IMMUNOHISTOCHEMICAL EVALUATION OF VASOENDOTHELIAL GROWTH FACTOR RECEPTOR-2 (VEGFR2) EXPRESSION IN RAT CARDIOMYOCYTES UNDER DOXORUBICIN AND BETULONIC ACID AMIDE ADMINISTRATION

Author: Lushnikova E.L.¹, Mzhelskaya M.M.², Koldysheva E.V.³, Klinnikova M.G.⁴

Abstract

The aim of the study is to perform an immunohistochemical analysis of the expression of the vascular endothelial growth factor receptor 2 (VEGFR2) in the myocardium of Wistar rats in doxorubicin-induced cardiomyopathy and the administration of betulonic acid amide as an agent with a polytarget effect in comparison with the severity of destructive processes in heart muscle cells. Material and methods. Wistar male rats (total 51 animals) were used in the work with isolated and combined administration of a single sublethal dose of doxorubicin (7 mg/kg) and course administration of betulonic acid amide at doses of 50 mg/kg/day and 100 mg/kg/day for 3 and 14 days. Immunohistochemical evaluation of VEGFR2 expression in the myocardium was carried out with the calculation of the index of VEGFR2-positive cardiomyocytes; the volume density of cardiomyocytes with lytic changes was estimated using stereological analysis. Results and discussion. A single administration of doxorubicin (at a dose of 7 mg/kg) caused destructive changes in cardiomyocytes and hemodynamic disorders in myocardium, which increased with the duration of the experiment (14 days). The complex of the revealed structural and metabolic lesions reflected the development of regenerative-plastic insufficiency of cardiomyocytes, which is the basis of cardiac insufficiency in anthracycline cardiomyopathy. Morphological changes in the myocardium with isolated administration of betulonic acid amide were largely determined by the dose used. At a dose of 100 mg/kg, myocardial changes were similar in severity with doxorubicin-induced changes. Combined use of doxorubicin and betulonic acid amide revealed the most significant structural damage of the myocardium. It was shown that the increase in the volume density of lytic altered cardiomyocytes correlated with a significant increase in VEGFR2 expression (correlation coefficient varied from 0.620 to 0.980).

Key words

myocardium, cardiomyocytes, doxorubicin, betulonic acid amide, vascular endothelial growth factor receptor 2, immunohistochemistry

References

Brazil D.P., Park J., Hemmings B.A. PKB binding proteins: getting in on the Akt // Cell. 2002. 111. 293-303.

Cardinale D., Bacchiani G., Beggiato M., Colombo A., Cipolla C.M. Strategies to prevent and treat cardiovascular risk in cancer patients // Semin. Oncol. 2013. 40. 186-198.

Chiusa M., Hool S.L., Truetsch P., Djafarzadeh S., Jakob S.M., Seifriz F., Scherer S.J., Suter T.M., Zuppinger C., Zbinden S. Cancer therapy modulates VEGF signaling and viability in adult rat cardiac microvascular endothelial cells and cardiomyocytes // J. Mol. Cell. Cardiol. 2012. 52. 1164-1175.

Choi J.Y., Youn H.J., Kang J.H. Transthoracic echocardiographic assessment of adriamycin-induced cardiomyopathy in rats with a 15 MHz transducer // J. Korean Soc. Echocardiogr. 2000. 8. 78-86.

Cichewicz R.H., Kouzi S.A. Chemistry, biological activity, and chemotherapeutic potential of betulinic acid for the prevention and treatment of cancer and HIV infection // Med. Res. Rev. 2004. 24. 90-114.

Compernolle V., Brusselmans K., Acker T., Hoet P., Tjwa M., Beck H., Plaisance S., Dor Y., Keshet E., Lupu F., Nemery B., Dewerchin M., Van Veldhoven P., Plate K., Moons L., Collen D., Carmeliet P. Loss of HIF-2 alpha and inhibition of VEGF impair fetal lung maturation, whereas treatment with VEGF prevents fatal respiratory distress in premature mice // Nat. Med. 2002. 8. 702-710.

Feng Y., Rhodes P.G., Bhatt A.J. Dexamethasone pre-treatment protects brain against hypoxic-ischemic injury partially through up-regulation of vascular endothelial growth factor A in neonatal rats // Neuroscience. 2011. 179. 223-232.

Gianni L., Herman E.H., Lipshultz S.E., Minotti G., Sarvazyan N., Sawyer D.B. Anthracycline cardiotoxicity: from bench to bedside // J. Clin. Oncol. 2008. 26. (22). 3777-3784.

Li F., Zhao H., Liao Y., Takashima S., Asano Y., Shintani Y., Hori M., Kitakaze M. Higher mortality in heterozygous neuropilin-1 mice after cardiac pressure overload // Biochem. Biophys. Res. Commun. 2008. 370. 317-321.

Lim C.C., Zuppinger C., Guo X., Kuster G.M., Helmes M., Eppenberger H.M., Suter T.M., Liao R., Sawyer D.B. Anthracyclines induce calpain-dependent titin proteolysis and necrosis in cardiomyocytes // J. Biol. Chem. 2004. 279. 9. 8290-8299.

Nepomnyashchikh L.M., Lushnikova E.L., Goldstein D.V. Whether modern cell technologies can break down biological limitations of tissue-specific regeneration of the myocardium. Bull. Exp. Biol. Med. 2005. 139. (4). 481–490.

Nepomnyashchikh L.M., Lushnikova E.L., Klinnikova M.G., Molodykh O.P. The effect of drugs with antitumor activity – doxorubicin and cyclophosphamide – on the structural reorganization of rat myocardium and the number of cardiomyocytes. Sibirskiy onkologicheskiy zhurnal = Siberian journal of oncology. 2011. (4). 30-35. [In Russian].

Nepomnyashchikh L.M., Lushnikova E.L., Molodykh N.A., Klinnikova M.G., Molodykh O.P. Ultrastructure and stereology of cardiomyocytes in the development of regenerative and plastic myocardial insufficiency during ontogeny. Bull. Exp. Biol. Med. 2011. 151. (1). 88–94.

Park S.Y., Kim H.J., Kim K.R., Lee S.K., Lee C.K., Park K.K. Betulinic acid, a bioactive pentacyclic triterpenoid, inhibits skeletal-related events induced by breast cancer bone metastases and treatment // Toxicol. Appl. Pharmacol. 2014. 275. 152-162.

Ruixing Y., Dezhai Y., Hai W., Kai H., Xianghong W., Yuming C. Intramyocardial injection of vascular endothelial growth factor gene improves cardiac performance and inhibits cardiomyocyte apoptosis // Eur. J. Heart Fail. 2007. 9. (4). 343-351.

Shiojima I., Walsh K. Role of Akt signaling in vascular homeostasis and angiogenesis // Circ. Res. 2002. 90. 1243-1250.

Tolstikova T.G., Sorokina I.V., Tolstikov G.A., Fekhter O.B. Biological activity and pharmacological prospects of lupane terpenoids: I. Natural lupane derivatives. Russ. J. Bioorgan. Chem. 2006. 32. (1). 37–49.

Westenbrink B.D., Ruifrok W.P., Voors A.A., Tilton R.G., van Veldhuisen D.J., Schoemaker R.G., van Gilst W.H., de Boer R.A. Vascular endothelial growth factor is crucial for erythropoietin-induced improvement of cardiac function in heart failure // Cardiovasc. Res. 2010. 7. (1). 30-39.

Zentilin L., Puligadda U., Lionetti V., Zacchigna S., Collesi C., Pattarini L., Ruozi G., Camporesi S., Sinagra G., Pepe M., Recchia F.A., Giacca M. Cardiomyocyte VEGFR-1 activation by VEGF-B induces compensatory hypertrophy and preserves cardiac function after myocardial infarction // FASEB J. 2010. 24. (5). 1467-1478.

Zhang X., Wang X., Zhu H., Kranias E.G., Tang Y., Peng T., Chang J., Fan G.C. Hsp20 functions as a novel cardiokine in promoting angiogenesis via activation of VEGFR2 // PLoS One. 2012. 7. (3). e32765.

About Authors (Correspondence):

Lushnikova E.L. – doctor of biological sciences, professor, head of the laboratory of cytology and cell biology, e-mail: pathol@inbox.ru

Full Text

snmzh_6-2018_lushnikova_5-12.pdf

Received: 18/12/2018

Accepted: 18/12/2018