Enio R Vsqus Estl RR Figuir Jol A Roh-Filho Cinthi Lnhott Jorg LS Ximns Hln B Nr Ivrn LS Trsriol Mrlo A Lim Tigo Rorigus José EM Cunh Elzr Chi f Luiz AC D’Aluqurqu f Flávio HF Glv?o f

a Laboratorio de Investiga?ao Medica 37, Departamento de Gastroenterologia, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de S?o Paulo, S?o Paulo 01246-903, Brazil

b Departamento de Gastroenterologia, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de S?o Paulo, S?o Paulo, Brazil

c Disciplina de Anestesiologia, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de S?o Paulo, S?o Paulo, Brazil

d Departamento de Bioquimica, Universidade Federal de S?o Paulo, S?o Paulo, Brazil

e Universidade Federal do ABC, Santo Andre, S?o Paulo, Brazil

f Servi?o de Transplante de Figado e Orgaos do Aparelho Digestivo, Departamento de Gastroenterologia, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de S?o Paulo, S?o Paulo, Brazil

TotheEditor:

Ischemia-reperfusion injury following surgery and transplanta- tion can lead to irreversible multiorgan failure. Intracellular cal- cium overload is associated to cellular death during ischemia- reperfusion. A recently discovered heparin fragment (HF), trisul- fated disaccharide (TD), that acts on sodium-calcium exchanger (NCX) decreasing intracellular Ca2+, showed effectiveness on pro- tecting hepatocytes from ischemia-reperfusion injury [1] , such as protection during cardiac arrhythmias [2] . HF of different molec- ular weights and origins have TD in their basic unit; however, calcium dynamics is heterogeneous due to differences in molec- ular weight and structural conformation [ 3 , 4 ]. We evaluated a new bovine HF (BHF001) of ～8000 kDa, presenting similarity to frag- ments of porcine and synthetic origin; however, without anticoag- ulation properties [5] . We designed a model to study the intracel- lular Ca2+behavior with administration of BHF001 in cell culture andinvivoexperiments to analyze its cellular and systemic pro- tection in rats submitted to liver ischemia-reperfusion injury.

Invitro, Rattus norvegicus BRL3A liver cells (Merck KGaA, Damstadt, Germany) cultured in modified Dulbecco’s medium were supplemented for 24 h and exposed to the Fura-2/AM cal- cium fluorescent marker (4μmol/L) for 30 min at 37 °C in 5% CO 2 . Cells were washed twice with 1.0 mL of CaCl 2 , 130 mmol/L NaCl, 5.6 mmol/L KCl, 0.8 mmol/L MgSO4, 1 mmol/L Na2HPO4, 25 mmol/L glucose, 2 mmol/L HEPES, and 2.5 mmol/L NaHCO 3 (pH 7.3), analyzed and digitally photographed. Changes in cytosolic cal- cium levels were measured at 37 °C continuously for 15 min. To decrease cytosolic calcium, 50μmol/L of BHF001 was added after 150 ms of the action of 200 μmol/L thapsigargin. Finally, the cul- ture was submitted to ionophore action (0.15μmol/L) for the for- mation of hydrophilic pores allowing elevation of the intracellular calcium of the extracellular medium and cytosolic calcium levels were measured again.

Invivo, 27 male Wistar rats (weighing 290–350 g) were housed in accordance with the Guide for the Care and Use of Laboratory Animals. The protocol was approved by Institutional Review Board (No. 138/13). Rats were allocated into three groups (n= 9 each group). In the control group, animals were subjected to liver is- chemia for 40 min; in the preconditioning group, animals received 2 mg/kg of intravenous HBF001, 10 min before ischemia; and in the postconditioning group, animals received HBF001, 10 min before reperfusion. Anesthesia and surgical procedures were performed as described previously [ 6 , 7 ]. Briefly, after standard anesthesia, a midline laparotomy and blood flow through common pedicle of median and left liver lobes was interrupted, inducing partial nor- mothermic ischemia for 40 min. After liver reperfusion, right and caudate non-ischemic lobes were resected immediately. At 4 h of reperfusion animals were re-anesthetized for carotid arterial blood sampling. Aspartate aminotransferase (AST) and alanine amino- transferase (ALT) levels were determined by the optimized ultra- violet method (Cobas Mira, Roche Diagnostics, Switzerland). Bicar- bonate (BIC), pH, base excess (BE), lactate, ionized calcium (iCa), potassium, and glucose levels were measured by ABL800 FLEX blood gas analyzer (Radiometer Medical ApS, Copenhagen, Den- mark). Prism-6 software (GraphPad, San Diego, CA, USA) was used for statistical calculations. Data expressed as mean ± standard deviation (SD) were compared with unpaired Student’st-test. AP＜0.05 was considered statistically significant.

Invitro, addition of BHF001 decreased intracellular Ca2+levels, with maximum effect ～600 ms, lasting up to 240 ms, when the fall stabilized. Around 800 ms the intracellular Ca2+, under BHF001 effect, dropped to levels similar to baseline before TAP ( Fig. 1 A).

Fig. 1. A: HBF001 effect on thapsigargin-induced cytosolic calcium increase in liver cell culture. Red line: control group; black line: thapsigargin effect. B and C: AST ( B ) and ALT ( C ) levels at 4 h after reperfusion. D-F: Arterial levels of bicarbonate ( D ), base excess ( E ) and lactate ( F ). *P ＜ 0.05, compared to control group.

Invivo, after 4 h of reperfusion, AST and ALT levels were sig- nificantly decreased in the postconditioning group (5595 ± 1878 and 4825 ± 1943 UI/L, respectively) compared to those in the con- trol group (10 466 ± 5643 and 9972 ± 6191 UI/L, respectively). There were no significant differences in AST (7297 ± 4380 UI/L) and ALT (6744 ± 4112 UI/L) between the preconditioning and other groups ( Fig. 1 B, C). Arterial BIC (23.13 ± 3.55 mmol/L) and BE (-2.57 ± 4.75 mmol/L) were significantly increased in the post- conditioning group compared to those in the control group (BIC: 18.99 ± 3.19 mmol/L and BE: -7.83 ± 4.47 mmol/L) ( Fig. 1 D, E). There were no significant differences in BIC (20.59 ± 2.99 mmol/L) and BE (-4.67 ± 4.05 mmol/L) in the preconditioning group com- pared to those in other groups. Lactate was significantly decreased in the preconditioning group (27.67 ± 8.36 mg/dL) and postcondi- tioning group (28.44 ± 18.70 mg/dL) compared to that in the con- trol group (44.38 ± 20.49 mg/dL), with no differences between the preconditioning and postconditioning groups ( Fig. 1 F). Additionally, there were no differences in arterial glucose, potassium and ion- ized calcium levels among the three groups ( Table 1 ).

Table 1 Arterial levels of glucose, potassium and ionized calcium 4 h after reperfusion.

Heparins of different origins have different efficacy, and the an- ticoagulant action may differ due to differences in their chem- ical structures [8] . BHF001 is a bovine HF with mean weight of 4274 kDa synthesized in our laboratory, similar to enoxa- parin (4084 kDa). These structures of different molecular weights present repetitive sequences of disulfide disaccharide units as in enoxaparin. However, the TD itself influences NCX, altering ex- change inhibitory peptide structure and accelerating the intracel- lular calcium output in situations of Ca2+ overload [4] . Therefore, HBF001, which is composed of repetitive TD units, may trigger a similar effect. In the present study, animals receiving HBF001 pre- ischemia maintained elevated levels of AST and ALT, as well as changes in BIC and BE suggestive of liver damage and metabolic acidosis, such as in the control group. In the preconditioning group, only lactate presented a statistically significant reductionas com- pared to the control group, but in the same magnitude as that ob- served with the post-ischemic strategy. The significant reduction in AST and ALT levels, the increase in BIC and BE, and the decrease in lactate levels in the postconditioning group, when compared to the control group, suggest a protective effect of HBF001 when administered before reperfusion, demonstrating a possible role of HBF001 in preventing injury and cell death that occurs during cal- cium overload determined by organic reperfusion after ischemia.

Possibly, the action of HBF001 related to calcium metabolism is time dependent, since its infusion in the pre-ischemic period only determined lactate reduction, without altering other param- eters of injury. On the contrary, when infused before reperfusion it allows prevention of cellular injury with normalization of eval- uating tests. The dynamics of the action time determined by theinvitrostudy shows an immediate action of HBF001 on reduction of intracellular calcium with maximum outflow of intracellular ion occurring around 60 s, with remaining effect for ～240 s, until the ionophore leads to a new calcium overload. We showed that TD pretreatment, before the intracellular calcium overload determined by thapsigargin, resulted in a slight decrease in cytosolic calcium. Although this effect was not statistically significant it resulted in lowering calcium overload, when added immediately after thapsi- gargin, and the effect of TD occurred around 60 s and lasted for 250 s until the use of ionophore [ 1 , 9 ]. This behavior of the iso- lated use of TD may explain the inefficacy of HBF001 infusion dur- ing pre-ischemia in theinvivoexperiment, and the good results with HBF001 infused prior to reperfusion. Similar values regarding the times of action of HBF001 and TD are reported in the liter- ature, which may indicate similar pharmacological and pharmaco- dynamic behaviors relative to its NCX action [10] . In conclusion, we observed that the new HBF001 heparin fragment decreases hepa- tocellular injury with attenuation of metabolic imbalance probably due to regulation of calcium homeostasis.

Acknowledgments

None.

CRediTauthorshipcontributionstatement

EnioRVasques:Data curation, Formal analysis, Writing - origi- nal draft.EstelaRRFigueira:Data curation, Formal analysis, Writ- ing - original draft.JoelARocha-Filho:Data curation, Formal analysis, Writing - original draft.CinthiaLanchotte:Data cura- tion, Formal analysis, Writing - original draft.JorgeLSXimenes:Methodology, Writing - original draft.HelenaBNader:Method- ology, Writing - original draft.IvarneLSTersariol:Methodology, Writing - original draft.MarceloALima:Methodology, Writing - original draft.TiagoRodrigues:Methodology, Writing - origi- nal draft.José EMCunha:Conceptualization, Supervision, Writing - review & editing.EleazarChaib:Conceptualization, Supervision, Writing - review & editing.LuizACD’Albuquerque:Conceptual- ization, Supervision, Writing - review & editing.FlávioHFGalv?o:Conceptualization, Supervision, Writing - review & editing. Concep- tualization, Supervision, Writing - review & editing.

Funding

None.

Ethicalapproval

This study was approved by the Ethics Committee on Animal Use of University of Sao Paulo School of Medicine (No. 138/13 in 04/24/2013).

Competinginterest

No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the sub- ject of this article.