NOSTRIN is an emerging negative regulator of decompensated cirrhotic patients with portal hypertension

doi:10.21203/rs.3.rs-4327525/v1

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NOSTRIN is an emerging negative regulator of decompensated cirrhotic patients with portal hypertension

https://doi.org/10.21203/rs.3.rs-4327525/v1

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Background and aims: Decreased nitric oxide (NO) bioavailability in a cirrhotic liver contributes to high intrahepatic vascular resistance (IHVR) and portal hypertension (PHT). Nostrin is an inhibitory protein of NO synthesising enzyme endothelial NO synthase (eNOS), shown to increase in cirrhosis with PHT, however, the precise molecular mechanism is poorly documented. This study aimed to elucidate the role of Nostrin and associated derangement in hepatic NO generation in cirrhotic liver. Further, we investigate whether Nostrin could be a biomarker in the progression of cirrhosis. Methods: The study was conducted in sixty healthy subjects and 120 cirrhotic patients (both compensated and decompensated) to analyze the blood Nostrin, cGMP and cytokine levels. In addition, liver tissue samples collected from cirrhotic patients were used for the analysis of gene and protein expression of Nostrin, eNOS and inflammatory markers.

Results:When compared to healthy controls, systemic levels of Nostrin and cGMP were elevated in compensated cirrhosis. In decompensated cirrhosis, further robust increases in Nostrin and cGMP were noted. Furthermore, hepatic Nostrin expression was considerably higher whilst reduced eNOS activity and hepatic cGMP levels in cirrhotic liver compared to control liver. Hepatic iNOS and NF kB protein expression were significantly increased in cirrhotic liver compared to control liver.

Conclusions: In decompensated cirrhotic patients, a robust increase in hepatic Nostrin expression was associated with inflammation and thus, reduced eNOS activity with concomitant local NO generation. Furthermore, Blood Nostrin concentration was higher and parallel to disease severity and could be a key diagnostic and prognostic biomarker in cirrhotic patients with exacerbated PHT.

Cirrhosis

eNOS trafficking inducer

inflammation

nitric oxide and portal hypertension

prognosis

In the human cirrhotic liver, we observed markedly elevated nitric oxide synthase trafficking inducer (Nostrin) expression and significantly decreased peNOS protein expression (a measure of eNOS activity).
Blood Nostrin levels were associated with 90-day mortality in patients with decompensated cirrhosis.
Elevated blood Nostrin levels were associated with the severity and prognosis of decompensated cirrhosis with exacerbated PHT.

Nitric oxide (NO) is a major vasoactive molecule synthesized from an amino acid L-arginine by the enzyme endothelial nitric-oxide synthase (eNOS) in the endothelium of the plasma membrane. NO has been recognized as critical in the biology of multiple liver processes and diseases. Reduced NO bioavailability results from a defect in hepatic eNOS activity in the cirrhotic liver, which appears to contribute to high intrahepatic vascular resistance (IHVR) and, thus, portal hypertension (PHT). eNOS activity is regulated by various factors, which include extensive posttranslational modification and intracellular localization of the enzyme eNOS trafficking inducer (Nostrin). In advanced cirrhosis with inflammation, a robust increase in Nostrin, sequestering eNOS from the caveolae plasma membrane into other sub-cellular compartments like Golgi apparatus or different vesicular-like structures, thereby reducing eNOS activity and NO generation. Furthermore, in decompensated cirrhosis, increased blood Nostrin concentration was associated with progression of disease pathogenesis.

Cirrhosis progresses over time from a compensated to a decompensated stage, which is concurrent with a conspicuous deterioration in life expectancy [1]. Despite significant healthcare and socio-economic burdens, the management of decompensated cirrhotic patients remains suboptimal and non-specific [1]. Portal hypertension (PHT) is a major hallmark feature of clinical decompensation of cirrhosis, which is widely prevalent and causes one million deaths annually worldwide [2]. The consequences of which include ascites, hepatorenal syndrome (HRS), hepatic encephalopathy (HE), bacterial infections and bleeding from gastro-esophageal varices resulting in impaired quality of life, hospitalization and high mortality [2]. Compelling evidence shows that progressive PHT, systemic inflammation, and liver failure lead to poor disease outcomes [1, 3]. Hepatic venous pressure gradient (HVPG) > 10 mmHg corresponds to clinically significant PHT and is the strongest predictor of decompensation [4]. Consequently, correcting PHT might positively impact the disease severity and form a rationale for therapy in cirrhotic patients. Furthermore, over the past decade, many studies have attempted to elucidate the key mechanistic role of inflammation on the pathogenesis and severity of PHT in cirrhosis. However, the precise pathomechanisms of vascular dysfunction and the role of inflammation in cirrhosis remain unclear.

Nitric oxide (NO) is a natural vasoactive compound regulating vascular tone in the intrahepatic microcirculation [5]. NO is synthesised from an amino acid, L-arginine, by the action of endothelial NO synthase (eNOS), which is crucial for active adaptive vascular remodelling to chronic changes in flow [5, 6]. NO activates soluble guanylyl cyclase (sGC) with concomitant generation of cyclic guanosine monophosphate (cGMP) and vasodilation. Moreover, substantially reduced hepatic NO bioavailability (i.e. reduction in NO production or increased breakdown) and increased vasoconstrictors are the consequence of endothelial dysfunction (ED), which causes increased intrahepatic vascular resistance (IHVR) to portal blood flow, thus PHT in cirrhosis [5, 7–10]. PHT, in turn, causes ED in the splanchnic and systemic circulation [11]. Furthermore, in cirrhotic liver, considerably decreased eNOS activity and increased oxidative stress and inflammation are the dynamic components responsible for diminished NO synthesis [7, 10].

Previous human studies have shown evidence that either uncoupling or deregulation of eNOS has been associated with decreased NO and triggers PHT [8, 12, 13] however, the molecular mechanism remains largely undetermined. Thus, maintaining effective eNOS function is central to the pathogenesis of PHT in cirrhosis. eNOS activity is tightly regulated at various levels, including complex post-translational modification, phosphorylation and protein-protein interactions [14–16]. Furthermore, previous studies have to date, focused on inhibitors of eNOS activity such as caveolin-1 [17] and asymmetric dimethylarginine (ADMA) [8] or on processes affecting posttranslational modification [12]. In this context, previous studies have established that interaction and co-localisation of eNOS with either Nostrin (eNOS traffic-inducing protein) or NOSIP (eNOS interacting protein) result in displacing eNOS from the plasma membrane caveolae to other vesicular-like structure thereby inhibiting NO generation [12, 15, 16]. Our recent study highlights compelling evidence that Nostrin overexpression is associated with dysfunctional eNOS enzyme with local NO generation, leading to elevated portal pressure in cirrhotic mice [10]. This is further confirmed with Nostrin siRNA transfections to HUVECS showing improved eNOS function and NO bioavailability [10]. Since Nostrin expression was identified in highly vascularized human tissues [18], this may influence abridged NO generation of hepatic vasculature in the pathogenesis of cirrhosis. Indeed, not many human studies in cirrhotic patients have focused on the underlying pathological factors, such as the inflammation-mediated interaction of Nostrin and eNOS and associated NO synthesis. Consequently, we aimed to establish whether Nostrin overexpression is associated with reduced eNOS function and related NO synthesis in human cirrhosis. In addition, we assessed whether blood Nostrin levels were associated with the severity and prognosis of decompensated cirrhosis.

Human Cirrhosis

The current cross-sectional study was performed on 120 cirrhotic patients who visited the outpatient unit or were admitted to the Departments of Medical Gastroenterology and Surgical Gastroenterology wards, Jawaharlal Institute of Post Graduate Medical Education and Research (JIPMER) between 2016 and 2018. All cirrhotic patients (cases) and healthy subjects (controls) signed written informed consent before they were enrolled. All the study participants were aware of concerning the background and procedure of this study. In addition, the study protocol was approved by the JIPMER Ethics Committee for Human Studies (JIP/IEC/2016/30/971) and was conducted in accordance with the revised (1983) Ethical Guidelines of the Helsinki Declaration.

Study Population

Both males and females aged 18–60 years were recruited and were diagnosed as cirrhosis (both compensated and decompensated n = 60, respectively) based on the combination of clinical, biochemical, endoscopic and/or ultrasound findings or a liver biopsy, according to recent EASL guidelines [3]. Sixty age and gender-matched healthy volunteers were included as controls (n = 60) based on standard hepatic function tests and without a history of any recent infections or other co-morbidities. All patient's demographic and clinical data were collected at admission. Furthermore, 8ml of blood samples collected in heparinized/EDTA-coated tubes were centrifuged (3000 rpm for 10 minutes), and the plasma separated was stored at -80^oC until analysis. Blood samples from healthy volunteers in the control group were collected and processed under the same conditions as the patient samples. All the biochemical parameters (liver and kidney function tests) were measured in the plasma samples using an autoanalyzer (Beckman Coulter, Inc. AU 5000, USA). In addition, liver specimens obtained during surgery of cirrhotic patients were fixed in formalin for histological evaluation, immunohistochemistry and immunofluorescence findings. Pieces of liver specimens were also snap-frozen in liquid nitrogen and stored at -80^oC for further analysis.

ELISA

As previously described [10], Nostrin and cGMP levels in both plasma and liver tissue supernatants were assessed by employing human ELISA kits (Ray Biotech, USA.). Furthermore, circulating cytokine levels were measured in plasma using human ELISA kits (TNFα, Ray Biotech, USA; IL-1β and IL-10, Diaclone, France.), according to manufacturer’s instructions.

Histopathological and immunohistochemistry (IHC) analysis

Hematoxylin-eosin (H&E) and IHC staining methods were used to stain the liver, which was prefixed with 10% normal buffered formalin. 5µm thick sections were cut from paraffin-embedded liver slices, deparaffinated and rehydrated with respective xylene and ethanol, and mounted onto silane-coated slides using an automated microtome (Leica). H&E staining was performed in tissue-embedded slides to assess liver parenchyma, as described earlier [10]. EVOS cell imaging system (Invitrogen, UK) was used to acquire the images. For IHC, silane-coated slides were treated with antigen retrieval and 3% H₂O₂ solutions to inhibit endogenous peroxidase activity. Tissues were incubated with 5% goat serum for 1 hr and incubated overnight at 4^oC with primary antibodies against rabbit polyclonal anti-human Nostrin (1:100; cat: sc-134803, Santacruz) and rabbit polyclonal anti- human eNOS (1:100; cat: PA-1712-1, Bosterbio). Sections were washed with TBS-tween-20 and incubated for 30 minutes at RT with universal secondary antibody (Cat: MP-7500, Vector lab). Antibody binding was revealed using hydrogen peroxide as substrate and ImPACT diaminobenzidine (DAB) as chromogen (Cat: SK-4105, Vector lab). Hematoxylin was used as a counterstain, and then the slides were mounted with Histamount (Invitrogen) and analyzed using the EVOS-FLC imaging system (Invitrogen, UK). For negative control, tissue slides were omitted with primary antibody and incubated only with the corresponding secondary antibody as described above.

Immunofluorescence

4% paraformaldehyde-fixed liver tissues were sectioned and mounted on silane-coated slides. Following antigen retrieval treatment, the tissue slides were blocked with appropriate normal blocking serum and then incubated with specific primary antibody against anti-human Nostrin for 1hr (1:100 dilution, respectively). For double immunostaining, sections were treated with anti-human Nostrin and anti-human eNOS antibodies. The sections were washed with TBST and then incubated with fluorescence-tagged secondary antibody such as goat anti-rabbit IgG (Alexa Fluor 488, Cat: A11034, Thermo Fisher Scientific, USA) for 1 hour in the dark. After washing, nuclear counterstaining was done using DAPI (1 µg/ml, 1 minute) (Cat: D9542, Sigma) and mounted with a coverslip using Histamount (Invitrogen) mounting medium. For negative control, tissue slides were omitted with primary antibody and incubated only with the corresponding secondary antibody as described above.

PCR

As previously described [10], total cellular RNA was extracted from frozen human liver tissues using the RNeasy mini Kit (catalogue: 74104, Qiagen, Germany). RNA purity and integrity were assessed by agarose gel electrophoresis after RNA quantification using the NanoDrop system (Thermo Scientific, USA). PrimeScript RT Reagent (catalogue: RR037A, Takara) was used for cDNA synthesis from quantified RNA samples. The primers specific for each gene including Nostrin (F: 5'-GGCAGAGCAATCCAGGTTC-3'; R: 5'-AGGCCTTGCAAAGTCTGCT-3'), eNOS (F: 5'-CGGCATCACCAGGAAGAAGA-3' R: 5'-CATGAGCGAGGCGGAGAT-3', and a housekeeping gene, glyceraldehyde-3phopshate dehydrogenase (GAPDH) (F: 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' R5'-CATGTGGGCCATGAGGTCCACCAC-3') were designed using the PrimerQuest tool (IDT) and obtained from Integrated DNA Technologies. Gene expression was analysed using CFX96 qPCR analysis software (Biorad Laboratories, USA). The same samples were tested in triplicates. Melting curve analysis was performed to verify the specificity of the PCR product. The target mRNAs were normalised with GAPDH, and their relative quantitation was analysed using the comparative Ct method.

Western blotting

Western blot analysis was performed on protein extraction obtained from liver homogenates, as described earlier [10]. Following protein estimation by employing an improved Bradford assay kit (Cat: 23236, Pierce™, Thermo Fischer Scientific, UK), liver protein samples were loaded onto 4–12% NuPAGE Bis-Tris protein Gels, then transferred onto polyvinylidene difluoride

(PVDF) membranes (Invitrogen, UK). Membranes were blocked in 5% non-fat dry milk powder (Sigma-Aldrich, Merck, India) diluted in TBST for 1hr at RT. The blots were incubated overnight with different primary antibodies, including rabbit anti-human NOSTRIN (1:1000; Santacruz, USA), mouse anti-human eNOS, iNOS and p-eNOS (Ser1177) (1:500, 1:10,000 and 1:1000 respectively; BD transduction laboratories, CA and Sigma, USA), and NFkB, (1:1000; Cell signaling, UK), and after being extensively washed with TBST, incubated with respective horseradish peroxidase (HRP) secondary antibodies diluted 1:1000 for 1 hr at RT. The blots were washed again, and the bands were visualized using an enhanced ECL detection kit (Clarity™ Western ECL substrate). ChemiDoc MP System (Bio-Rad, Hercules, CA, USA) was used for image acquisition and quantification. Rabbit anti-β-actin antibody was used to assess the loading accuracy after stripping the blots with Western blot stripping buffer (Thermo Fisher, USA).

Statistics

Data were presented as mean ± SEM. The Mann-Whitney U test or ANOVA, followed by the Kruskal-Wallis multiple comparison test were performed for significant differences between control and disease groups; p-value < 0.05 was defined to denote statistical significance. The correlation coefficient (Rho) was calculated using Spearman’s correlation. ROC curve analysis was done using sensitivity and specificity for possible cut-off values for plasma Nostrin levels. Software used included Microsoft Excel 2021, Version 16.8 (Microsoft Corp., Redmond, WA) and GraphPad Prism 10.0 (GraphPad Software, Inc., San Diego, CA).

Demographic and clinical features of the study population

The patient’s demographic and baseline clinical characteristics and the comparison between compensated and decompensated cirrhosis are presented in Tables 1&2. A total of 120 patients were included, most of whom were male (75%). When compared to compensated cirrhosis, the MELD score was significantly higher in decompensated cirrhosis, and there was no difference in age between these two groups. In addition, there was no difference in BMI between controls and compensated cirrhosis, but was considerably decreased in decompensated cirrhosis (Table 2). The most common etiology of decompensated cirrhosis was alcohol consumption (62%). Overall, 83% of patients in the decompensated group had developed ascites. Most of the decompensated cirrhotic patients belong to Child-Pugh class B and C compared to compensated cirrhosis (child class A) (Table 1). Moreover, the severity of liver disease was higher (with higher PT, INR, Child-Pugh and MELD scores) in decompensated cirrhosis than in compensated cirrhosis (Table 2).

Table 1

Clinical and radiological characteristics of cirrhotic patients with and without decompensation.
	Compensated cirrhosis		Decompensated cirrhosis
Characters	N	%	N	%
Alcoholic Non-alcoholic	25 35	42 58	37 23	62 38
PHTN Present Absent	28 32	47 53	60 0	100 0
Pedal edema Present Absent	0 60	0 100	34 26	57 43
Ascites Absent Mild Moderate Severe	60 0 0 0	100 0 0 0	10 26 13 11	17 43 22 18
Esophageal varices Absent Grade I Grade II Grade III Grade IV	60 0 0 0 0	100 0 0 0 0	43 7 7 3 0	73 11 11 5 0
Hepatitis infection No hepatitis infection HBs Ag positive HCV positive	48 9 3	80 15 5	54 4 2	90 7 3
Encephalopathy Absent Grade 0 Grade I Grade II Grade III Grade IV	60 0 0 0 0 0	100 0 0 0 0 0	44 0 10 6 0 0	73 0 17 10 0 0
Child class A B C	50 10 0	83 17 0	6 27 27	10 45 45
Total	60	100	60	100

Table 2

Demographic, biochemical parameters and disease severity in cirrhotic patients with and without decompensation.
Variable	Control subjects (n = 60)	Compensated cirrhosis (n = 60)	Decompensated Cirrhosis (n = 60)	One way ANOVA
				p-value
Gender (M/F)	4 45/15	44/16	53/7
Age (Years)	44.80 ± 9.68	48.63 ± 11.38	48.73 ± 9.01
BMI (Kg/M²)	26.11 ± 2.96	25.82 ± 4.48	23.91 ± 4.21^$
Total bilirubin (mg/dL)	0.61 ± 0.24	1.28 ± 0.67^****	3.47 ± 4.9^$$	< 0.0001
Direct bilirubin (mg/dl)	0.19 ± 0.06	0.51 ± 0.24^****	1.10 ± 1.37^$$$$	< 0.0001
Total protein (gm/dl)	7.79 ± 0.36	6.8 ± 0.53^****	6.39 ± 0.55^$$$$	< 0.0001
Albumin (gm/dl)	4.31 ± 0.31	3.42 ± 0.50^****	2.81 ± 0.54^$$$$	< 0.0001
ALT (IU/L)	24.08 ± 7.87	51.05 ± 19.78^****	77.21 ± 77.03^$$$	< 0.0001
AST (IU/L)	27.08 ± 7.61	79.53 ± 35.36^****	135.5 ± 86.72^$$$$	< 0.0001
γGT (IU/L)	23.97 ± 8.17	60.12 ± 54.22^****	82.83 ± 89.23^ns	< 0.0001
ALP (IU/L)	142.06 ± 38.8	236.03 ± 80.3^****	316.83 ± 130.6^$$$$	< 0.0001
Urea (mg/dl)	19.48 ± 0.5.4	24.3 ± 9.23^**	31.61 ± 11.00^$$	< 0.001
Creatinine (mg/dl)	0.87 ± 0.09	1.10 ± 0.27^****	1.59 ± 0.80^$$$$	< 0.0001
Sodium (mEq/L)	139.8 ± 2.95	137.7 ± 2.58^***	134.5 ± 3.98^$$$$	< 0.0001
Potassium (mEq/L)	4.27 ± 0.25	4.19 ± 0.46	4.10 ± 0.48	< 0.080
PT (Sec)	14.37 ± 1.07	17.28 ± 2.46^****	22.79 ± 6.35^$$$$	< 0.0001
INR	1.06 ± 0.08	1.31 ± 0.20^****	1.82 ± 0.60^$$$$	< 0.0001
Child-Pugh Score	-	5.76 ± 0.85	9.3 ± 2.33^$$$$	< 0.0001
MELD Score	-	11.93 ± 3.01	20.70 ± 8.58^$$$$	< 0.0001
MELD-Na	-	13.40 ± 0.38	21.90 ± 1.07^$$$$	< 0.0001

Biochemical parameters of the study population

The biochemical parameters such as bilirubin, ALT, AST, ALP and GGT were significantly increased, whilst plasma total protein and albumin concentrations were significantly decreased in compensated cirrhosis as compared to control subjects (p < 0.0001). In decompensated cirrhotic patients, the concentrations of bilirubin, ALT, AST, and ALP were further significantly increased compared to compensated cirrhotic patients. In contrast, plasma albumin and total protein concentration were further significantly decreased. Moreover, urea and creatinine levels were increased in compensated cases compared to control subjects and were further considerably elevated in decompensated cirrhosis when compared to compensated cases. Serum sodium concentrations was considerably low in compensated cirrhosis compared to control subjects and was further reduced in decompensated cirrhosis. However, potassium levels did not alter between groups (Table 2).

Diagnostic and prognostic potential of Nostrin concentrations in cirrhosis

Firstly, we analysed the blood concentrations of Nostrin in control subjects and cirrhotic patients with and without decompensation (Fig. 1A). Nostrin levels were significantly elevated in compensated cirrhosis as compared to healthy controls (2.23 ± 0.09 Vs 5.85 ± 0.22; p < 0.0001). Moreover, plasma from decompensated cirrhotic patients showed further considerably elevated Nostrin levels when compared to compensated cirrhotic patients (11.13 ± 0.42 Vs 5.85 ± 0.22; p < 0.0001). Subgroup analyses showed that Nostrin levels were elevated extensively in a stepwise manner from Child-Pugh A over Child-Pugh B to Child-Pugh C (6.21 ± 0.28 Vs 9.93 ± 0.64 Vs 11.72 ± 0.52) (Fig. 1B). Table 3 shows the correlation of Nostrin with biochemical parameters and disease severity markers. We found plasma Nostrin levels was positively correlated with bilirubin, ALT, AST, ALP, GGT, urea, creatinine, PT, INR, child-pugh score, MELD, IL1β and IL-10 whilst negatively correlated with total protein, albumin and sodium. The prognostic value of Nostrin was also noted as it was substantially elevated with the MELD score > 10 compared to the MELD score < 10 (Fig. 1C). This is further confirmed by Pearson correlation analysis of Nostrin with MELD score (Fig. 1D; r = 0.4194; p < 0.0001). The prognostic potential of Nostrin was also evidenced by the correlation between Nostrin and MELD-Na score (Fig. 1E; r = 0.4082, p < 0.0001). A receiver operating characteristic (ROC) curve was plotted to predict the diagnostic efficiency of blood Nostrin concentration in both compensated and decompensated cirrhosis (Fig. 1F). The optimal cutoff value was ≥ 7. 388 ng/ml for Nostrin, exhibiting a sensitivity of 90% and specificity of 90%. AUC _Nostrin = 0.9371 with p < 0.0001; +ve likelihood ratio of 6.191 and a negative likelihood ratio of 0.084 to discriminate decompensated cirrhosis from compensated cirrhosis. Interestingly, the noted blood Nostrin level was higher (p < 0.01) in alcoholic cirrhotic patients compared to non-alcoholic cirrhosis (Fig. 1G). In addition, we found increased blood Nostrin concentration in patients with non-survivors when compared to survivors (6.072 ± 0.24 Vs 12.92 ± 0.75; p < 0.0001).

Table 3

Correlation of serum Nostrin with biochemical parameters, inflammatory markers and disease severity in cirrhotic patients.
Parameters	Pearson Correlation coefficient	p value
Creatinine	0.6702	< 0.0001
Urea	0.7421	< 0.0001
Total protein	-0.6685	< 0.0001
Albumin	-0.7212	< 0.0001
AST	0.7252	< 0.0001
ALT	0.4248	< 0.001
γGT	0.3739	< 0.01
ALP	0.5127	< 0.0001
Total bilirubin	0.7863	< 0.0001
Direct bilirubin	0.7863	< 0.0001
Sodium	0.6913	< 0.0001
Potassium	0.7227	< 0.0001
INR	-0.5022	< 0.0001
PT	-0.1980	0.1055
Child-Pugh score	0.3877	< 0.05
MELD-Na	0.6343	< 0.0001
	0.7077	< 0.0001
MELD score	0.4194	< 0.0001
	0.4082	< 0.0001

Measurement of blood cGMP levels in cirrhosis

As expected, cGMP (surrogate index of NO bioavailability) plasma levels were increased considerably in cirrhosis as compared to controls (p < 0.0001). When compared to compensated cirrhosis, plasma cGMP was significantly upregulated in decompensated cirrhosis (Fig. 2A). In addition, increased plasma cGMP concentration observed was positively correlated with elevated Nostrin in cirrhosis (Fig. 2B; r = 0.3675, p < 0.001).

Expression of Nostrin and eNOS in control and cirrhotic liver

Figure 3A shows histopathological changes and expression of Nostrin and eNOS in control and cirrhotic livers. The control liver specimen shows normal hepatic architecture, whereas the cirrhotic liver shows moderate inflammatory infiltrate in the portal zone and extensive bridging of fibrous tissue (A, i). Furthermore, IHC (A, ii) and immunofluorescence (A, iii) findings revealed increased hepatic expression of Nostrin in the cirrhotic liver as compared to the control liver. By contrast, the control liver showed high eNOS positivity but was absent in the cirrhotic liver (A, iv). The co-localization of Nostrin with eNOS was also identified in cirrhotic liver by double immunostaining (A,v). Moreover, Nostrin protein and mRNA expressions were significantly increased in the cirrhotic liver compared to the control liver (p < 0.05 for both). Subsequently, we analysed hepatic peNOS protein (D) and mRNA (E) expressions in the control and cirrhotic liver tissues. eNOS activity, as indicated by phosphorylation (peNOS-ser1177) was significantly (p < 0.05) decreased when compared to the normal liver, but eNOS mRNA was insignificantly increased in the cirrhotic liver compared to the control liver. Furthermore, decreased hepatic cGMP levels (surrogate index of NO bioavailability) were observed in the cirrhotic liver compared to the control liver (F, p < 0.01).

Assessment of systemic and hepatic inflammation in controls and cirrhosis

As systemic inflammation plays a vital role in predicting the pathophysiology of cirrhosis, we evaluated well-established pro-inflammatory cytokines such as plasma TNF α and IL1β (Figs. 4A&B), which were significantly increased in compensated cirrhosis compared to controls. Moreover, these levels were further upregulated in decompensated cirrhosis when compared to compensated cirrhosis alone (p < 0.0001). The anti-inflammatory cytokine IL-10 concentrations were also considerably higher in compensated cirrhosis compared to controls and further up-regulated in decompensated cirrhosis compared to compensated cirrhosis alone (Fig. 4C). Furthermore, hepatic iNOS and NFκB protein expression significantly increased in the cirrhotic liver compared to the control liver (Figs. 4D&E).

Understanding the pathophysiological mechanisms that regulate eNOS-driven NO generation in the hepatic vasculature plays a fundamental role in developing PHT in cirrhosis [5, 9]. Our recent in vivo study shows convincing evidence that hepatic Nostrin overexpression in cirrhotic mice results in reduced eNOS activity with concomitant NO generation and, thus, elevated portal pressure [10]. However, the role of Nostrin in regulating eNOS activity in human cirrhotic patients is limited. Furthermore, there is no data to relate the association of elevated Nostrin levels with disease severity and decreased eNOS function on the background of inflammation in cirrhotic patients with PHT. Notably, studies on biomarker-based mortality prediction in cirrhosis with PHT are also limited. This study identified that blood Nostrin concentration was higher in compensated cirrhotic patients and further elevated in decompensated cirrhosis. Moreover, blood Nostrin levels were shown to increase in parallel to the disease severity of cirrhotic patients. The prognostic value of Nostrin was noted as it was substantially elevated with MELD score > 10. Increased blood Nostrin levels were also correlated with MELD-Na, and CTP revealed its prognostic potential in the progression of cirrhosis. Notably, increased Nostrin concentration was observed in patients with non-survivors compared to survivors. This finding might represent a new indication for Nostrin as a novel prognostic biomarker in the progression of cirrhosis with PHT. Beyond this observation, we also established hepatic tissue Nostrin expression and its negative connotation with eNOS in the context of cirrhosis with PHT.

The increased IHVR and altered portal blood flow are the key determinants that synergistically ascertain and worsen the severity of PHT [8, 12]. In the cirrhotic liver, reduced NO synthesis was well correlated with a defect in hepatic eNOS activity, which contributes to high IHVR and PHT [7, 10, 12]. In alcoholic hepatitis patients, increased Nostrin has been shown to displace eNOS from the endothelial plasma membrane into the Golgi and other vesicular-like structures, thereby inhibiting its activity [12]. In support of this notion, the data presented herein provides insight into the pivotal role of Nostrin in altering eNOS activity and concomitant decreases in local NO generation. We found substantially elevated Nostrin mRNA and protein expression in the cirrhotic liver compared to the control liver. These observations were further confirmed with IHC and immunofluorescence (IF) findings. Furthermore, as expected, decreased phosphorylated eNOS protein expression and hepatic cGMP levels were observed in the cirrhotic liver. These findings might implicate posttranslational regulatory mechanisms resulting in defective NO production in cirrhosis. In this context, our very recent study shows evidence that Nostrin gene knock-down improved eNOS activity with concomitant elevation of NO synthesis in human endothelial cells [10]. Our results corroborated with an earlier observation that Nostrin colocalizes extensively with eNOS in the plasma membrane of HUVEC cells [19]. In addition, Mookerjee et al. demonstrated an association of Nostrin with the hepatic vascular endothelium of human cirrhotic liver [12]. The NO downstream signaling target soluble guanylate cyclase (sGC) causes vasodilation by synthesising cGMP from GTP [20]. Our results demonstrate that reduced cGMP concentration (a surrogate marker of NO generation) in the cirrhotic liver due to reduced eNOS activity may allow for augmented IHVR and PHT (Fig. 5). In support of this concept, a previous report shows in alcoholic hepatitis patients with PHT, that reduced eNOS activity and increased hepatic Nostrin expression and higher portal pressure [12].

Hepatic inflammation contributes as a dynamic component to PHT, driving both the progression and regression of cirrhosis [21]. In this context, inflammatory markers were shown to be positively correlated with hepatic venous pressure gradient (HVPG) and negatively associated with eNOS activity [8], corroborating that there is a positive association with PHT progression. Hepatic Nostrin overexpression was also noted in acute on chronic liver failure (ACLF) and alcoholic hepatitis (AH) patients compared to decompensated cirrhotic patients alone [12]. In the placenta of women with pre-eclampsia, elevated Nostrin expression was found to be associated with diminished eNOS activity [22], a condition also exemplified by heightened inflammation [18]. Our results build on similar contributions on this line of observation in the literature. In addition, this study highlights augmented hepatic inflammation as evidenced by increased inducible NOS (iNOS) and NFκB expression in the cirrhotic liver, where elevated Nostrin expression and diminished eNOS activity were observed. Furthermore, systemic inflammatory markers such as serum CRP and IL-6 are elevated in advanced cirrhosis and correlate with PHT and mortality [23, 24]. In this context, recombinant HDL treatment has been shown to neutralise LPS-induced systemic inflammation, improving eNOS-driven NO synthesis and abrogating PHT [25]. Here, we were able to show increased systemic TNF-α, IL-6 and IL-10 in cirrhotic patients compared to controls. In this regard, Mookerjee et al. emphasize direct inhibition of TNF- α with infliximab in alcoholic hepatitis (AH) patients results in a sustained reduction in portal pressure [26]. Consequently, both hepatic and systemic inflammatory responses induce Nostrin overexpression and reduce hepatic eNOS activity in cirrhosis.

In conclusion, our study shows evidence that inflammation drives Nostrin overexpression in human cirrhotic liver, decreasing eNOS activity and associated local NO generation, thereby increasing portal pressure in cirrhosis. In addition, our data suggest that elevated blood Nostrin could be a key diagnostic and prognostic biomarker in cirrhosis with PHT. Indeed, further prospective studies are warranted to delineate the prognostic role of Nostrin in advanced cirrhosis.

This is a single-centre study. Patient follow-up and survival analysis were not executed.

AH, alcoholic hepatitis; cGMP, cyclic guanosine monophosphate; IHC, immunohistochemistry; iNOS, inducible nitric oxide synthase; IL-1β interleukin-1β; IHVR, intrahepatic vascular resistance; peNOS, phosphorylated endothelial nitric oxide synthase; PHT, portal hypertension; NO, nitric oxide; NOSTRIN nitric oxide synthase trafficking inducer; NFkB, nuclear factor-kB; TNF, tumor necrosis factor.

Funding information

BV was awarded the Ramalingaswami re-entry fellowship grant (BT/RLF/Re-entry/25/2012) from the Department of Biotechnology, Government of India.

Acknowledgements

We appreciate Dr Palanivel C, Department of PSM, JIPMER, for assisting with sample size calculation and Dr Sundhar M for his help in technical assistance in IHC and IF staining. We thank all the patients and healthy volunteers for participating in this study.

Conflict of interest

The authors declare that they have no conflict of interest.

Institutional Review Board statement

The study was reviewed and approved by the JIPMER Scientific Advisory Committee (JSAC) and Institute Ethics Committee (IEC) for human studies (Human Studies #JIP/IEC/2016/30/971). Written informed consent was obtained from all the study participants during recruitment.

Author contributions

Balasubramaniyan V: Conceptualization, Methodology, Investigation, Resources, Writing – original draft, Review & editing.

Ravikumar TS: Resources, Supervising, Review & editing.

Amit KR: Methodology, Investigation.

Pazhanivel M: Conducted the study of clinical samples - Review & editing.

Pottakkat B: Conducted the study of clinical samples - Review & editing.

Data availability statement

All data used were included in this article.

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NOSTRIN is an emerging negative regulator of decompensated cirrhotic patients with portal hypertension

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Abstract

Figures

Highlights

Lay Summary

Introduction

Material and methods

Human Cirrhosis

ELISA

Histopathological and immunohistochemistry (IHC) analysis

Immunofluorescence

PCR

Western blotting

Statistics

Results

Demographic and clinical features of the study population

Biochemical parameters of the study population

Diagnostic and prognostic potential of Nostrin concentrations in cirrhosis

Measurement of blood cGMP levels in cirrhosis

Expression of Nostrin and eNOS in control and cirrhotic liver

Assessment of systemic and hepatic inflammation in controls and cirrhosis

Discussion

Conclusion

Limitation

Abbreviations

Declarations

References

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