V and p = 0.0183 for CCl4-V vs. E-VEGF-C. (C) Mesentery tissue of CCl4-V and E-VEGF-C rats. Arrowheads indicated proliferation and branching of mLVs in the mesentery of E-VEGF-C-treated rats. (D) Quantitative analysis of branching points in the mesentery in control, CCl4-V, and E-VEGF-C rats. n = 3–4 rats in each group. p >0.05 for each comparison. (E) Whole-mount images of mLVs 2 h after BODIPY FL-C16 administration in control, CCl4-V, and E-VEGF-C rats. Scale bar: 309.1 μm. Representative graphs for characterisation of functional mLVs. Mean of three points from each field was taken. (F) Diameter of LVs was measured using ImageJ diameter plugin. (G) Drainage of LVs was measured by quantifying fluorescence intensity inside the vessels using ImageJ. (H) Leakage from mLVs was quantified by measuring fluorescence intensity in the extraluminal space of mLVs using ImageJ. n = 4 or 5 rats in each group. (I) Whole-mount images of mLVs 2 h after BODIPY FL-C16 administration in TAA-V, TAA + SAR, and TAA + E-VEGF-C rats. Scale bar: 200 μm Representative graphs for characterisation of functional mLVs. A mean of three points from each field was taken. (J) Diameter of LVs was measured in μm using ImageJ diameter plugin. (K) Drainage of LVs was measured by quantifying fluorescence intensity inside the vessels using ImageJ. (L) Leakage from mLVs was quantified by measuring fluorescence intensity in the extraluminal space of mLVs using ImageJ. n = 4 or 5 rats in each group. Data expressed as mean ± SD. One-way ANOVA with Tukey’s post hoc test was performed. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001. CCl4, carbon tetrachloride; E-VEGF-C, engineered VEGF-C; LV, lymphatic vessel; mLV, mesenteric LV; ns, not significant; TAA, thioacetamide; VEGF-C, vascular endothelial growth factor-C.

E-VEGF-C reduces ascites and ameliorates PP in cirrhotic and non-cirrhotic portal hypertensive rats

We next probed whether an improvement in mesenteric lymphatic drainage by E-VEGF-C has any effect on ascitic fluid volume in cirrhotic rats. No ascites were observed in the control group, whereas all CCl4-V rats displayed severe ascites (Fig. 5A). E-VEGF-C rats showed a marked reduction in ascites volume compared with that in CCl4-V rats (p <0.05; Fig. 5A and B, and Table S3). Along with ascites reduction, Evans blue staining showed a significant increase in plasma volume in the E-VEGF-C group compared with CCl4-V rats (p <0.05; Fig. 5C). To assess whether E-VEGF-C could also prevent formation of ascites, we developed a BDL animal model of liver cirrhosis (Fig. S5A). One week after surgery, a prophylactic dose of 600 μg/kg E-VEGF-C was administered orally twice a week for the next 3 weeks. BDL rats were sacrificed at the end of the fourth week. In the BDL-V group, five of six rats showed the presence of ascites, whereas only one animal developed mild ascites in BDL + E-VEGF-C rats (Fig. S5B). Next, we analysed whether this reduction in ascitic fluid volume after E-VEGF-C treatment was also associated with hepatic haemodynamic changes. In CCl4-V rats, PP was markedly increased as compared with that in controls (p <0.001; Fig. 5D). However, compared with vehicle, E-VEGF-C treatment significantly attenuated PP (p <0.001). We also monitored these parameters to ascertain whether the observed reduction in PP was caused by the change in portal blood flow (PBF) or intrahepatic resistance (IHR). Compared with CCl4-V, E-VEGF-C treatment resulted in significantly decreased PBF, in turn increasing the mean arterial pressure (MAP) (p <0.05; Fig. 5E and F). However, IHR in E-VEGF-C-treated rats was similar to that in CCl4-V rats (p >0.05; Fig. 5G). Compared with those in control rats, liver weights in E-VEGF-C and CCl4-V rats were similar (Fig. S6). In TAA-V and TAA + SAR rats, PP and PBF were increased compared with those in controls, whereas PP and PBF were significantly reduced in E-VEGF-C-treated rats (p <0.05; Fig. S7). Moreover, MAP was reduced in TAA + E-VEGF-C rats compared with TAA-V and TAA + SAR rats (p <0.05). In BDL + E-VEGF-C-treated rats, PP was decreased significantly (p <0.05; Fig. S8), and MAP was increased compared with those in BDL-V rats (p >0.05). We next evaluated changes in histological and biochemical parameters of the liver in cirrhotic rats, including CCl4, TAA, and BDL after E-VEGF-C. Masson’s trichrome staining of liver tissues revealed that in CCl4 cirrhotic rats, there was no improvement in liver fibrosis after E-VEGF-C treatment. However, BDL + E-VEGF-C and TAA + E-VEGF-C rats showed a slight reduction in liver fibrosis compared with that in BDL-V and TAA-V rats, respectively (Fig. 5H and Fig. S9). CCl4 rats showed significantly increased serum alanine aminotransferase levels and decreased albumin levels compared with those in control rats (Table S3). There was no significant improvement in serum albumin and alanine aminotransferase levels in E-VEGF-C-treated CCl4 rats compared with CCl4-V rats. Kidney functions were normal in CCl4-V rats as well as in E-VEGF-C CCl4 rats.

Fig. 5.

Fig. 5

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Effect of E-VEGF-C treatment on ascitic fluid volume and haemodynamic parameters in cirrhotic and non-cirrhotic portal hypertensive rats.

(A) Representative computed tomography scan slices of control, CCl4-V, and E-VEGF-C rats showing abdominal cavity. Regions of interest are marked with red dotted outline and correspond with fluid accumulation. (B) Dot plots showing ascitic fluid volume (ml) in CCl4-V and E-VEGF-C-treated rats. n = 6 for each group. p = 0.0168. (C) Histograms showing plasma volumes (ml) using the Evans blue dye dilution technique. n = 4 each. Bar diagrams showing hepatic haemodynamic parameters (D) PP, (E) PBF, (F) MAP, and (G) IHR in study groups. n = 6 each. p <0.05. (H) MT stained images of liver tissues in different animal groups. Liver fibrosis was assessed using the Laennec fibrosis scoring system. Bar diagrams showing hepatic haemodynamic parameters (I) PP, (J) PBF, and (K) IHR in control, PPVL vehicle (VEH), and PPVL + E-VEGF-C rats. n = 5 each Data expressed as mean ± SD. Unpaired two-tailed t-tests were performed. One-way ANOVA with Tukey’s post hoc test was performed. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001. CCl4, carbon tetrachloride; E-VEGF-C, engineered VEGF-C; IHR, intrahepatic resistance; MAP, mean arterial pressure; MT, Masson’s trichrome; ns, not significant; PBF, portal blood flow; PP, portal pressure; VEGF-C, vascular endothelial growth factor-C.

We also investigated whether E-VEGF-C treatment improved PP irrespective of liver cirrhosis by measuring hepatic haemodynamics in non-cirrhotic portal hypertensive (PPVL) rats (Fig. S10A). Haemodynamic analysis revealed a significant reduction in PP and PBF of E-VEGF-C-treated PPVL rats compared with vehicle-treated PPVL rats (p <0.01; Fig. 5I–K). Histology of these animals displayed portal inflammation in the liver but no significant fibrosis (Fig. S10B). Compared with PPVL-V rats, PPVL + E-VEGF-C rats also showed increased Pdpn+ mLV number and VEGF-C protein expression with reduced inflammation in mesentery. Furthermore, compared with vehicle, E-VEGF-C-treatment reduces the mRNA expression of iNOS and eNOS in the mesentery (p <0.05; Fig. S10C–G).

E-VEGF-C increases the trafficking of immune cells and clearance of bacteria load in MLNs

Cirrhosis increases gut bacterial translocation to MLNs and other organs.24 Therefore, to test the effect of E-VEGF-C on endogenous bacterial load/clearance in MLNs and other organs, 100 mg of each tissue was extracted from cirrhotic rats in sterile conditions and plated on Luria Bertani agar (Fig. 6A). In CCl4 and TAA rats treated with E-VEGF-C, we observed a decrease in bacterial load in MLNs and other organs studied (Fig. 6B and C; p <0.05). In TAA + SAR rats, there was a significantly increased bacterial load in Peyer’s patches and liver compared with that in TAA-V rats (p <0.001 each). To ascertain the role of E-VEGF-C in the reduction/clearance of bacterial load from MLNs, we next looked at the immune cell trafficking in MLNs.18,25 Cells were isolated from MLNs of control, CCl4-V, and E-VEGF-C rats and labelled with antibodies for T-cell subsets and dendritic cells for quantification using flow cytometry (Fig. S11A and B). Total T cell, helper T cell, and cytotoxic T cell populations in MLNs did not change significantly in control and CCl4-V rats as previously reported (p >0.05; Fig. 6D and E).26 Treatment with E-VEGF-C also led to no change in the populations mentioned above. We further quantified recently activated T cells with CD134 and regulatory T cells with CD25 expression. No significant change was observed in CD134 expression in varied groups (p >0.05; Fig. 6D and E), but we observed a significant decrease in CD8 regulatory T cells after E-VEGF-C treatment in comparison with CCl4-V (p <0.05; Fig. 6F and G). Dendritic cells, along with the expression of T cell activation coreceptor CD80, were increased after treatment with E-VEGF-C compared with that observed after vehicle treatment (p <0.05; Fig. 6H).

Fig. 6.

Fig. 6

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Effect of E-VEGF-C on clearance of bacterial load in different organs.

(A) Bacterial load in different organs extract from CCl4-V, E-VEGF-C, TAA-V, TAA + SAR, and TAA + E-VEGF-C rats. (B) Quantitative analysis of CFU/gm in MLNs, Peyer’s patches, liver, spleen, and lung of CCl4-V and E-VEGF-C rats. n = 3 each. (C) Quantitative analysis of CFU/gm in MLNs, Peyer’s patches, liver, spleen, and lung of TAA-V, TAA + SAR, and TAA + E-VEGF-C rats. n = 3 each. (D) Immune cell quantifications from MLNs of control, CCl4-V, and E-VEGF-C rats. Dot plots of T-cell subsets in each study groups. (E) Percentage population of CD3 T cells, CD4 Th cells, CD8 Tc cells, and CD134+ recently activated Th and Tc cells. (F) Dot plots of CD25+ regulatory helper T cells in each study groups. (G) Percentage population of Treg cells positive for CD3, CD4/CD8, and CD25. (H) Percentage population of DCs positive for CD11c, CD103, and CD80. p <0.05. Data expressed as mean ± SD. One-way ANOVA with Tukey’s post hoc test was performed.∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001 (comparison with CCl4-V or TAA-V). CCl4, carbon tetrachloride; CFU/gm, colony forming unit–granulocyte/macrophage; DC, dendritic cell; E-VEGF-C, engineered VEGF-C; MLN, mesenteric lymph node; TAA, thioacetamide; Tc, cytotoxic T; Th, helper T; Treg, regulatory T; VEGF-C, vascular endothelial growth factor-C.

As we observed no significant changes in T-cell subsets in MLNs of either CCl4-V or E-VEGF-C rats, we investigated changes in the priming of immune responses after antigen challenge. To analyse the effect of activated T cells on bacterial clearance in MLNs after E-VEGF-C treatment, we gavaged rats with GFP-labelled Salmonella typhimurium in the CCl4 cirrhotic and PPVL non-cirrhotic models of portal hypertension (Fig. 7A and Fig. S11C).24 After 48 h of gavaging, GFP-labelled bacteria were found only in the MLNs of control rats. In contrast, in CCl4-V and PPVL-V rats, GFP-labelled bacteria were present in all the collected tissues, including blood in CCl4-V rats (Fig. 7B and Fig. S11D and E). Interestingly, in E-VEGF-C-treated CCl4 rats, bacterial translocation was confined only to MLNs with reduced live bacteria compared to MLNs in CCl4-V rats (p <0.001; Fig. 7B and C), suggesting clearance of bacterial load. In PPVL + E-VEGF-C rats, bacterial translocation was reduced in MLN and other organs (Fig. S11F). After the bacterial challenge, we found a significant increase in recently activated helper T cells in MLNs of E-VEGF-C-treated rats compared with CCl4-V rats, which indicates an active immune response in MLNs in the presence of antigen (Fig. 7D–G). We also assessed the levels of endotoxins and inflammatory cytokines in serum and ascites. Endotoxin levels were reduced in ascites of E-VEGF-C rats (p <0.05); however, no change was observed in serum endotoxins (Fig. S12A and B). Serum IL-6 levels were significantly reduced in E-VEGF-C rats compared with CCL4-V rats (p <0.05); however, no change was observed in serum tumour necrosis factor-alpha levels in the E-VEGF-C-treated rats compared with CCl4-V rats (Fig. S12C and D).

Fig. 7.

Fig. 7

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Effect of E-VEGF-C on priming of immune cells after bacterial challenge.

(A) Schema of workflow, 109 GFP + bacteria were given to rats orally, and tissue was collected in sterile conditions after 48 h of gavage. Afterward, 100 mg of MLN tissue was used for bacterial load quantification. Cells were isolated from MLNs for quantification using flow cytometry. (B) GFP+ Salmonella typhimurium colonies in 100 mg of MLN tissue extract of each group were visualised using a UV transilluminator. (C) Quantitative analysis of CFU/gm of MLN tissue for GFP + Salmonella typhimurium. n = 3 each. p <0.001. (D) Dot plots of T-cell subsets in cells isolated from MLNs of control, CCl4-V and E-VEGF-C treated rats after bacterial challenge. (E) Percentage population of CD4 Th cells, CD8 Tc cells, and CD134+ recently activated Th and Tc cells. (F) Dot plots of CD25+ regulatory Th cells. (G) Percentage population of Treg cells positive for CD3, CD4/CD8, and CD25. n = 4 each. Data expressed as mean ± SD. One-way ANOVA with Tukey’s post hoc test was performed. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001 (comparison with CCl4-V). #p <0.05 (comparison with control). CCl4, carbon tetrachloride; CFU/gm, colony forming unit–granulocyte/macrophage; E-VEGF-C, engineered VEGF-C; GFP, green fluorescent protein; MLN, mesenteric lymph node; ns, not significant; Tc, cytotoxic T; Th, helper T; Treg, regulatory T; VEGF-C, vascular endothelial growth factor-C.

E-VEGF-C treatment modulates gene expression in LyECs of mesentery and lymph nodes

To gain mechanistic insights into VEGF-C-induced lymphangiogenesis in mLVs, LyECs from the mesentery and MLNs were isolated using FACS, and the expression of relevant genes was examined (Fig. 8A). The mRNA level of differentiation and proliferation markers of LyECs, that is, Prox1 and LyVE1, were upregulated in LyECs of E-VEGF-C-treated rats compared with CCl4-V rats (p <0.05; Fig. 8B). The mRNA levels of VCAM1 were decreased in LyECs of CCl4-V rats, indicating reduced cell adhesion in LyECs of mLVs. No significant change was observed in vascular endothelial (VE)-cadherin in CCl4-V rats compared with controls. Treatment with E-VEGF-C led to increased expression of both VCAM1 and VE-cadherin in LyECs (p <0.05). LyECs also act as antigen-presenting cells, presenting Ag to T cells for tolerance or induction of immune responses. The costimulatory marker CD86 was significantly upregulated in LyECs of treated rats. In contrast, no significant change was observed in MHC-II levels in the vehicle vs. treated group (p >0.05). The inflammatory marker COX2 was markedly increased in LyECs of CCl4-V rats, suggesting an inflamed lymphatic endothelium, but was further reduced in LyECs of the treated group (p <0.05). CCL21 and CCL19 chemokine mRNA levels were increased in LyECs of E-VEGF-C-treated rats compared with vehicle rats, resulting in increased immune cell trafficking to MLNs (p <0.05). Programmed death-ligand 1 (PD-L1) was increased in LyECs of CCl4-V rats but was further reduced in the treated group (p <0.01). We next asked whether these transcriptional changes in LyECs, specifically VE-cadherin, could lead to differences in the ability to allow passage of BODIPY (labelled fatty acid) through their cell–cell junctions (i.e., permeability). To answer this question, we used a barrier Transwell assay in which a confluent monolayer of mesenteric LyECs was isolated from control, CCl4-V, and E-VEGF-C-treated rats and plated on the upper chamber of the Transwell. From initial time points onwards (4–24 h), there was increased permeability in LyECs of CCl4-V rats compared with controls, as measured by an increase in the amount of BODIPY fluorescence in the lower chamber (p <0.001; Fig. 8C). In LyECs of E-VEGF-C rats, BODIPY could not pass through the monolayer; therefore, its reduced amount was observed in the lower chamber compared with that in CCl4-V rats (p <0.001).

Fig. 8.

Fig. 8

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Effect of E-VEGF-C on gene expression profiling and permeability of LyECs.

(A) Schema of workflow. LyECs were isolated from the mesentery and MLNs using FACS. RNA isolation and qRT-PCR analysis were performed for different marker genes of LyECs. (B) Relative gene expression of LyVE1, Prox1, VCAM1, VE-Cad, MHC-II, CD86, CCL21, and COX2 genes in CCl4-V and E-VEGF-C-treated rats were plotted. Dotted lines represent control (C) Transwell permeability assay using BODIPY in cultured mesenteric LyECs from control, CCL4-V, and E-VEGF-C-treated rats. The concentration of BODIPY in collected media after 4, 6, 10, and 24 h n = 3 each. Data expressed as mean ± SD. One-way ANOVA with Tukey’s post hoc test was performed. ∗p <0.05; ∗∗p <0.01 (comparison with CCl4-V). CCl4, carbon tetrachloride; E-VEGF-C, engineered VEGF-C; LyEC, lymphatic endothelial cell; MLN, mesenteric lymph node; ns, not significant; qRT-PCR, quantitative reverse-transcription PCR; VE-Cad, vascular endothelial-cadherin; VEGF-C, vascular endothelial growth factor-C.

Discussion

We investigated morphological and molecular alterations of mLVs and MLNs in experimental cirrhosis and portal hypertension. A significantly increased number of dilated and leaky mLVs with reduced drainage were observed in cirrhotic portal hypertensive rats, implying gut lymphatic dysfunction similar to that observed in intestinal lymphangiectasia and IBD27,28 Increased dysfunctional mLVs in cirrhosis may be attributed to compensatory lymphangiogenesis response to lymphatic occlusion.27 Therefore, we focused on increasing the number of new functional lymphatic channels via VEGF-C treatment.

Owing to the short half-life and systemic effects of VEGF-C,21 we constructed recombinant VEGF-C molecules using RM-based nanolipocarriers, which are nanoparticle-sized water-in-oil microemulsions with controlled size. A recent report illustrated the therapeutic potential of a human fusion protein F8-VEGF-C for targeted delivery of VEGF-C in mouse models of chronic inflammatory skin disease.29 However, the study did not report sustained release of VEGF-C. In another study, VEGF-C mRNA was encapsulated in lipid nanoparticles for sustained release in experimental lymphoedema.30 In our study, RM-based lipid nanocarriers allowed VEGF-C to be carried in chylomicron-sized particles in LVs. Encapsulation also ensured their sustained and programmable release.31 In vitro, release studies indicated more than 80% VEGF-C release in 4 h. In vivo, the release profile showed a biphasic peak, first at 10 min and then at 5 h. Biphasic drug release is a characteristic feature of drugs encapsulated in nanolipocarriers.32 In addition, cirrhotic rats showed maximum expression of VEGF-C in the mesentery, indicating efficacy of our delivery vehicle. We did not follow any conjugation chemistry to prepare the E-VEGF-C molecule focused on preparing simple solid lipid nanoparticles with an aqueous template using a modified multiple emulsification technique.33

In both CCl4-and TAA-induced cirrhosis, treatment with E-VEGF-C enhanced expression of VEGF-C protein in the mesentery, along with concomitant increase in the sprouting of mLVs, in both the mesentery and MLNs, suggestive of VEGF-C-driven proliferation of LyECs.34 Dilations in mLVs of cirrhotic rats were also attenuated with E-VEGF-C treatment. Specific genes, such as Prox1 and LyVE1, were significantly increased in mesenteric LyECs of E-VEGF-C rats, indicating differentiation and proliferation of LyECs. Expression of inflammatory genes, such as Cox2, was decreased, whereas VCAM-1 and VE-cadherin, which govern cell adhesion and vessel permeability, increased in LyECs of E-VEGF-C rats as compared with cirrhotic rats. The functional implications of enhanced gene expression were evident in terms of reduced permeability, lymph leakage, and improved drainage in treated rats. In addition, there was a conspicuous decrease in mesenteric tissue inflammation in E-VEGF-C treated rats. This is in accordance with the previously reported drainage-promoting function of VEGF-C.35,36 We did not detect any significant changes in CD31+ blood vessels with E-VEGF-C treatment, indicating that LV-specific VEGF-C (Cys156Ser) did not affect mesenteric blood vessels.37

An improvement in lymphatic drainage and functionality of the mLVs in cirrhotic rats was also associated with a reduction in ascites in CCl4 rats. The decrease in ascites was accompanied by an increase in the plasma volume of treated rats.11 Compared with the 4-week BDL vehicle, E-VEGF-C treatment also prevented ascites formation in BDL models when given as a prophylactic treatment. We, however, did not follow these models beyond 4 weeks. Along with a reduction in ascites, there was also a decrease in PP in CCl4-and TAA-induced portal hypertensive rats treated with E-VEGF-C, which was associated with attenuated PBF and increased MAP. However, there was no change in IHR, indicating that improvement in PBF and not hepatic resistance (fibrosis) led to improved PP after treatment. It has been postulated that removal of ascites plays a role in post-paracentesis systemic haemodynamic changes through mechanical decompression of the splanchnic vascular bed.38 However, reduction in PP caused by increased LVs and decreased interstitial fluid pressure may also have led to decreased ascites.11 This decrease in PP was also observed in the non-cirrhotic PPVL animals after E-VEGF-C treatment, validating the favourable effects of VEGF-C on PBF. An improvement in PP was not associated with an improvement in liver pathology in the treated CCl4 and TAA rats, indicating no protective effects of E-VEGF-C treatment on the hepatic compartment per se. In these models, we administered therapeutic E-VEGF-C treatment when liver cirrhosis had already been established. A slight reduction in liver fibrosis was however seen in E-VEGF-C-treated BDL rats compared with BDL-V rats, in which an early treatment was given.

A reduction in permeability and improved drainage of mLVs was associated with reduced endogenous bacterial translocation in cirrhotic animals treated with E-VEGF-C. When we challenged these cirrhotic animals with live bacteria, we observed live bacteria in MLNs and other organs including blood, indicating impaired immune responses in the MLNs.39 In cirrhotic animals treated with E-VEGF-C, bacteria remained confined to MLNs only. Translocation of live bacteria was reduced in MLNs of non-cirrhotic PPVL animals treated with E-VEGF-C. This decrease in bacterial translocation could explain the observed reduction in PP of PPVL animals.24

VEGF-C increases immune cell trafficking to the draining lymph node.15 We did not observe any change in T-cell recruitment in MLNs of E-VEGF-C-treated animals compared with CCl4 rats. There was, however, an increase in costimulatory markers, such as CD86, in the LyECs of E-VEGF-C-treated CCl4 rats, suggesting active antigen presentation by these cells.40,41 In MLNs of CCl4 animals, VEGF-C treatment increased recently activated CD4 T cells, indicative of an appropriate immune response. An earlier study documented that stimulation of cardiac lymphangiogenesis with VEGF-C improved trafficking of immune cells to draining lymph nodes after myocardial infarction resolving inflammation.25 Along with improved gut immune responses, we also observed a decrease in systemic endotoxins and inflammatory cytokines after E-VEGF-C treatment.42

In summary, our study underscores the use of nanolipocarriers incorporating LV-specific VEGF-C as a novel therapy for improving lymphatic drainage, gut immunity, and portal hypertension by providing an efficient exit route for ascites. Gut LV-targeted delivery of E-VEGF-C may open new and exciting avenues for treating and preventing decompensation in cirrhosis.

Financial support

The study was funded by the Department of Science and Technology (DST/NM/NT/191/G) and Science and Engineering Research Board (SERB, SPG/2021/002451), Government of India.

Authors’ contributions

Conceptualised and designed the work: SK, DMT, SB. Collected the data: PJ, SNRR, DJ, AM, IK, PR, SR. Analysed and interpreted the data: PJ, , SNRR, SK, DMT, SB, IK, PR. Drafted the article: PJ, SNRR, SK, SB. Did critical revisions of article: PJ, DJ, AM, VT, SK, SB, VN, SKS. Read and approved the final article: all authors.

Data availability statement

All the data supporting the findings of this study are available within the article and its Supplementary information files and from the corresponding authors upon reasonable request.

Conflicts of interest

All authors declare no competing interest.

Please refer to the accompanying ICMJE disclosure forms for further details.

Acknowledgements

The authors would like to thank the Department of Science and Technology (DST /NM/NT/2019/191) and Science and Engineering Research Board (SERB, SPG/2021/002451) Government of India, for the financial support and Center for Nanotechnology (CNT) and the North East Centre for Biological Sciences and Healthcare Engineering (NECBH) of IIT-Guwahati, Assam, India, for providing the facility for AFM and FE-SEM analysis. We acknowledge Dr. Sheetal Gandotra from Institute of Genomics and Integrative Biology, New Delhi for providing GFP-labelled Salmonella typhimurium.

Footnotes

Author names in bold designate shared co-first authorship

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jhepr.2023.100816.

Contributor Information

Subham Banerjee, Email: subham.banerjee@niperguwahati.ac.in.

Savneet Kaur, Email: savykaur@gmail.com.

Supplementary data

The following are the supplementary data to this article.

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Associated Data

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Supplementary Materials

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Data Availability Statement

All the data supporting the findings of this study are available within the article and its Supplementary information files and from the corresponding authors upon reasonable request.

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