Role of complement factors, ADAMTS13 and Von Willebrand factor in pathogenesis of non cirrhotic intrahepatic portal hypertension (NCIPH) in humans
Hypothesis: Cytokine induced up regulation of Von Willebrand factor (VWF) on microvascular endothelial cells, coupled with shear stress, altered complement activation. Deficiency of ADAMTS13 protease facilitates formation of microvascular thrombi within the liver resulting in portal hypertension.
- Aim 1: Examine circulating levels of inflammatory cytokines and complement factors in patients with NCIPH
- Aim 2: Evaluate induction of VWF on microvascular endothelial cells in response to levels of inflammatory cytokines seen in NCIPH patient circulation under static conditions in comparison to shear stress.
- Aim 3: Examine interaction of complement factors with endothelial expressed VWF in a milieu of ADAMTS13 deficiency under static and shear conditions.
Idiopathic non- cirrotic interahepatic portal hypertension (NCIPH) is caused due to thrombosis in venules of hepatic circulatory system by unknown mechanism. Altered complement activity can induce Inflammatory response that can cause thrombosis. There is a need for proper investigation on these cases for proper diagnosis and treatment. So the inflammatory response components and other inter leukins that provoke thrombosis can be measured and validated.
Secondly, irrespective of cytokine measurements the activated complements in patient samples will be assessed by invitro study using liver microvascular endothelial cells. The endothelial cells will be activated for anchoring ULVWF protein on cell surface by treating it with patient plasma/ serum platelet adhesion can be tested.
Finally the liver endothelial alteration by these inflammatory components will be assessed by comparing liver endothelial with endothelial cells of different origin. It is suspected that P selectin activity will be a key controller in anchoring ULVWF in liver endothelial cells than others.
By assessing inflammatory cytokines and endothelial cell alteration we can understand the complementary role in activation of thrombosis.
NCIPH is a liver disorder of vascular origin is defined by a portal venous pressure of exceeding 5mm Hg between portal vein and inferior vena cava (sanyal 2008). Characterized by occlusion in 3rd /4th order branches of hepatic circulatory system ( madhu 2008)
In our study clinical studies, between 30-40% of patients with cryptogenic chronic liver disease, liver biopsy confirms diagnosis as NCIPH(Madhu, Avinash 2009, Goel, madhu 2013).
The pathogenesis of NCIPH is still challenging, in this proposal it is hypothesized that activation of vWF factors and down regulation of ADAMTS13 may be provoked by inflammatory cytokines by the inflammatory responses. Inflammatory cytokines during acute inflammation or infection will inhibit ADAMTS13 biosynthesis and results in thrombotic microangiopathy in the hepatic venules that leads to portal hypertension.
As a background, in our patients, there was a imbalance in vWF and ADAMTS13 but the reason driving this disparity is not clear. It is hypothesized that cytokines were involved as due to the change in complement pathway. Cytokines will inhibit the ADAMTS13 level( Goel, Ramakrishna 2011) and low ADAMTS13 can stimulate the alternative complement pathway (Turner 2013) in pathogenesis of thrombotic microangiopathy activation of alternative complement pathway stated as the main contributory for the disease (George 2014). In a study 1/3rd of NCIPH patients who underwent spleno-renal shunt developed glomerulonephritis (Dash 1997). This suggest that activation of alternate pathway may be a reason in these patients. Based on these observations, we also aim to study this interaction of low ADAMTS13 and complement activation in patients with NCIPH.
Key words: Portal Hypertension, Endothelium, ADAMTS13, Complement, Von-Willebrand factor, Cytokines,
Cases : Patients with idiopathic non-cirrhotic intrahepatic portal hypertension (NCIPH) as diagnosed by the presence of intrahepatic portal hypertension (i.e. patent portal vein and hepatic venous outflow tract on Doppler with gastroesophageal varices on gastroscopy), absence of etiology of chronic liver disease (e.g. alcohol, Hepatitis B/ C etc.) and absence of advanced fibrosis (i.e. bridging fibrosis/ cirrhosis) on liver biopsy)
Healthy control group: Healthy volunteers and patients with functional bowel disease who do not have any known liver disease
Sample size: We will enroll 50 patients with NCIPH in a 3 year period. We would enroll 50 subjects as healthy control groups as well.
Methods: All cases and controls will undergo a baseline history, clinical examination and laboratory evaluation, which will include tests for liver disease diagnosis and severity assessment. Besides routine tests, tests for vWF-ADAMTS13 will be done in all groups. Citrated (0.105 M) whole blood will be collected by clean venepuncture with minimal stasis using the Vacutainer system. Following blood collection, platelet-poor plasma will be prepared by centrifugation at 2,500 g for 20 min. The plasma will then be aliquoted and frozen at -80°C until assay.
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Human blood will be collected from the NCIPH patients and the control individuals who don’t have the history of thrombosis and had not been medicated for atleast 2 weeks. All the donors will be signed consent forms. To isolate the platlets the blood will be drawn in tubes containing (ACD; 85 mM sodium citrate, 111 mM glucose, and 71 mM citric acid, 10% vol/vol) anticoagulant and will be centrifuged at 150g for 15 minutes at room temperature to separate the platelets rich plasma. Further, centrifugation of this PRP at 9oog for 20 minutes will gives the platlet pellet. Then the platelet pellets will be washed once with a CGS buffer (13 mM sodium citrate, 30 mM glucose, and 120 mM sodium and used on the cultured endothelial cells.
To measure the level of circulating inflammatory cytokines and complement factors in NCIPH patients. Inflammatory cytokines like TNFα, IL6, IL8, and IL10 in NCIPH the amount of interleukins in NCIPH cases and controls will be determined using ELISA.
An enhanced protein-binding ELISA plate will be coated with 2 pg/ml purified rat anti-mouse monoclonal antibody to each cytokine (50 pl/ well). The plate will be covered and incubated overnight at 4°C. It will be then will be washed twice with phosphate-buffered saline solution (PBS) supplemented with 0.05% Tween 20 . Then wells will be blocked with 200 μl PBS supplemented with 10% Fetal calf serum. The plates will be incubated at room temperature for 2 hours and washed twice with PBS/ Tween. The plate will be incubated overnight at 4°C. Again the he wells will be washed four times with PBS/Tween. Biotinylated anti-mouse ILs or TNFα monoclonal antibodies l (100 μl, 1 μg/ml), and the plate was incubated at RT for 45 minutes. The wells were washed six times with PBS/ Tween. Avidin-peroxidase diluted with PBS/FCS (100 pl, 2.5 μg/ml) will be added to each well, and the plate will be incubated at room temperature for 30 minutes. The wells were then washed eight times with PBS/Tween. Immediately after mixing 10 μl 30% H202 into a solution of 10 ml of 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS, 0.3 μg/ml) in 0.1 mol/L citric acid, 100 μl of the resulting ABTS substrate was added to each well. The plate was incubated at room temperature to develop the color reaction, and the plate will be read at 490nm
The functional relationship of ADAMTS13 in proteolytic cleavage of UL-VWF on endothelial cells to maintain sufficient number of UL-VWF is not clear. Whereas the induction of VWF on microvascular endothelial cells in response to levels of inflammatory cytokines seen in NCIPH patient circulation should be analysed. This will be done under comparison of shear stress. So these proteins in NCIPH patients will be investigated thoroughly for understanding the biochemical system. Normal and NCIPH patient serum (1:2) in buffer (20 mM HEPES, pH 7.4, 150 mM NaCl and 5 mM CaCl2) after 5 minutes incubation on HUVEC cell lines were tested for surface bound UL-vWF polymers in presence and absence of shear stress..
The experimental setup will be done by culturing the microslides consist of micro channels
Effect of ADAMTS13 deficiency on complement activation: In vitro study to analyse plasma from NCIPH patients: In vitro experiments using HUVECs (human umbilical vein endothelial cells) in culture will be carried out to recreate the proposed intrahepatic vascular milieu in NCIPH, where absence of ADAMTS13 cleavage results in accumulation of ULWF (ultralarge von Willebrand factor) on endothelial cells. Towards this, HUVECs grown in culture will be stimulated with histamine for 2 minutes to induce secretion of ULVWF, which will be immuno-stained prior to their cleavage by plasma derived ADAMTS13. These cells will then be treated with serum from controls and NCIPH patients under normal as well as orbital shear conditions. After 15 minutes incubation, cells will be washed and immunostained for complement proteins. In the second set of experiments it tested for platelet adhesion to these cell lines which purified from the peripheral blood of the patients and control individuals. It is anticipated that plasma from NCIPH patients will show an increased complement and Platelet binding to ULVWF as illustrated in figure 1.
HUVEC cells will be seeded on glass coverslips for microscopy experiments and grown in Endothelial Basal Medium (EBM, Lonza, Hopkinton, MA), supplemented with 3% penicillin-streptomycin (P/S), 0.2 mM L-glutamine and Low Serum Growth Supplement (Invitrogen). For imaging experiments, HUVECs seeded on glass cover slips will be washed with PBS and stimulated with 100 mM histamine in 1 ml of PBS for 2 min followed directly by immunostaining for ultra-large vWF strings (ULVWF). Cells will then be incubated with plasma from either NCIPH patients or controls under normal as well as orbital shear conditions for 12 minutes, following which cells will be fixed and immunostained for complement components such as C3. Cell nuclei will be stained with DAPI.
The function of ATAMTS13 and the folding structure of UL-vWF will vary under static and flow condition. Hence, to mimic the physiological condition the role of ADAMTS13 from NCIPH samples will be evaluated invitro using micro-channels. This experiment will delineate the biophysical stress that involved in the alteration of micro channels in the hepatic circulation. The HUVEC cells will be cultured inside the microfluidic channels of NCIPH and the defined media will be injected at a defined flow rate, the shear stress rate can be adjusted either by the flow rate or by the micro channel size.
The shear rate can be calculated using the formulae
Were f – injection flow rate (μl/min)
R – diameter of the tube (mm)
Similarly various flow rates will be adjusted and calculated. The HUVEC cells as mentioned above cultured in these channels under different flow rate. Similar to static culture the cells will be stimulated using histamine and imaged for ULvWF. Cells will then be incubated with plasma from either NCIPH patients or controls under flow rate for 12 minutes, following which cells will be fixed and immunostained for complement components such as C3 and platelet adhesion. Cell nuclei will be stained with DAPI.
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