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Gut Lymphangiopathy: Adding Fuel to the Fire in Chronic Liver Disease

The splanchnic inflammation inchronic liver disease increases intestinal angiogenesis. In the current study our aim was demonstrating that the small bowel lymphangiogenesis is associated with angiogenesis in chronic cholestasis in the rat. A stereological study of the lymphatic microcirculation in the small intestine was performed in cholestatic rats. Portal enteropathy in cholestasis increases lymphatic microvessels in the mucosa and submucosa layers. Thus, the lymphatic microvessel volume fraction was superior (p < 0.001) in the mucosa (0.16 ± 0.01) and submucosa (0.16 ± 0.01), in regard to the muscle layer 0.015 ± 0.01. The lymphatic microvessel length density was higher in the mucosa (76.89 ± 2.86 mm −2 ; p < 0.001) and submucosa (14.87 ± 2.86 mm −2 ; p < 0.01), in relationship to the muscle layer (5.04 ± 2.92 mm −2 ). These alterations predominate in the duodenum (volume fraction: 0.10 ± 0.01 and length density: 33.55 ± 5.98 mm −2 ) and ileum (volume fraction: 0.16 ± 0.01 and length density: 38.62 ± 6.07 mm −2 ). This study demonstrates the predominance of an increased lymphangiogenic response in both end sides of the small bowel associated with chronic liver disease. Since the porto-systemic venous collateral circulation in the chronic liver insufficiency is developed in the ends of the gastrointestinal tract, the excessive duodeno-ileal lymphangiogenesis could suggest the development of amesenteric-systemic lymphatic bypass in the chronic portal hypertension.


Introduction
The basic structural impairment found in the gastrointestinal tract in portal Advances in Bioscience and Biotechnology hypertension is vascular and consists of a greater number and larger blood vessels [1] [2]. Consequently, the name proposed for this kind of enteropathy is "hypertensive portal intestinal vasculopathy" [3].
In addition, sustained portal hyperpressure increases mesenteric lymph flow and produces impairment of lymphatic drainage, which is considered an important etiopathogenic mechanism of ascites [4] [5]. In turn, chronic functional failure of the gastrointestinal lymphatic vascular system in portal hypertension induces a compensatory lymphangiogenic response [5] [6]. At present, the characteristics of intestinal lymphatic vascular alterations typical of portal hypertension are unknown, especially when associated with chronic progressive liver failure [6]. Consequently, one of the objectives of this study is to demonstrate the distribution of intestinal lymphatic vascular changes in an experimental model of chronic portal hypertension secondary to biliary fibrosis.
Since splanchnic alterations associated with chronic liver failure are considered inflammatory in nature [7] [8], it could be proposed that, similar to the mesenteric venous vasculopathy, a hypertensive intestinal lymphangiopathy is developed associated with biliary fibrosis in rats, which would also have a similar pathogenesis.

Animal Management
Male Wistar rats, with an average weight between 218 and 334 g, corresponding to an age from 8 to 10 weeks from the Animal Facility of the experimental surgery unit of La Paz University Hospital were used.
The animals were fed a standard laboratory rodent diet (rat/mouse A 04 maintenance diet; Panlab. Barcelona. Spain) and water ad libitum. They were housed in a light/dark-controlled room (light cycles: 7:30 a.m. -7:30 p.m.) with an average temperature (22˚C ± 2˚C) and humidity (65% -70%), in groups of three to four animals. The experimental procedures employed in this study were based on the principles and practices of the 1986 European Guide for the Care and Use of Laboratory Animals, in accordance with the Ethical Guidelines from European Community Council Directive (86/609/EEC) and published in Spanish Royal Decree 1201/2005. All procedures were approved by the La Paz Hospital Animal Welfare Committee.

Experimental Design
Forty-one male rats were randomized and divided into two groups: Sham-operated (SO; n = 11), microsurgical cholestasis (MC; n = 30). In the SO-group, all the animals survived and in the MC-group, five rats died by gastrointestinal hemorrhage related to hepatic failure in the seventh week of postoperative evolution. Therefore, thirty-six of the rats from the beginning of the study survived until the time of sacrifice: Sham-operated (SO; n = 11) and microsurgical cholestasis (MC; n = 25).
In the SO rats, the bile duct and its lobular branches were dissected without resection. In the MC rats, an extrahepatic biliary tract resection was performed using a binocular operatory microscope (OPMI 1-FR; Zeiss, Madrid, Spain), as previously described [9] [10]. Briefly, the common bile duct was ligated (silk 4/0) and sectioned and the lobular biliary branches are ligated and then sectioned.
All the rats were sacrificed at 8 th week of intervention. The following aspects were studied:

Ascites Volume Determination
The extraction of ascitic fluid was performed in all the cholestatic rats by aspiration using a 10 ml syringe.

Portosystemic Collateral Circulation
First, a middle abdominal incision was performed. Next, the areas where the collateral venous circulation develops, that is, splenorenal, gastroesophageal, and colorectal, were studied for the presence of collateral veins [7].

Serum Biochemical Tests
In the blood obtained by puncture of the infrahepatic inferior vena cava, bilirubin, alkaline phosphatase, Aspartate-aminotransferase (AST), Alanine-aminotransferase (ALT) and Albumin serum concentrations were measured with commercially available kits (Sigma), using a Hitachi U-2000 spectrophotometer (Hitachi Nissei Sangyo Co. Ltd., Japan).

Immunolabelling of the Intestinal Lymphatic Vessels with LYVE-1
Lymphatic endothelial cells share many markers in common with vascular endothelial cells [11]. Cell surface markers specific of lymphatic endothelium have been developed. Lymphatic endothelial cells have been isolated by positive selection using antibodies to podoplanin, vascular endothelial growth factor receptor (VEGFR)-3 or lymphatic vessel hyaluronan receptor (LYVE)-1, and by a negative selection with antibodies to CD34 [12]. Furthermore, the transcriptional factor prospero-related homeodomain transcription factor (Prox)-1 is essential for establishing lymphatic endothelial cell identity; its expression is controlled by Advances in Bioscience and Biotechnology Sox 18 transcription factor [13] [14]. Lymphatic endothelial cells express specific molecules such as Prox-1, VEGFR-3, the membrane glycoprotein podoplanin and LYVE-1 [15]; the latter was used as a cellular lymphatic marker in the present stereological study ( Figure 1).

Quantitative Methods for Studying the Intestinal Lymphatic Vessels
The following parameters were assessed in all groups of animals: 1) Volume fraction occupied by lymphatic vessels (V v vasc).
points on vessels vasc points on reference spa ce 2) Length density of lymphatic vessels (L v ). Dissector frame was used to sample immunostained vascular profiles eligible to be counted by the Sterio rule [18]. The length density is calculated using the formula: where Q− = number of eligible immunopositive vascular profiles and A ∑ = stromal area sampled, i.e., area of dissector frame, multiplied by the number of selected frames.
L v = was expressed in mm −2 (mm length of vessel/mm 3 reference volume).
3) Average vascular diameter (AVD) calculated according to the formula and expressed in µm [18]:

Statistical Analysis
The data was analyzed using a statistical package for social sciences (SPSS, ver-

Body and Organ Weights
Liver and spleen weight in the animals with MC increased (p < 0.001) with respect to the SO animals. On the contrary, the testicular weight of rats with MC decreased (p < 0.05) with respect to the SO rats (Table 1).

Ascites Volume
The mean volume of ascitic fluid in the group with MC was 10.5 ± 8.6 ml.

Liver Function
The animals with MC had an increase (p < 0.001) of total bilirubin, alkaline phosphatase, AST and ALT serum levels with respect to the SO-rats (Table 2).
Albumin serum level was lower (p < 0.001) in MC-rats compared to SO-rats (Table 2).

Stereological Study of the Lymphatic Microcirculation in Microsurgical Obstructive Cholestasis
The and Figure 3(b)). Thus, these parameters increased in the duodenum (V v vasc:    ; the results are expressed as mean ± SD. **p < 0.01; ***p < 0.001: statistically significant value in regards to SO rats; p < 0.05; P < 0.001: statistically significant value in regards to MC rats. 6.26 ± 6.07 mm −2 ; p < 0.001), of the cholestatic rats in relationship to SO-rats, although these increases are more significant in duodenum and ileum ( Figure   1(b) and Figure 1(e); Figure 2(b) and Figure 3(b)).
Finally, the average vascular diameter (AVD) was similar either in all the layers as in each anatomical area (duodenum, jejunum and ileum) of the small bowel in the SO-rats and in the cholestatic rats.

Discussion
This experimental study used stereological methods in combination with a cellular surface marker for lymphatic endothelial cells to estimate such parameters as volume fraction, length density, and average vascular diameter of lymphatic vessels [19] [20] in the small intestine of microsurgical cholestatic rats.
Lymphatic endothelial cells share many markers [11], which have been iso- The outstanding role of lymphatic vessels in the etiopathogeny of inflammation has been explained in several excellent reviews [29] [30] [31]. The growth of lymphatic vessels or lymphangiogenesis is actively involved in multiple pathological conditions, including tissue and organ inflammation [32]. In particular, lymphangiogenesis could be advantageous in decreasing inflammation related to lymphedema [24].
The stereological study shown in this paper suggests that the alterations found in the mucosal and submucosal lymphatic microcirculation of cholestatic rats could be attributed to an activation of lymphangiogenesis in response to the intestinal inflammation, mainly in the mucosa. However, further studies would be needed to prove this hypothesis. It has been demonstrated that two members of the VEGF family, VEGF-C and VEGF-D, produce lymphangiogenesis by activation of VEGFR-3, which is expressed by lymphatic endothelial cells in normal adult tissues [33] [34].
The increased volume fraction and length density of lymphatic vessels in the duodenum and ileum, with respect to the jejunum, could correspond to the predominance of the inflammatory response that occurs in portal hypertension in both ends of the small bowel. This characteristic location of the intestinal lymphangiogenesis would be similar to the special intestinal location of the angiogenic response that is developed in portal hypertension. Thus, the excessive angiogenesis in the proximal and distal ends of the intra-abdominal intestinal Advances in Bioscience and Biotechnology tract, i.e. esophagus and rectum, respectively, induces the development of porto-systemic collateral circulation. This is the reason why it seems appropriate to consider that the excessive lymphangiogenesis in the proximal and distal ends of the small bowel could also represent the development of a mesenteric-systemic lymphatic collateral circulation (Figure 4). The existence of a porto-systemic venous collateral circulation allows noxious substances of intestinal origin, like damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), to reach the systemic circulation [28] [29]. Since the presence of these noxious substances in the systemic circulation severely aggravates the prognosis of the patients with chronic liver disease, the existence of a mesenteric-systemic collateral circulation would therefore represent a new pathophysiological factor in these patients ( Figure 5).
In portal hypertension, the excessive angiogenic response and the lymphangiogenic response could be related, among other factors, with the increased mast cells infiltration in the small bowel and mesenteric lymphatic nodes [35] [36].
The human mast cells are a major source of both angiogenic and lymphangiogenic factors. Moreover VEGF-C and VEGD-D produced at sites of inflammation and tumors can amplify mast cells infiltration by interacting with VEGFRs.
In addition, mast cell-derived proteases, such as tryptase, MMP2 and MMP9 chymases, can modulate angiogenesis and lymphangiogenesis [35] [37]. Cells of the immune system and their mediators need consideration for future therapeutic manipulation of angiogenesis/lymphangiogenesis in chronic inflammatory disorders and tumors [34].
Portal hypertension related to biliary fibrosis in rats causes extensive mast cell infiltration of the mesenteric lymph complex (MLC) [36]. This massive recruitment   [38]. In this way, the hypothesis that the mesenteric-lymphatic collateral circulation is developed through the incidence of two consecutive hits in portal hypertensive rats could be proposed. The first hit would be related to the inflammation produced by portal hypertension, with edema and lymphatic flow increase, while the second hit would be the intestinal lymphatic hypertension, which in turn favor lymphangiogenesis and mesenteric adenitis [38]. Hence, either intestinal angiogenesis or lymphangiogenesis in portal hypertension would have a complementary etiopathogenic role [39].
Lastly, this intestinal lymphatic pathology associated with portal hypertension Advances in Bioscience and Biotechnology could have an etiopathogenic role in ascites production. Thus, the coupling between portal venous hypertension and mesenteric lymphatic hypertension would induce an intestinal inflammatory response, with being a harmful effect. Maybe this intestinal lymph hyperproduction overwhelms the drainage ability of the neoformed lymphatic collateral circulation in the cholestatic rats. If so, the overloading of lymphatic volume in the interstitium-lymphatic-peritoneum axis would finally cause ascites [6] [39] [40].

Conclusion
In conclusion, this study demonstrated the development of a lymphatic vascular enteropathy in rats with chronic hepatic disease related to cholestasis. The excessive lymphangiogenesis in the duodenum and ileum could represent the pre-