Alterations of actin cytoskeleton and arterial protein level in patients with obstructive jaundice

Abstract Vascular hypo-responsiveness to vasopressors in patients with obstructive jaundice (OJ) is a common anesthetic event, which leads to perioperative complications and increased mortality. The cause of this clinical issue remains unclear. In this study, we estimated the actin cytoskeleton and arterial protein level in the artery of OJ patients by proteomic analysis. Ten patients with OJ due to bile duct diseases or pancreatic head carcinoma were enrolled, while another ten non-jaundice patients with chronic cholecystitis or liver hemangioma as the control group. Vascular reactivity to noradrenaline was measured before anesthesia on the day of surgery. Artery samples in adjacent tissues of removed tumor were collected and evaluated by 2-dimensional electrophoresis. Proteins with differential expression were detected by MALDI-TOF mass spectrometry with immunoblot confirmation. The results confirmed the phenomenon of vascular hypo-reactivity in OJ patients as suppressed aortic response to noradrenaline were existed in these patients. We also found that actin cytoskeleton and several actin-binding proteins were up- or down-regulated in the artery of OJ patients. These proteins changed in OJ patents might be the basic mechanism of vascular hypo-reactivity, further studies to uncover the role of these proteins in OJ is critical for clinical treatment of these patients.


Introduction
Obstructive jaundice (OJ) is a common clinical symptom which could attributed to gallstones, malignant tumors and other ailments.Most OJ patients need surgery for treating of primary disease.However, the incidences of perioperative complications and mortality are higher in OJ cases compared with non-jaundice patients (Sewnath et al., 2002).This is mainly due to perioperative hypotension, acute renal failure and other complications caused by endotoxemia (Binah et al., 1985;Green and Better, 1995).What's more, hypotension is a highly rated and prominent clinical mark for anesthesia in OJ during operation (Rege, 1995).
Our previous study found that impaired arterial baroreflex sensitivity may play a role in enhancing susceptibility to perioperative complications encountered in OJ patients during anesthesia (Song et al., 2009), consistent with other studies (Finberg et al., 1981;Bomzon et al., 1986).However, the molecular mechanisms behind vascular function change in OJ remain unclear, and morbidity and overall mortality of low blood pressure, renal failure or multiple organ dysfunction syndrome cannot be improved until suitable treatment for vascular injury in jaundice is developed.
In the current work, changes in the artery of OJ patients were evaluated.This study aimed to assess the arterial proteome for differentially expressed proteins in various databases, to provide insights into the molecular mechanisms underpinning vascular hypo-responsiveness in patients with OJ.

Patients
Eligibility criteria were based on our previous work (Song et al., 2009).This trial had approval from the Shanghai ethics committee (Register Number: ACTRN12611000980932).All subjects participating in the current trial provided signed informed consent.The trial was registered prior to patient enrollment in ANZCTR (Australia New Zealand Clinical Trials Registry;Research Register Number, U1111-1124-2612;Principal investigator, Feixiang Wu;Registered Data, 09/2011).Ten patients with obstructive jaundice due to bile duct or pancreatic head carcinoma were enrolled, while ten non-jaundice patients with chronic cholecystitis or liver

Vascular reactivity measurement
Vascular reactivity was measured prior to anesthesia on the day of surgery.Upon arrival to the operating room following fasting (8 to 10 h), electrocardiography monitoring (lead II), finger pulse oximetry and non-invasive blood pressure assessment were carried out.Under local anesthesia, the left radial artery was punctured and catheterized, and connected via a sensor to a waveform analyzer (Vigileo system, Edwards life sciences, USA).Parameters were set with no adjustment, for continuous monitoring of cardiac output (CO), cardiac output index (CI), each-volume (SV), stroke volume index (SVI) and stroke volume variation (SVV).After puncturing the right internal jugular vein and indwelling a three-cavity central venous catheter under local anesthesia, central venous pressure (CVP) was simultaneously recorded.Body circulation resistance (SVR) and body circulation resistance index (SVRI) were calculated by inputting the obtained CVP values.Recorded values were averaged from three separate measurements.Ringer lactate solution was continuously instilled at 2 ml/kg/h.The patients were placed in the supine position for 20 minutes or more after entering the surgical room, and vascular reactivity was observed upon tension relief.After recording baseline values for mean arterial pressure (MAP), HR, CO, CI, SVR and SVRI, continuous infusion of increasing concentrations of noradrenaline for 5 min (30 and 60 ng.kg -1 min -1 ) was performed, with a 20-minute interval between the two concentrations to allow recovery.Systolic blood pressure did not exceed 160 mmHg at the maximum dose.Stable for 3 minutes during each administration, systolic blood pressure and the heart rate returned to pre-test levels, fluctuating up and down within 5%.

Preparation of total protein extracts
Artery samples near or surrounding the removed tissues were collected.About 100 mg of these samples were washed with cold PBS buffer three times.After PBS removal, 1 ml of lysis buffer (9.5 M Urea, 65 mM DTT, 4% CHAPS and 0.2% IPG buffer, mixed with enzyme inhibitors at 50:1 v/v) was added.The samples were then homogenized with a Dounce homogenizer after vertexing.Next, ultrasonication of the specimens was performed 5 times at 15-second intervals on ice (80 W, 10 seconds).Supernatants were collected and stored at -80 ℃ after centrifugation (12,000 g, 4 ℃) for 45 minutes.Quantitative analysis of total protein in artery samples was performed with Bio-Rad protein assay reagents.

Two-dimensional electrophoresis (2-DE)
Proteins (100 μg) were firstly resolved by isoelectrofocusing (IEF) with gel strips measuring pH values between 3 and 10 based on a non-linear gradient (IPG strip, 18 cm; GE Healthcare, Waukesha, WI, USA).Strip rehydration was carried out in a Protein IEF Cell (GE Healthcare) in the passive (4 h) and active (12 h) modes at room temperature.Simultaneous IEF was carried out on an Ettan IPGphor Isoelectric Focusing system as follows: (a) 30 V for 12 h; (b) voltage gradient to 500 V in 1 h; (c) gradient to 1000 V in 1 h; (d) gradient to 8000 V in 8 h; (e) 500 V for 4 h.Next, IPG strips were placed onto vertical 12.5% polyacrylamide gels in an Ettan-DALT Six system (GE Healthcare).Second-dimension SDS-PAGE was performed on a Hofer SE 600 system (GE Healthcare) at 5 W/gel for 30 min followed by 180 W for 4 h.Protein spots were detected after Coomassie blue dyes on an UMax Powerlook 2110XL scanner (GE Healthcare).

Trypsin digestion and gel extraction
Spots of interest were excised from Coomassie Brilliant Blue stained gels after 2 Milli-Q water (15 min) and 4 NH4HCO3 (25 mM in 50% acetonitrile; 30 minutes) washes.After washing, the gel pieces were submitted to acetonitrile (50 mL) dehydration until opacity and further placed at 60º C to total dryness; rehydration was carried out in 50 mM NH4HCO3.Next, the samples were digested with trypsin (12.5 ng/mL; Promega, Madison, USA) in 10-mL reactions.The samples were chilled on ice for 45 min followed by addition of 25 mM NH4HCO3 and overnight incubation (37º C).After centrifugation, supernatants were submitted to 3 extractions with 50% acetonitrile containing 0.1% trifluoroacetic acid.The resulting mixtures were vacuum-dried.In MALDI-TOF/ TOF, peptide elution was performed with 0.7 mL α-cyano-4-hydroxy-cinnamic acid (Sigma, St. Louis, MO, USA) in 0.1% trifluoroacetic acid/50% acetonitrile.

Mass spectrometry
A 5800 MALDI TOF/TOF analyzer (Applied Biosystem, Framingham, MA, USA) was employed for assessments.Mass spectrum (m/z 800-4000) acquisition was in the positive ion reflector mode.The top 20 ions in terms of intensity were submitted to MS/MS sequencing in the 2 kV mode.The NCBI protein database was queried for matched MS/MS spectra by Matrix Science (http://www.matrixscience.com)for the identification of proteins: taxonomy, Green Plants; peptide tolerance, 1.2 Da; MS/MS tolerance, 0.6 Da; 1 incomplete cleavage allowed; modification, methionine oxidation.Based on MASCOT probability assessment (P<0.05),significant hits were used for protein identification.The identified proteins were examined for sub-cellular localization and function with TargetP (http://www.cbs.dtu.dk/services/TargetP/) or PSORT (http://psort.hgc.jp/).Further, potentially related pathways and biochemical reactions were queried in Uniprot (http://www.uniprot.org/)and Genome-Net (http://www.genome.ad.jp/ kegg/).The STRING toll (https://string-db.org/)was used to make the protein-protein interaction networks and analysis the biological process.

Western blot
Aorta segment lysis was performed with chilled lysis buffer in presence of protease inhibitors.Lysate centrifugation was carried out at 12,000 rpm for 15 min at 4 °C.Total protein in the supernatant was quantitated with a BSA assay kit (P0006, Beyotime, Jiangsu, China).Protein separation was performed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and protein bands were electro-transferred onto polyvinylidene difluoride (PVDF) membranes.The samples were incubated overnight at 4 °C with anti-tropomyosin β, anti-transgelin, anti-annexin, anti-gelsolin, anti-HSP-27, anti-cofilin-1 and anti-GAPDH (1:1000; Abcam, Cambridge, UK) primary antibodies.Further incubation was performed with goat anti-mouse or anti-rabbit secondary antibodies (1:10,000; Santa Cruz Biotechnology, USA) for 1h in ambient conditions.Development was carried out with the BeyoECL kit (Beyotime, China) and a Tanon 5200 system.

Statistical analysis
Spot intensities for differentially expressed proteins in D gels were obtained from three independent experiments.One-way analysis of variance and Duncan's multiple range test were employed for comparisons.P<0.05 at two tails was deemed statistically significant.

General conditions
Age, gender distribution, height, weight and BSA were comparable in both groups.Serum total bilirubin and bile acid amounts were markedly elevated in OJ patients compared with controls (all P<0.01; Table 1).

Vascular reactivity
Baseline values for hemodynamic data such as MAP, heart rate (HR), CVP, CO, CI, SVR and SVRI were comparable in both groups (Figure 1).Increases in doserelated MAP, SVR and SVRI were observed in both groups compared with baseline values (Figure 1).After continuous infusion of 30 ng.kg -1 min -1 norepinephrine for 5 min, MAP increase in jaundice patients (from 80.7±5.9 mmHg to 85.5± 6.6 mmHg) was lower than that of the non-jaundice group (from 84.1±6.7 mmHg to 94.5±7.4 mmHg, p<0.05).Similarly, after continuous pumping of 60 ng.kg -1 min -1 norepinephrine for 5 min, MAP increase in patients with jaundice was significantly lower than of the non-jaundice group (from 81.7±6.3 mmHg to 88.8±6.5 mmHg and from 85.7±6.9 mmHg to 102.3± 8.4 mmHg, respectively; p<0.05).

Proteomics findings
In order to determine quantitative expression differences in proteins between obstructive jaundice and non-jaundice patients, the proteomic profiles of both groups were analyzed.In this comparison, thirty-three differentially expressed spots were identified by 2-DE image analysis (Figure 2A).

Confirmation of proteomics findings
We further used Western blot to confirm seven actin or actin related proteins in the cytoskeleton signaling pathway.As shown in Figure 2B, the expression levels of actin, tropomyosin β, transgelin, annexin, gelsolin, HSP-27 and cofilin-1 were in accordance with proteomics data.

Bioinformatics findings
A total of 24 proteins with differential expression between obstructive jaundice and non-jaundice patients were identified by MS/MS.They included 17 upregulated and 7 downregulated proteins (Table 2).Functional annotations for all proteins are based on Uniport (https://www.uniprot.org/).
As described by Bevan et al. (1998), the above proteins were grouped by functional category.They belonged to the categories of Cytoskeleton, Metabolism, Signal transduction, Transport, Proliferation/Apoptosis/ Differentiation, Stress response, DNA repair, Transcription regulation, Cell adhesion and Immune response, respectively.An important fraction of proteins was associated with the cytoskeleton and were involved in metabolism.Protein-protein interaction maps of the differentially expressed protein spots in artery of obstructive jaundice patients (Figure 3), with an average node degree of 4.75, an average local clustering coefficient of 0.405, an expected number of edges of 7, and with a PPI enrichment p-value of less than 1.0e -16 .The enrichment results indicates that the proteins are at least partially biologically connected, as a group.

Discussion
Previous studies have reported vascular hypo-reactivity in OJ cases as well as reduced aortic response to vasoactive products; further animal studies revealed that this effect was attributed to deregulated expression of receptors or related proteins in vascular smooth muscle cells and endothelial cells.To confirm this clinical phenomenon, we compared vascular reactivity to norepinephrine in obstructive jaundice and non-obstructive jaundice patients by non-invasive and invasive monitoring methods.Then, differential protein expression was assessed in blood vessels of these two groups by proteomic analysis.
In the current clinical trial, we identified dose dependent hemodynamic changes after norepinephrine infusion, and changes in HR, CVP, CO and CI showed no marked differences between the OJ and control groups, reflecting similar cardiac contractility and blood volume in both groups.What's more, differential responses of MAP, SVR and SVRI to the vasoconstrictor were found in both groups, confirming vascular hypo-reactivity in the artery and aorta of jaundice patients to certain vasoactive agents.Vascular hypo-reactivity in OJ has long been reported clinically, however, this seems to be the first prospective research to analyses the circulatory functions and their reactions toward norepinephrine in these patients.
By using 2-DE separation of proteins from whole extracts and followed by mass spectrometry, we compared proteomic profiles between artery samples from jaundice and non-jaundice patients, and revealed a total of 24 differentially expressed proteins.2-DE technology provides protein separation by its hydrophobicity, which could identify the first axis of isoelectric point, and thus provides good mixture simplification, with the fractionated proteins intact and in the liquid phase for easy isolation and analysis with other analytical techniques such as MS, and Western blot analysis.Previous studies have already proved that 2-DE chromatography analysis performed well in the identification of novel proteins with high confidence in human amniotic fluid (Michaels et al., 2007;Bujold et al., 2008).We further investigated seven abnormally expressed proteins which were closely related to vascular hypo-reactivity of jaundice patients, independently confirming their expression trends by immunoblot.
Functional annotation of proteins with differential expression in jaundice patients revealed approximately 33% were associated with the cytoskeleton, while 17% were involved in metabolism.These findings validate our hypothesis, since lower reactivity occurs during cytoskeleton structure modifications in VSMCs and/or endothelial cells (Akata, 2007), as well as changes in cellular metabolism.Actin cytoskeleton signaling emerging from the above proteomics data depicting proteins with differential expression in OJ, seemed to have an important function in vascular hyporeactivity in jaundice patients (Schildmeyer et al., 2000).These findings suggested that vascular hypo-reactivity in OJ patients is probably due to an impairment of actin-mediated muscle contractile activity.The contractile system of vascular smooth muscle comprises thin and thick filaments made of actin and myosin, respectively.Actin polymerization and cytoskeletal dynamics regulate force development in vascular smooth muscle, which results in blood pressure and blood flow regulation.In α-actin deficient mice, vascular contractility and blood pressure homeostasis are impaired, with compromised vascular contractility, decreased blood pressure, and reduced blood flow after α-actin gene transcription in the smooth muscle is inactivated (Tang and Anfinogenova, 2008).
As shown above, actin was impaired in the artery of OJ patients; meanwhile, several actin-binding proteins were also downregulated in OJ patients.Tropomyosin is known to bind actin filaments and interact with multiple actins binding proteins, playing significant roles in muscle contraction and thin-filament assembly in both muscle and non-muscle cells.Greenberg MJ demonstrated that the intrastrand reinforcement by smooth muscle tropomyosin increases persistence length by 1.5-fold (Greenberg et al., 2010).The conserved periodic sites of tropomyosin are also important in regulating actin-myosin interaction that produces force generation between actin filaments, leading to contractions.Hence, beta-tropomyosin upregulation in the present study in blood vessels may probably lead to vascular hypo-reactivity in OJ patients.According to the above results, annexin A2 and A5 were also upregulated in OJ.Annexin represents a calciumdependent phospholipid-binding protein that contributes to the exocytosis of intracellular proteins (Burger et al., 1996), and modulates several cell events, including motility, membrane-associated protein complex binding to the actin cytoskeleton, endocytosis and fibrinolysis (Bharadwaj et al., 2013).Recombining actomyosin, annexins, and caveolar fats in presence of calcium ions generates a well-arranged precipitate; such arrangement ensures efficient transmission of contractile activity in smooth muscle cells (Babiychuk et al., 1999).Another increased protein was gelsolin, a calcium-regulated actin filament severing and nucleating protein found in many cell types which is involved in various cellular processes, including regulation of actin dynamics, cell motility, control of apoptosis and cellular signal transduction.Knockdown of the gelsolin-like protein CapG in pulmonary arterial smooth muscle cells (PASMCs) causes reduced proliferation, enhanced apoptosis and cell cycle arrest, while its downregulation attenuates pulmonary hypertension.This indicates CapG contributes to pulmonary vascular remodeling (Xu et al., 2016).CapG promotes monomer assembly into filaments and also severs existing filaments.Further studies to elucidate the physiological role of gelsolin are under way in our laboratory.Of note, stress resistance and actin organization related heat shock protein (HSP) were also overexpressed in OJ patients.HSP regulates the actin cytoskeleton in differentiated smooth muscle cells due to elevated amounts of this protein as well as co-localization with contractile proteins (Bitar, 2002).Stretch causes remarkable structural disruption of the actin cytoskeleton and decreases the F/G-actin ratio in bladder smooth muscle cells (BSMCs) in vitro with HSP27 over-expression and knock-down, indicating an important role for HSP27 in bladder smooth muscle contraction (Smoyer et al., 1996).In smooth muscle samples after permeabilization, agonist-associated contraction is suppressed by anti-HSP27 antibodies, reducing HSP27 phosphorylation inhibits vasoconstriction caused by angiotensin II (Webb, 2019).In the present study, HSP27 was decreased in OJ patients as assessed by proteomic analysis and further confirmed by Western blot.The last upregulated actin binding protein in OJ was cofilin-1.Actin depolymerizing factors (ADFs)/cofilins are widely found in eukaryotes, with diverse functions based on highly complex associations with both monomeric and filamentous actins (Remedios et al., 2003).Cofilin binds to F-actin and is involved in the regulation of actin cytoskeleton dynamics and cell morphology (Ostrowska and Moraczewska, 2017).Transgelin is an actinbinding protein but was lowly expressed in OJ patients.This protein is also known as smooth muscle 22 α (SM22α).It is specifically found in the contractile smooth muscle of mammalians alongside additional differentiation markers such as α-actin, calponin and smooth muscle myosin.In SM22a(-/-) mice, contraction of aortic rings in response to angiotensin II (Ang II) is significantly decreased in vitro, and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylated levels are reduced, suggesting SM22α promotes AngII-associated contraction via maintenance of the ERK1/2 pathway (Webb, 2019), meanwhile, actin amounts are reduced by 10-25% in vessels from transgelin knockout mice (Schildmeyer et al., 2000).In OJ patients, whether transgelin downregulation occurs through actin or actin upregulation is through transgelin requires further investigation.
Besides actin binding proteins, proteins related to actin regulation were decreased in the artery of OJ patients.LIM and SH3 domain protein 1 (LASP) has a critical function in regulating the dynamic activities of the actin cytoskeleton.Agonist-associated LASP1 phosphorylation alterations might also modulate actin-related ion transport in parietal cells as well as other secretory epithelial cells expressing high F-actin levels.This novel actin-binding protein could control a pathway contributing to cell cytoskeleton organization (Schreiber et al., 1998).Adapter proteins, such as Sorbin and SH3 domaincontaining protein 2 (SORBS), play roles in signaling complex assembly, linking ABL kinases and the actin cytoskeleton.SORBS2 is an adapter protein that functions in cytoskeletal organization, cell adhesion and signaling pathways.
Other non-actin related proteins significantly altered in OJ included vimentin, one of the main structural constituents of IFs in multiple cells, plays a critical role in essential cell functions, including contractility, migration, stiffness, stiffening and division.Vimentin-deficient cells exhibit reduced contractility, indicating vimentin IFs are essential in preserving mechanical interactions among cells (Wang and Stamenovic, 2002).Considering the apparent upregulation of this protein, we also propose that it may have a critical function in vascular hypo-reactivity in OJ, which deserves further animal experiments.

Conclusions
The present study confirmed vascular hypo-reactivity to norepinephrine in OJ patients, and several deregulated proteins were further determined by proteomic analysis.The present findings suggested that several actin or actin-related proteins in the actin cytoskeleton signaling pathway may probably be largely involved in vascular hypo-reactivity in OJ patients.Although with several limitations, for instance, smooth muscle cells and epithelial cells of the artery were not separated in this analysis, we believe that this study provides valuable evidence and directions for further investigations.

Figure 1 -
Figure 1 -Vascular reactivity after agent administration.A. Measured MAP values in OJ and non-OJ patients (P=0.013,two-way ANOVA with post hoc Bonferroni correction, N=10 in Jaundice group while 9 in Control).B. Measured SVR in OJ and non-OJ patients (P=0.08,two-way ANOVA with post hoc Bonferroni correction, N=10 in Jaundice group while 9 in Control).C. Measured SVRI in OJ and non-OJ patients (P=0.11,two-way ANOVA with post hoc Bonferroni correction, N=10 in Jaundice group while 9 in Control).

Figure 2 -
Figure 2 -Differentially expressed protein spots in artery of obstructive jaundice patients.A. Representative analytical proteome map of obstructive jaundice.B. Western blot analysis for seven actin or actin-related proteins identified by DIGE analysis.

Figure 3 -
Figure 3 -Protein-protein interaction maps of the differentially expressed protein spots in artery of obstructive jaundice patients.The STRING toll (https://cn.string-db.org) was used to make the networks and analysis the biological process.The line color indicates the type of the interaction evidence.Network nodes represent proteins splice isoforms or post-translational modifications are collapsed.
BSA: body surface area.Data are presented as Mean ± SD.

Table 2 -
Identification results of proteins expressed in obstructive jaundice vs. non-jaundice patients.