Monaminergic Mechanisms of Hematopoiesis Regulation under Cytostatic Effects

The dissertation is dedicated to the study of monaminergic mechanisms of hematopoiesis regulation under cytostatic effects. In the work, the role of the adrenergic, dopaminergic, and serotonergic systems in the development of myelosuppression and regeneration of hematopoietic tissue has been investigated in a comparative aspect on models of cytostatic effects caused by an alkylating agent (cyclophosphamide) and an antimetabolite (5-fluorouracil). It has been established that the regulatory influence of central monaminergic structures is realized through adrenergic, dopaminergic, and serotonergic receptors on committed hematopoietic precursors and cellular elements of the hematopoiesis-inducing microenvironment, as well as indirectly — through the erythropoietin and granulocyte colony-stimulating factor systems. The specific role of the serotonergic system in the regulation of erythropoiesis and the adrenergic and dopaminergic systems in the control of granulopoiesis has been demonstrated. The obtained data make a substantial contribution to the understanding of monaminergic mechanisms of hematopoiesis control under myelosuppressions and substantiate the development of pathogenetically justified methods of pharmacological correction of hematological disorders.

Relevance of the problem. The study of the patterns of functioning of the blood system under normal and pathological conditions is an urgent task of modern experimental hematology and pathophysiology, the solution of which creates prerequisites for the development of adequate pathogenetically justified methods of pharmacological prevention and correction of hemodepressive states of various etiology [Moore M.A.S., Warren D., 1987; Dexter T.M., Heyworth C.M., 1994; Carde P., 1994; Drize N. et al., 1996; Khlusov I.A. et al., 1997; Prow D., Vadhan-Raj S., 1998; Dygai A.M. et al., 1999; Zhdanov V.V. et al., 2000; Vorobiev A.I., 2002; Goldberg E.D. et al., 2007]. In recent years, fundamentally new data have been obtained that have largely made it possible to decipher the mechanisms of local regulation of hematopoiesis under myeloinhibitory effects [Dygai A.M. et al., 1992; Zhdanov V.V. et al., 1998; Goldberg E.D. et al., 2001, 2007; Uduy E.V. et al., 2008], which, in turn, served as the basis for the creation of a number of highly effective hemostimulators based on glycosaminoglycan derivatives and recombinant forms of cytokines [Asano S., 1991; Ruef S., Coleman D.L., 1991; Abels R.I., 1992; Ganser A., Hattori K. et al., 1996; Zhdanov V.V. et al., 1997; Steward W.P. et al., 1998; Dygai A.M. et al., 1999; Gabrivole J., 2000; Zak K.P., Grytsyuk S.N., 2001; Ptushkin V.V., 2002; Egrie J.C. et al., 2003; Goldberg E.D. et al., 2007; Beutel G., Ganser A., 2007; Littlewood T.J., Collins G.P., 2007; Xu Y.J. et al., 2007]. A promising approach to the creation of new hemostimulators could be considered the search for agents capable of affecting distant mechanisms of control of hematopoietic processes. In particular, methods for the therapy of cytostatic myelosuppressions using drugs that modulate the activity of the sympathetic division of the autonomic nervous system (ANS) have been proposed and tested in clinical practice [Goldberg E.D. et al., 1997; Khlusov I.A. et al., 1997].

Meanwhile, the accumulated data to date on the participation of neurotransmitter systems in ensuring hematopoiesis indicate that the spectrum of neuropharmacological agents capable of stimulating the processes of restoration of hematopoietic tissue under suppressive effects (including upon administration of cytostatics) is much broader [Chatelain S. et al., 1989; Yang M. et al., 1996; Maestroni G.J. et al., 1997; Tsao C.W. et al., 1997; Broome C.S., Miyan J.A., 2000; Devoino L. et al., 2001]. Evidence for this is the fact that in the parenchyma of the bone marrow, in addition to sympathetic, sensitive, purinergic, and cholinergic nerve endings have been detected [Yamazaki K., Allan T.D., 1990; Weihe E. et al., 1991; Tabarowski Z. et al., 1996; Artico M. et al., 2002; Lakshmi C. et al., 2005; Aquila H.L., 2006]. Secretion by them of adrenaline, noradrenaline, dopamine, tachykinins, and a number of other highly active biological substances has been traced [Maestroni G.L., 2000; Zakharov Yu.M., 2004]. It has been established that hematopoietic precursor cells of various degrees of maturity, as well as morphologically recognizable hematopoietic and stromal cells, are capable of carrying on the plasma membrane receptors to various ligands of mediator nature (acetylcholine, catecholamines, serotonin, substance P, neurokinins A and B, opioids, etc.) [Morley A. et al., 1971; Bosing-Schneider R., Haug M., 1976; Brown J.E., Adamson J.W., 1977; Fedorov N.A., 1979; Van Furth R., 1980; Depelchin A., Letesson J.J., 1981; Hall N.R., 1981; Miles K. et al., 1984; Mladenovic J., Adamson J.W., 1984; Setchenska M.S. et al., 1986; Zahniser N.R. et al., 1989; Kharkевич D.D., 1989; Broome C.S., Miyan J.A., 2000]. A direct (receptor) and mediated through cellular elements of the hematopoiesis-inducing microenvironment (HIM) regulatory influence of neurotransmitters on the processes of proliferation and differentiation of committed hematopoietic precursors has been demonstrated [Mladenovic J., Adamson J.W., 1984; Setchenska M.S. et al., 1986; Maestroni G.L., Conti A., 1994; Zakharov Yu.M., 2004; Yang M. et al., 2007]. Of no small importance is the ability of hematopoietic cells and cellular elements of the HIM not only to synthesize and secrete neurotransmitters but also to involve them in autoregulation [Marino F. et al., 1997, 1999]. All these and many other facts presuppose the existence of a monaminergic local control of the processes of damage and regeneration of hematopoietic tissue in response to myeloinhibitory effects. Moreover, the activity of adrenergic, dopaminergic, and serotonergic mechanisms with respect to various cellular populations is, most likely, of a specific nature.

According to current concepts, hematopoietic growth factors and inhibitors play a crucial role in the vital activity of hematopoietic cells [DePace D.M., Weber R.H., 1974; Sieff C.A. et al., 1987; Fibbe W.E. et al., 1989; Metcalf D., 1989; Dorshkind K., 1990; Nathan D.G., Ziff K.A., 1994; Carde P., 1994; Grassinger J. et al., 2006; Kertesz Z. et al., 2006]. Selective sensitivity of multipotent stem cells and more mature precursors, including morphologically recognizable ones, to certain hemopoietins is known [Metcalf D., 1989; Heyworth S.M. et al., 1992; Karger A.G., 1993; Nathan D.G., Ziff K.A., 1994; Carde P., 1994; Cross M.A. et al., 1997; Vozianov A.F. et al., 1998; Chertkov I.L., Drize N.I., 1998]. At the level of committed precursors, a pronounced synergy of action of growth factors is observed, and for an optimal response, the presence of a combination of "early" and "late" cytokines is extremely important [Sieff C.A., 1987; Ikebuchi K. et al., 1987; Rennick D. et al., 1989; Tanaka R. et al., 1992; Necas E. et al., 1993; Cross M.A. et al., 1997; Vorobiev A.I., 2002; Grassinger J. et al., 2006]. It has been established that under extreme conditions (immobilization, administration of cytostatics, neurotropic effects, etc.), erythroid and granulocytic precursors acquire sensitivity to catecholamines and adrenotropic preparations, to which they are capable of responding outside the structures of the hematopoietic microenvironment [Maestroni G.L. et al., 1997; Goldberg E.D. et al., 1997, 2004]. At the same time, in none of the known experiments have the researchers examined the combined influence of hematopoietic growth factors and ligands of adrenergic, dopaminergic, and serotonergic receptors on hematopoietic precursors under pathological conditions.

Another aspect of the problem under consideration is the question of the role of monoamines of the central nervous system (CNS) in the regulation of hematopoiesis. There is information about the possible effector impact of various neurotransmitter systems of the brain (adrenergic, dopaminergic, serotonergic, M-cholinergic, GABA-ergic) on the cellularity of individual hematopoietic buds under neurotropic effects [Goldberg E.D. et al., 2002, 2004]. Activation of central links of the dopaminergic system causes immunostimulation accompanied by accumulation of CD4+ T-cells in the bone marrow, an increase in the number of antibody-forming and rosette-forming cells in the spleen of animals, abolition of stress-induced depression of the immune response, as well as enhancement of the proliferative response of T-lymphocytes to mitogens [Devoino L.V., Ilyuchenok R.Yu., 1993; Idova G.V., 1994; Tao S.Zh. et al., 1997; Idova G.V. et al., 2001]. However, the presented literature data do not allow the formation of a clear picture of the regulatory influence of central monoamines on the pool of committed hematopoietic precursor cells, as well as the function of the HIM under normal and various pathological conditions (including cytostatic hemodepressions).

Taking into account the above, the study of the role of central and peripheral monoamines in the development of myelosuppression and subsequent regeneration of hematopoietic tissue under cytostatic effects, as well as the features of adrenergic, dopaminergic, and serotonergic control of the processes of proliferation and differentiation of hematopoietic precursors and functional activity of the hematopoiesis-inducing microenvironment, is of undoubted interest. Research in this context represents not only theoretical but also practical interest, since the obtained results will serve as the basis for the development of new original methods of correction of hematological disorders under myelosuppressions caused by the administration of antitumor preparations.

Goal of the research: to study the mechanisms of regulatory influence of the adrenergic, dopaminergic, and serotonergic systems on hematopoietic tissue under cytostatic myelosuppressions.

Tasks of the research:

1. To assess the participation of the adrenergic, dopaminergic, and serotonergic systems in the development of disorders of the blood system under conditions of administration of cyclophosphamide and 5-fluorouracil.

2. To investigate the features of monaminergic regulation of division and maturation of hematopoietic cell precursors upon prescription of cytostatics.

3. To reveal the mechanisms specific to the adrenergic, dopaminergic, and serotonergic systems for the restoration of functional activity of elements of the hematopoiesis-inducing microenvironment under modeling of cytostatic myelosuppressions.

4. To assess the contribution of monaminergic systems to the realization of regulatory effects of hemopoietins (erythropoietin, G-CSF) under conditions of administration of cyclophosphamide and 5-fluorouracil.

5. To develop pathogenetically justified methods of correction of disorders in the erythroid and granulocytic hematopoietic buds under cytostatic effects, based on modulation of the activity of central and peripheral monaminergic structures.

Propositions to be defended:

1. Under hypoplastic conditions modeled by administration of an alkylating agent (cyclophosphamide) or a fluoropyrimidine antimetabolite (5-fluorouracil) at 1/3 MPD, proliferation and differentiation of committed hematopoietic precursors and functional activity of cellular elements of the HIM, the CSF system, and erythropoietin are under the control of the adrenergic, dopaminergic, and serotonergic systems. In this case, the serotonergic system is to a greater extent responsible for changes on the side of the erythron, while the adrenergic and dopaminergic systems are responsible for changes in the granulocytic hematopoietic bud.

2. The main causes of various tempos and nature of regeneration of bone marrow tissue upon prescription of an alkylating agent and a fluoropyrimidine antimetabolite are the specific activity of central adrenergic, dopaminergic, and serotonergic structures and features of their interaction with hematopoietic precursors and cells of the HIM, as well as the selective sensitivity of erythropoietin and G-CSF to the regulatory influence of monoamines.

3. Under conditions of cyclophosphamide administration, the inhibitory action of the adrenergic system on the granulocytic and erythroid hematopoietic buds is mediated by depression of the functional activity of adhering cells of the HIM (formation of HIM, production of EPA) and a decrease in the rate of division of granulomonocytic precursors (associated with G-CSF and peripheral adrenergic mechanisms). Acceleration of hematopoietic tissue regeneration by the adrenergic system upon prescription of 5-fluorouracil is due to the formation of HIM, production of EPA by non-adhering cells of the HIM, and an increase in the level of CSA in the blood serum. In this case, stimulation of the proliferative activity of hematopoietic precursors is due to G-CSF, erythropoietin, and alpha- and beta-adrenergic structures.

4. The dopaminergic system deepens disorders of the structural-functional organization of the predominantly erythroid compartment of hematopoiesis caused by the alkylating agent, which leads to a delay in erythropoiesis regeneration and a more pronounced reticulocytopenia in the peripheral blood. At the same time, stimulation of G-CSF- and dopamine-dependent mechanisms of control of proliferation of granulomonocytic precursors is observed, preventing the development of neutropenia.

5. Upon prescription of 5-fluorouracil, the dopaminergic system, on the one hand, increases the rate of erythron regeneration (due to an increase in the serum EPA level and erythropoietin-dependent activation of proliferation of CFU-E), on the other hand, delays the restoration of the granulocytic hematopoietic bud (due to a decrease in CSA production by adhering cells of the HIM).

6. Under cytostatic effects, the serotonergic system aggravates the development of depression of erythropoiesis: under conditions of administration of an alkylating agent, this is associated with depression of the formation of erythroid HIM and functional activity of erythroid precursors (mediated by the erythropoietin system) and a decrease in the level of EPA in biologically active media; upon prescription of a fluoropyrimidine antimetabolite — with a decrease in EPA secretion by cells of the adhering fraction of the HIM. At the same time, upon use of 5-fluorouracil, serotonin of the CNS increases the rate of regeneration of the granulocytic hematopoietic bud by enhancing the formation of granulocytic and mixed HIM and stimulating the CSF-mediated division and maturation of granulomonocytic precursors.

7. Under conditions of cytostatic myelosuppression caused by administration of cyclophosphamide, depletion of catecholamine depots by reserpine significantly increases the granulopoiesis-stimulating activity of G-CSF due to the organization in the bone marrow of de novo granulocytic and mixed HIM. Disruption of serotonergic mediation in the CNS by cyproheptadine has a potentiating effect on the erythropoiesis-stimulating effects of erythropoietin largely due to the restoration of the antimetabolite-disrupted structural-functional organization of the erythroid compartment of hematopoiesis.

Scientific novelty. In the work, for the first time in a comparative aspect, the role of the adrenergic, dopaminergic, and serotonergic systems in the development of myelosuppression and regeneration of hematopoietic tissue was studied on models of cytostatic effects caused by an alkylating agent and an antimetabolite. It has been demonstrated that the regulatory effects of central monaminergic systems are realized through adrenergic, dopaminergic, and serotonergic structures on committed hematopoietic precursors and cellular elements of the HIM, as well as indirectly — through the erythropoietin and G-CSF systems.

It has been revealed that under cytostatic effects, the sensitivity of hematopoietic precursors to the direct action of monaminergic ligands increases, however, in the dynamics of development of the hypoplastic state, not only stimulation of division and maturation of CFU-E but also inhibition of these processes is traced. In vitro, it has been shown that N-acetylneuraminic acid increases the binding capacity of adhering cells of the HIM, subjected to the action of an alkylating agent, with respect to pretreated with isadrinine or serotonin erythropoiesis precursors.

Depletion of catecholamine depots (reserpine) prevents the development of depression of bone marrow granulopoiesis and accelerates the restoration of hematopoietic tissue after administration of cyclophosphamide, which is predominantly associated with the formation of de novo hematopoietic islands in the bone marrow and production of EPA by adhering nucleated cells. At the same time, the coupled with G-CSF (inhibition period) and adrenergic structures (regeneration period) division and maturation of granulomonocytic precursors increase, as well as erythropoietin (inhibition period) — and catecholamine (1st–7th days) — dependent differentiation of erythroid cells. Inhibition of hematopoiesis by a sympatholytic under conditions of antimetabolite administration is caused, first of all, by disorders of HIM functions (formation of HIM and secretion of EPA by non-adhering myelocardiocytes) and a decrease in the serum CSA level, and, secondarily, by depression of the functional activity of hematopoietic precursors mediated by hemopoietins and catecholamines (inhibition period) and beta-adrenergic structures, erythropoietin (regeneration period).

Pharmacological blockade of postsynaptic dopamine D2 receptors (haloperidol) aggravates hypoplasia of granulopoiesis under conditions of administration of an alkylating agent, which is due to destruction of HIM of granulocytic and erythroid-granulocytic types and reduction of the pool of proliferating CFU-GM. In contrast to this, haloperidol, by increasing the functional activity of adhering cells, the level of EPA in the blood serum, and the intensity of differentiation of CFU-E, increases the rate of erythropoiesis regeneration. Upon prescription of an antimetabolite, the basis of haloperidol-stimulated granulopoiesis lies in the increase in CSA secretion by adhering cells of the HIM and the high proliferative activity of granulomonocytopoiesis precursors mediated by peripheral dopaminergic mechanisms and G-CSF, while inhibition of the erythron is associated with depression of division and maturation of erythroid cells mediated by erythropoietin and dopaminergic structures.

Disruption of serotonergic mediation in the CNS by cyproheptadine increases the level of EPA in the blood serum and in conditional media of adhering myelocardiocytes during the inhibition period and depresses peripheral serotonergic mechanisms of regulation of erythroid precursors during the period of restoration of hematopoietic tissue upon administration of cyclophosphamide, which correspondingly affects the content of morphologically recognizable erythroid cells in the blood system (increase in their number during the depression of the erythron and decrease — during the regeneration period). Upon administration of 5-fluorouracil, the main condition for acceleration of hematopoiesis regeneration by an antiserotonergic drug (predominantly the erythroid hematopoietic bud) is the stimulation of formation of de novo erythroid and granulocytic HIM, as well as serotonergic and cytokine mechanisms of control of proliferation of granulomonocytic precursors.

For the first time it has been demonstrated that under conditions of cyclophosphamide administration, a decrease in catecholamine depots potentiates the granulopoiesis-stimulating effect of G-CSF, and pharmacological blockade of postsynaptic serotonergic 5-HT2 receptors enhances the erythropoiesis-stimulating effect of erythropoietin upon prescription of 5-fluorouracil, which is associated with the ahead-of-schedule restoration of the structural-functional organization of the bone marrow (formation of HIM).

Under conditions of optimal vital activity of the organism (intact animals) in vitro, stimulation by dopamine of colony growth of predominantly granulomonocytic type, by serotonin — erythroid type, was established.

Depletion in vivo of catecholamine depots leads to a decrease in the output of CFU-GM under the action of G-CSF, mesaton, and isadrinine, but stimulates in vitro formation of CFU-E in culture with erythropoietin and adrenomimetics. Disruption of dopamine mediation in the CNS selectively decreases the number of granulocyte-macrophage colonies (in vitro — dopamine), pharmacological blockade of postsynaptic serotonergic 5-HT2 receptors of the brain depresses erythroid colony formation (in vitro — serotonin).

Practical significance of the work. The important role of the adrenergic, dopaminergic, and serotonergic systems in the development of myelosuppression and regeneration of bone marrow tissue under cytostatic effects has been demonstrated, the bases of the regulatory influence of central monaminergic structures on committed hematopoietic cells and the hematopoiesis-inducing microenvironment have been revealed. The obtained data make a substantial contribution to the understanding of specific monaminergic mechanisms of control of erythropoiesis and granulopoiesis under modeling of myelosuppression by administration of cytostatics with different mechanisms of action.

The results of the experimental research provide an opportunity to determine pathogenetically justified methods of pharmacological correction of hypoplastic states of hematopoiesis using drugs that modulate the activity of central and peripheral monaminergic structures.

Materials of the preclinical study of the granulocyte colony-stimulating factor drug Neustimra, developed by the Research Institute of Pharmacology of the SB RAMN jointly with FGUN GC VB "Vector" (Novosibirsk), have been submitted to the Pharmacological Committee of the Ministry of Health and the Russian Federation; permission for its clinical use and production has been obtained (Registration No. LR-010185/08).

Defense of the work. Materials of the dissertation work were reported and discussed at scientific conferences of the Research Institute of Pharmacology of the Tomsk Scientific Center of the SB RAMN: "Actual Problems of Pharmacology and Pharmacotherapy" (Tomsk, 1996), conferences of pharmacologists of Siberia and the Far East dedicated to the 15th anniversary of the Research Institute of Pharmacology of the TNC SB RAMN (Tomsk, 1999), "Actual Problems of Experimental and Clinical Pharmacology" (Tomsk, 2001, 2002), "Actual Problems of Pharmacology" (Tomsk, 2004); at the V, VI, VIII, IX Russian National Congress "Man and Medicine" (Moscow, 1998, 1999, 2001, 2002), at the conference of young scientists of the SB RAMN "Fundamental and Applied Problems of Modern Medicine" (Novosibirsk, 2000); at the International Scientific Conference "Search, Development and Implementation of New Drugs and Organizational Forms of Pharmaceutical Activity" (Tomsk, 2000); at the XII International Conference on Space Biology and Aviation-Cosmic Medicine (Moscow, 2002); at the conference "Actual Questions of Experimental and Clinical Morphology" (Tomsk, 2002); at the 4th Congress of Physiologists of Siberia (Novosibirsk, 2002); at the XIX Congress of the Physiological Society named after I.P. Pavlov (Yekaterinburg, 2004); at the conference "Pharmacological Regulation of Stem Cell" (Tomsk, 2005); at the 4th International Conference "Biological Foundations of Individual Sensitivity to Psychotropic Substances" (Moscow, 2006); at the Republican Scientific Conference "Creation of New Drugs" (Tomsk, 2007); at the conference "Problems of Oncopharmacology" (Tomsk, 2008).

Publications. 60 papers on the dissertation topic have been published, including 21 in journals recommended by the "List" of the VAK of the Ministry of Education and Science of Russia.

Patent obtained. Patent (1Sh) No. 2317541 "Method for studying hematopoiesis taking into account the features of higher nervous activity" (published 20.02.2008, Bull. No. 5); decision on granting a patent (1Sh) "Agent preventing the development of depression of the erythroid hematopoietic bud under cytostatic myelodepressions" (No. 2007137872 dated 12.09.2008) has been obtained.

Volume and structure of the dissertation. The dissertation is presented on 492 pages of typed text, illustrated with 39 figures, 147 tables, and consists of an introduction, 4 chapters, conclusions, a list of cited literature including 650 sources, of which 222 domestic and 428 foreign.