Endothelin as modulator of acute phase shear

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The most dramatic complication of liver surgery is postoperative liver failure (PLF), a clinical syndrome characterized by liver dysfunction with prolonged cholestasis and coagulopathy, portal hypertension, and if severe with ascites. Endothelin-1 (ET-1) plays a significant role in the regulation of hepatic microcirculation and is involved in the pathogenesis of liver fibrosis and cirrosis via sinusoidal constriction. ET-1 is produced not only in the ischemia-reperfused liver tissue but also in the splanchnic area subjected to portal congestion during hepatic inflow occlusion, taking part into the pathogenesis of small for size liver injury. Nowadays the only way to predict postoperative liver failure is analysis of biochemical parametres (total bilirubin and PT on POD5).

Aim

The present study was undertaken to measure ET-1 levels in peripheral blood of patients undergoing hepatic surgery and to investigate their correlation with pre-, intra-, and postoperative variables; it was also aimed to clarify ET-1 role in liver hemodynamics.

Patients and methods

From January 2009 to May 2009, 65 patients undergoing liver resection at Department of surgery - Liver Unit at Scientific Institute H. San Raffaele were entered in the study. Preoperatively, in POD1, POD2 and POD% peripheral blood samples were collected to determine ET-1 serum levels. Clinical and biochemical parameters of all the patients were prospectively collected.

Results

ET-1 levels were not significantly correlated to parenchymal liver disease and extent of liver resection. ET-1 levels were higher in patients who developed PLF, even thou this finding was not statistically significant. ET-1 peripheral levels in POD2 were strongly associated with portal ET-1 level at the end of resection and with bilirubin and thrombin time of POD5 (p<0,01).

Discussion

Since ET-1 levels change according to bilirubin and PT values they could be used as an earlier marker of PLF. An insufficient residual liver volume can't be considered the only determinant implied in ALF: this was confirmed comparing ET-1 levels patients grouped according to liver resection extent. Increased ET-1 may promote sinusoidal constriction, ET-1 receptors upregulation and further ET-1 production: then, ET-1 production is achieved by liver itself and leads to PLF.

INTRODUCTION

Postoperative liver failure (PLF) remains the most dramatic complication of liver surgery. It is a recognizable clinical syndrome, which occurs when liver mass is insufficient to maintain normal function. The syndrome is characterized by liver dysfunction with prolonged cholestasis and coagulopathy, portal hypertension, and if severe with ascites.

Although PLF is potentially reversible, survival and complete restitution of the liver are often not achieved. Recent studies suggest that hepatocellular damage in PLF is not only due to the insufficient residual liver volume but is aggravated by the drastic reduction of the microvascular bed upon major hepatectomy, exposing the remnant liver to excessive portal perfusion, known to induce irreversible sinusoidal endothelial injury as described in small-for-size liver grafts. The excessive Hyperperfusion, defined as acute phase shear stress, is known to induce the release of proinflammatory and cytotoxic mediators from activated, non-parenchymal sinusoidal-lining cells, such as Kupffer cells (KC), hepatic stellate cells (HSC), and sinusoidal endothelial cells . It has been suggested that the release of these mediators may additionally impair the hepatic microcirculation at the sinusoidal level, leading to local ischemia and increased leukocyte endothelial interaction.

The Endothelins, discovered in 1988, consist of a family of 3 unique 21 amino acid peptides termed ET-1, ET-2, and ET-3.

The importance of endothelins in liver disease has been emphasized by reports of elevated circulating ET-1/ET-3 levels in patients with cirrhosis. Importantly, the source of endothelin in cirrhotic patients is the injured liver itself. In the normal liver, ET-1 is produced primarily by sinusoidal endothelial cells (as for endothelial cells in the peripheral vasculature); after injury, however, endothelin is derived largely from stellate cells, and moreover, the synthesis of ET-1 by sinusoidal endothelial cells is reduced.

During liver injury, a remarkable paradigm is currently evolving: the proposed model revolves around the concept that a shift in the balance of vasoactive substances occurs after liver injury and alters intrahepatic resistance. However, in the injured liver, an imbalance occurs; in the model highlighted, ET-1 synthesis is increased and NO production decreased, leading to an "endotheliopathy" within the liver.

With this background, the present study was undertaken to measure ET-1 concentrations in the peripheral blood of patients undergoing hepatic resection and to investigate their correlation with preoperative, intraoperative, and postoperative variables.

Finally it analyzes Endothelin prognostic value in postoperative liver dysfunction assessment.

PATIENTS

From January 2009 to May 2009, 65 patients undergoing liver resection at Department of surgery - Liver Unit at Scientific Institute H. San Raffaele were entered in the study.

Patients who met one or more of the following criteria were excluded from the study: preoperative biliary drainage or stenting; previous vascular procedures like transarterial chemoembolization (TACE) and radiofrequency; laparoscopic resections; portal or hepatic vein thrombosis; Child-Pugh class B or C; patients under 18 years of age.

Patient profiles are summarized in Table 1.

Underlying liver disease states were confirmed with histopathological examinations; 7 patients (10,76%) had liver cirrhosis, 8 (12,3%) had chronic hepatitis and the remaining 50 (76,92%) patients had no apparent pathological parenchyma.

The cases included both primary and metastatic liver neoplasms and benign pathologies too, as shown in Table 2.

We performed 22 major (33,8%), 41 minor (63,1%) and 2 extended (3,1%) resections (according to Brisbane classification), as reported in Table 3.

METHODS

Clinical and Laboratory Variables

Markers of hepatocyte damage and recovery, including alanine aminotransferase (ALT) aspartate aminotransferase (AST), total bilirubin, alkaline phosphatase (ALP), and gamma glutamyl transpeptidase (GGT), were measured using a serum multiple biochemical analyzer. Plasmatic samples were collected preoperatively and on POD 1, 2, and 5. Antithrombin III (AT-III), prothrombin time-international normalized ratio, fibrinogen, D dimer, and platelets were measured the day before operation and on POD 1, 2, and 5. Data regarding preoperative variables, duration of surgery, intraoperative blood loss and transfusion, intraoperative and postoperative complication, and hospital stay were prospectively collected.

Endothelin-1 levels

Sisthemic levels of Endothelin-1 (ET-1) were assessed the day before operation and on POD 1, 2 and 5.

Blood levels were quantified using a sandwich enzyme immunoessay. The lower detection threshold of this assay was 2.30 pg. Assays were run in duplicate, and values were expressed in picograms per milliliter.

Statistics

The data were expressed as the means ± the standard error. Statistical analysis between parametric variables was performed using the Student t test. Statistical correlation and P values were determined by Pearson's correlation coefficient using SPSS software. A P value less than 0.05 was considered significant.

RESULTS

ET-1 time course

Peripheral ET-1 concentration was 18,27 ± 9,7 pg/mL before surgery and 17,28 ± 7,43 pg/mL in the first postoperative day (POD1). This difference was not significant. It kept increasing, reaching a peak of 22,14 ± 9,84 pg/mL in POD2. In POD5 ET-1 was 20,62 ± 10,69 pg/mL (Graph 1). None of these values was significantly different.

ET-1 and cirrhosis

ET-1 levels in liver cirrhosis were higher in POD2 and POD5 (respectively 25,12 ± 6,89 pg/mL and 22,88 ± 4,31pg/mL) compared to normal liver (21,82 ± 10,61 pg/mL in POD2 and 21,00 ± 11,66 pg/mL in POD5) and to chronic hepatopathy (22,10 ± 8,18 pg/mL in POD2 and 17,11 ± 7,30 pg/mL in POD5), but the difference between groups was not statistically significant (Graph 2).

ET-1 and liver resection extent

In Minor resection Group ET-1 level was 20,38 ± 10,85 pg/mL in POD2 and 20,26 ± 9,54 pg/mL in POD5. In Major resection Group was 29,97 ± 7,67 pg/mL in POD2 and 21,69 ± 13,61 pg/mL in POD5. In Extended resection Group ET-1 level reached 26,91 ± 2,5 pg/mL in POD2 and 19,59 ± 14,89 pg/mL in POD5. Despite extended resections had higher ET-1 levels in POD2, there was no statistically significant difference between groups (Graph 3).

ET-1 and postoperative complications

Fourty sevent patients (72,3%) had a postoperative stay without complications, while 18 (27,7%) developed one of them. Among these patients, Grade I complications (according to Dindo-Clavien classification) were 3 (4,6%), Grade II were 9 (13,8%), Grade III were 4 (6,2%) and Grade IV were 2 (3,1). There were no Grade V complications.

ET-1 time course was not significantly different in these groups of patients.

Four patients developed postoperative liver failure: ET-1 time course of these patients was compared with those without failure. The higher difference was evident in POD2 (21,04 ± 9,61 pg/mL in No Failure Group and 35,98 ± 7,67 pg/mL in Failure Group) and in POD5 (19,82 ± 9,75 pg/mL in No Failure Group and 37,0 ± 12,4 pg/mL in Failure Group). Indipendent sample T test demonstrated no significant difference between groups ( p=0,703 in POD2 and p=0,544 in POD5), as reported in Graph 4. Graph 5 gives a representation of distribution of ET-1 values in patients who developed liver failure compared to those who didn't developed it. It appears clearly how difference in mean ET-1 level is higher between clusters than among groups of the same cluster .

Bivariate correlation

Analysis of bivariate correlation using Pearson Coefficient was done between ET-1 levels and factors listed below.

Blood parametres

Inflammatory response

Coagulation

Clinical variables

 

 

Hb, Hct

TNF-a

PT, aPTT

Intraoperative blood loss

Crea

IL-6

ATIII

Lenght of surgery

Bilirubin

XDP

Postoperative stay

AST, ALT

Fibrinogen

 

ALP, gGT

 

 

 

Each variable was measured pre-operatively and in POD1, POD2 and POD5.

Pearson correlation demonstrated an association between ET-1 peripheral levels in POD2 and ATIII, XDP and IL-6 of POD2 (p<0,05). Finally they correlate with bilirubin and thrombin time of POD5 (Graph 6) with a p<0,05.

DISCUSSION

Although PLF is potentially reversible, survival and complete restitution of the liver are often not achieved especially in diseased patients. Despite considerable progress in research, the pathophysiology of the damage seen in PLF following liver resection is not entirely understood. Though the liver is unique with its ability to regenerate and to revert to its original function after resection, there is a limitation of the extent of liver resection.

Sato et al proposed that shear stress due to portal flow, which appears to be a simple mechanical force, may trigger liver regeneration and controls the volume of the liver after partial hepatectomy. On the other hand, portal hyperperfusion has been considered responsible for organ damage frequently observed after reperfusion of small-for-size liver grafts, resulting in hepatocyte ballooning with tremendous mitochondrial swelling, irregular large gaps between sinusoidal lining cells, and collapse of the space of Disse.

After hepatectomy the entire stream of portal blood is directed through the small remnant, leading to a relative directed through the small remnant, leading to a relative increase in portal flow per unit of liver tissue. Portal flow exceeding the capacity of the small remnant to accomodate it could produce flow injury to sinusoidal endothelial cells and activate KC to release inflammatory cytokines.

During liver injury, a remarkable paradigm is currently evolving: the proposed model revolves around the concept that a shift in the balance of vasoactive substances occurs after liver injury and alters intrahepatic resistance. For example, in normal liver, vasoconstrictors (i.e., ET-1) and vasodilators (i.e., NO) are "balanced." However, in the injured liver, an imbalance occurs; in the model highlighted, ET-1 synthesis is increased and NO production decreased, leading to an "endotheliopathy" within the liver. If liver results functionally and structurally impaired because of this damage, PLF develops.

Although this particular condition is of increasing frequency, a standardized definition of PLF is still lacking. None of the proposed definitions of liver failure established outside the context of surgery can be easily extrapolated to the early period following liver resection. Actually the most common and used criteria to predict PLF is the "50-50 Criteria" on POD5, proposed by Belghiti. The values of PT and serum bilirubin indicating a significant impairment of liver function were chosen: the value of 50% of normal for PT corresponds to an INR of 1.7, and a serum bilirubin of 50 mol/L corresponds to a value of 3 mg/dL. The conjunction of these two values on POD 5, the 50-50 criteria, could predict nearly 100% morbidity rate and 50% mortality rate.

In this study, Pearson Correlation has shown that ET-1 peripheral levels in POD2 are strongly associated with bilirubin and PT of POD5 (p<0,01).

It can then be assumed that ET-1 levels change according to bilirubin and PT values and that, since ET-1 levels increase before bilirubin and PT, they can be used as an earlier marker of PLF.

Anyway when ET-1 time course of patients with clinically evident ALF was compared with those without failure, the higher ET-1 liver difference was evident in POD2 (21,04 ± 9,61 pg/mL in No Failure Group and 35,98 ± 7,67 pg/mL in Failure Group) and in POD5 (19,82 ± 9,75 pg/mL in No Failure Group and 37,0 ± 12,4 pg/mL in Failure Group). Despite this statistical analysis demonstrated no significant difference between groups ( p=0,703 in POD2 and p=0,544 in POD5), suggesting that maybe other factors are implied in ET-1 dynamics.

An insufficient residual liver volume can't be considered the only determinant implied in ALF. To verify this hypothesis ET-1 time course after liver surgery was compared in groups of patients according to liver resection extent: despite extended resections seemed to have higher ET-1 levels in POD2, there was no statistically significant difference between groups.

This finding suggest that liver resection extent is not the only determinant of ET-1 levels after surgery: even if future remnant liver is still an important predictor of failure and any effort has to be made to strengthen compensatory hypertrophy techniques, PLF risk, according to "50-50 criteria", can't be defined only calculating the quantity of spared parenchyma.

It seems possible that the microcirculatory disorders related to ET-1 imbalance may contribute to PLF pathogenesis. ET-1 production by splanchnic areas could be induced by hyperperfusion and consequently by acute phase shear stress: this causes sinusoidal vasoconstriction and ET-1 secretion by liver itself.

Concluding, excessive ET-1 levels may cause functional and structural liver damage, leading to postoperative liver failure. Further studies should achieve the definition of a cut off value to distinguish patients with higher PLF risk.

Efforts shoul be done to study if ET-1 receptors blockade could really attenuate microcirculatory disorders and their morphologic sequelae by correcting the ET-1 balance and to display its potential clinical application for the prevention of SFS liver injury in recipients after reduced size liver transplantation, as well as in patients undergoing liver resections.

Table 1: Clinicopathological profile of patients

Sex Ratio (M/F)

 

37/28

Age (years)

 

64,57±11,8

Comorbidities

 

 

 

None

11

 

Hypertension

16

Arrhytmia

2

 

CHD

2

 

Previous CT

22

 

Kidney disease

1

 

Others

9

Associated parenchymal disease

 

 

 

Normal liver

50

 

Chronic hepatitis

8

 

Liver cirrhosis

7

Table 2: Diagnosis

Frequency

Percent

Colorectal metastases

39

60

Non colorectal metastases

2

3,1

Hepatocarcinoma

8

12,3

Cholangiocarcinoma

6

9,2

Gallbladder carcinoma

4

6,2

Hepatolithiasis

2

3,1

Hydatid cyst

2

3,1

Angioma

2

3,1

Table 3: Kind and extent of liver resection

Kind of resection

Frequency

Percent

 

Right hepatectomy

9

13,8

 

Left hepatectomy

7

3,1

 

Mesohepatectomy

2

3,1

 

Right lateral settoriectomy

4

6,2

 

Left lobectomy

5

7,7

 

Bisegmentectomy

8

12,3

 

Segmentectomy

20

30,8

 

Nodulectomy

5

7,7

 

Cystopericystectomy

1

1,5

 

Multiple nodulectomies>5

1

1,5

 

Extended right hepatectomy

1

1,5

 

Extended left hepatectomy

2

3,1

 

 

 

 

Resection extent

Frequency

Percent

 

Major

22

33,8

 

Minor

41

63,1

 

Extended

2

3,1

Graph 1: ET-1 time course

Graph 2: ET-1 time course according to associated parenchymal disease

Graph 3: ET-1 time course according to liver resection extent

Graph 4: ET-1 time course and PLF

Graph 5: ET-1 levels in patients with or without liver failure (represented in clusters). Patients with no failure are divided according to presence or absence of complications (represented in Groups).

Graph 6: Correlation of Pod2 ET-1 with Pod5 PT and bilirubin

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