**2. Secretory-mediatory hormone-dependent functions of eye membranes in the mechanisms of glaucoma development**

#### **2.1. The role of transforming growth factor β-2 (TgFβ-2), insulin-like growth factor-1 (IgF-1) and Е2 prostaglandins (PgE2) in pathogenesis of primary open-angle glaucoma**

Nowadays the role of TGF<sup>β</sup> produced in post-barrier membranes of an eye is considered to be of no less importance for realization of processes ensuring the drainage function of the eye anterior chamber-associated immune deviation (ACAID) *[Mansfield K. et al., 2004; Banh A. et al., 2006; Kim Y.S. et al., 2008; Dawes L.J. et al., 2009*]. According to scientific publications, TGFβ-2 produced in the post-barrier membrane of an eye (in cornea, ciliary body, retina) at some eye diseases takes an active part in the increase of intraocular pressure [*de Iongh R.U. et al., 2005; Stefan C. et al., 2008; Dawes L.J. et al., 2009; Hindman H.B. et al., 2010; Pattabiraman P.P, Rao P.V., 2010*].

To our mind, during the last years rather informative data signifying in favour of pleotropic potencies of TGFβ-2 produced in post-barrier membranes of the eye.

Heredity, general type changes in specific integrative systems of the organism (CNS, endo‐ crine, immune and cardiovascular) are among the general factors bringing forth disorders of

Amongst the local factors relatively persistent elevation of intraocular pressure, primary dystrophic and atrophic changes, including age-related shifts in the cornea, ciliary body and the trabecular meshwork, which cause the infringement of hydrodynamic and hydrostatic

As mentioned by A.P. Nesterov (2008) chronologically occurring processes, which might be conditionally subdivided into 2 stages, are engaged in the pathogenesis of glaucomas in anterior and posterior chambers of an eye. At the first stage mechanisms bringing forth the increase of intraocular pressure are triggered in the anterior chamber of an eye. At the second stage mechanisms localized in the posterior part of the eye chamber are initiated and in the long run become the cause of atrophy of the visual nerve. At that, the "glaucomatous process" firstly originates in the anterior chamber of an eye, while the dystrophic and atrophic processes

During the last years, rather informative evidences were obtained to discuss the role of biologically active substances produced *in situ*, i.e. in specific eye membranes, in mechanisms of anterior open-angle glaucoma origination using the clinical and experimental material.

We did not set the problem to analyze the current state of the art on the role of general pathogenetic factors engaged in induction and the course of anterior open-angle glaucoma.

The currently available data of scientific publications and results of our own investigations devoted to the role of *in situ* produced biologically active substances of cytokine, mediatory and hormonal origin in mechanisms of a stable increase of the intraocular pressure in case of

**2. Secretory-mediatory hormone-dependent functions of eye membranes**

**2.1. The role of transforming growth factor β-2 (TgFβ-2), insulin-like growth factor-1 (IgF-1)**

Nowadays the role of TGF<sup>β</sup> produced in post-barrier membranes of an eye is considered to be of no less importance for realization of processes ensuring the drainage function of the eye anterior chamber-associated immune deviation (ACAID) *[Mansfield K. et al., 2004; Banh A. et al., 2006; Kim Y.S. et al., 2008; Dawes L.J. et al., 2009*]. According to scientific publications, TGFβ-2 produced in the post-barrier membrane of an eye (in cornea, ciliary body, retina) at some eye diseases takes an active part in the increase of intraocular pressure [*de Iongh R.U. et al., 2005; Stefan C. et al., 2008; Dawes L.J. et al., 2009; Hindman H.B. et al., 2010; Pattabiraman P.P,*

**and Е2 prostaglandins (PgE2) in pathogenesis of primary open-angle glaucoma**

in the visual nerve are resulting from the exposure to high intraocular pressure.

anterior open-angle glaucoma will be analyzed in this work.

**in the mechanisms of glaucoma development**

*Rao P.V., 2010*].

the hematoophthalmic barrier and the increase of intraocular pressure.

properties of the aqueous humour, are considered.

76 Glaucoma - Basic and Clinical Aspects

In particular, in a post-surgery period in patients operated for complicated and senile cataracts *in situ* produced TGFβ-2 induces trans-differentiation of epithelial cells of crystalline lens capsule into fibroblasts; this latter was manifested as opacity of lens with all the subsequent after-effects [*Dawes L.J. et al., 2009*]. The modulatory effect of TGFβ-1 towards the processes of activation of cells of fibroblastic line in the cornea was also established. Thus, the authors [*Karamichos D. et al., 2010*] under conditions of cultivating cells of cornea using TGFβ-1 dosedependent mode activated *in situ* synthetic processes in fibroblasts, thus bringing forth intensification of collagen(ous) fibrilles synthesis and eventually to regional overgrowth of immature connective tissue with the resulting fibrosis.

TGFβ-2 high level was also revealed in cells of the trabecular meshwork of patients with openangle glaucoma [*Stefan C. et al., 2008*]. The authors consider that at the mentioned disease TGFβ-2 stimulates fibronectin synthesis in trabecular cells, thus predefining "profibrotic" effects of TGFβ-2 in post-barrier membranes of the eye.

Literature data is available [*Ochiai Y., Ochiai H., 2002*], according to which in patients with anterior open-angle glaucoma, diabetes complicated by anterior open-angle glaucoma the level of TGFβ-2 in aqueous / intraocular humor is markedly increased. As a control, the authors studied aqueous humour of patients with cataracts.

Processes reflecting the specific precise stages of TGFβ-2 and IGF-1 activity in post-barrier membranes of the eye are the subject of a wide discussion. Furthermore, the study on mech‐ anisms of their direct and/or mediated interaction in processes ensuring the drainage function of an eye is mainly emphasized.

In the organism of mammals, the post-barrier membranes of an eye also serve as a source of both cytocines. IGF-1 and its receptors, IGF-IR, were found in epitheliocytes of lens and cornea, epitheliocytes of retina meshwork, Muller's cells [*Shaw L.C. et al., 2006; Ko J.A. et al., 2009*]. TGFβ-2 is produced in post-barrier membranes of an eye and, first of all, in fibroblasts of cornea [*Streilein J. et al., 1992; Wilkbanks G. et al., 1992; Hollborn M. et al., 2000; Fleenor D. et al., 2006*].

According to C. Stefan et al. (2008), cells of the trabecular meshwork of the anterior angle of the eye chamber might serve as the source of TGFβ-2 synthesis.

S.H. Chung and associates used human lens epithelial cells (HLE B-3) to reveal the role of IGF-1 in processes of TGFβ-2 mediated fibronectin accumulation in lens cells [*Chung S.H. et al., 2007*]. Based on analysis performed by the authors (reverse polymerase transcriptase chain reaction, immune-fluorescent studies) mentioned researchers draw a conclusion that IGF-1 counteracts TGFβ-2 induced fibronectin accumulation in lens epitheliocytes.

J.A. Ko et al. (2009) studied the role of IGF-1 in intrercellular regulation in cultured fibroblasts and human corneal epitheliocytes. According to authors, the presence of epitheliocytes in the culture medium enhanced N-cadherin expression in fibroblasts. Similar effect of corneal epitheliocytes was also simulated by IGF-1, but not fibroblasts growth factor or epidermal growth factor. The authors conclude that IGF-1 produced in epitheliocytes regulates Ncadherin positive expression in corneal fibroblasts.

prostaglandins functioning in post-barrier membranes of the eye are realized exceptionally

Modern Aspects of Glaucoma Pathogenesis Local Factors for Development of Primary Open-Angle Glaucoma…

http://dx.doi.org/10.5772/55037

79

Taking into account the abovementioned, an assumption might be proposed according to which *in situ* produced Е<sup>2</sup> prostaglandins are engaged into the pathological process observed

Fibronectins are a group of cold-insoluble glycoproteids with the molecular mass 400.000– 450.000 D localized both on the surface of connective tissue cells and in the extracellular matrix. The following functionally active domains were revealed in fibronectin structure: NH2 terminal domain includes sites of fibrin binding; then collagen and heparin binding domains and domain ensuring cells adhesion are localized; at COOOH-cone there is one more heparin binding domain [*Kuznik B. et al., 1989*]. Fibronectins have an important part in cells prolifera‐ tion and differentiation, morphogenesis and embryogenesis of tissues. In particular, tissue fibronectin of fibroblastic genesis actively participates in collagen formation both at norm (at the stage-by-stage process of the connective tissue maturation), and in pathology state (different distrophic and inflammatory processes occurring at the site of the connective tissue. Thus, in particular, an important role is assigned to fibronectin in reparative processes, influence on cells migration, growth and proliferation. Fibronectin produced by cells of the connective tissue (fibroblasts, endotheliocytes, smooth-muscle cells of arterioles, etc.) in its turn actively participates in formation of the extra-cellular matrix, especially at early stages of the connective tissue formation. The following cells of different genesis serve as the main source of fibronectin synthesis: endothelium, hepatocytes, fibroblasts, smooth myocytes, Schwann's cells, alveolar and peritoneal macrophages, epitheliocytes, and thrombocytes

Currently the sources of fibronectin synthesis in the post-barrier membranes of an eye are disputable as well. According to H.B. Hindman et al. (2010), corneal keratocytes might be considered as probable sources of fibronectin synthesis. This fact was revealed under cultiva‐ tion of keratocytes localized in the anterior and posterior parts of cornea. Furthermore, in the process of cultivating corneal cells of fibroblastic line the authors established TGFβ-1-dependent activation of keratocytes, in which a marked activation of fibronectin synthesis occurred. Simultaneously, Thy-1 secretion increases in the same keratocytes. According to D. Karami‐ chos et al. (2010), *in situ* (in post-barrier membranes of an eye) produced TGFβ-2, under conditions of pathology might serve as a provoking factor binging forth fibrosis of the cornea. Therefore, we cannot exclude that realization of this TGFβ-2 related effect is mediated due to

C. Stefan et al. (2008) use the immune enzyme assay to determine TGFβ-2 and fibronectin concentration in aqueous humour of patients with anterior open-angle glaucoma. The authors revealed a significant increase of TGFβ-2 concentration in this cohort of patients and draw a conclusion that TGFβ-2 produced in post-barrier membranes of the eye should be considered as a "special" cytokine that increases fibronectin concentration in the trabecular meshwork;

at the area of post-barrier membranes of an eye at primary open-angle glaucoma.

**2.2. The role of fibronectin in pathogenesis of primary open-angle glaucoma**

according to the receptor mechanism.

[*Kuznik B. et al., 1989*].

the activation of fibronectin in the same keratocytes.

moreover, it might be considered as a local pro-fibrotic factor.

There is an opinion that IGF-1 and IGF-2 regulate the processes of proliferation and apoptosis in corneal epitheliocytes [*Yanai R. et al., 2006*]. According to K. Izumi and co-workers (2006), TGFβ produces an influence to corneal fibroblasts differentiation into myofibroblasts. More‐ over, IGF-1 is engaged in this mechanism. Thus, treatment with TGFβ-2 caused expression of IGF-1, mRNA, IGF ВР-3 and IGF ВR-3 protein in human corneal. According to N. Yamada et al. (2005), IGF-1, alongside with fibronectin, IL-6 and substance P, actively participate in stimulation of fibroblastic processes in cornea.

The analysis of above mentioned scientific publications signifies to the important role of *in situ* produced TGFβ-2 and IGF-1 in mechanisms of fibroblastic processes formation and their cellular metaplasia in specific eye membranes: in post-barrier eye membranes in ACAID mechanisms and withdrawal. One cannot exclude that locally produced cytokines possess short-distant range of activity; moreover, their realization might occur according to either the principles of intercellular interaction, i.e. through the paracrine mechanism, or on the basis of intercellular autocrine regulation. Apparently, both mechanisms have an important part in infringement of drainage function of an eye at different types of glaucoma.

As known, prostaglandins play an important role in integrative activity of the mammalian organism, in particular, in regulation of immunogenesis, hemostasis, non-specific resistance at the organism level [*Kuznik B. et al., 1989*].

However, until present the probability of prostaglandins synthesis in post-barrier membranes of an eye seems still disputable.

There are only sporadic communications related to the mentioned aspect; an attempt was made to reveal Е1, Е2 and F2α prostaglandins in post-barrier membranes of an eye and in the aqueous humour.

It is considered established that prostaglandins increase intraocular pressure and infringe the function of hematoophthalmic barrier [*Podos S.M. et al., 1972 a; b; Podos S.M., 1976a ; b; c*]. Moreover, the drainage function of an eye is simultaneously realized by prostaglandins in the aqueous humour.

According to C.B. Toris and associates, numerous prostaglandin-dependent effects in postbarrier membranes of an eye are realized according to the receptor mechanism associated at the level of mRNAs [*Toris C.B. et al., 2008*]. Similar receptors were revealed in the trabecular meshwork, celiar muscle, and sclera.

Prostaglandin-dependent receptors in post-barrier eye membranes were revealed not only in humans, but also in rats, mice, rabbits, pigs and monkeys. Considering the vasoactive properties of prostaglandins, as well as their role in sustaining the drainage function of an eye, the sysnthetic analogs of prostaglandins are widely applied in ophthalmological practice for treatment of glaucoma [*Bucci F.A., Waterbury L.D., 2008; Toris C.B. et al., 2008*].

Therefore, it is no excluded that Е2 prostaglandins might participate in maintenance of the drainage function and, appropriately, the intraocular pressure as well; similar mechanisms of prostaglandins functioning in post-barrier membranes of the eye are realized exceptionally according to the receptor mechanism.

Taking into account the abovementioned, an assumption might be proposed according to which *in situ* produced Е<sup>2</sup> prostaglandins are engaged into the pathological process observed at the area of post-barrier membranes of an eye at primary open-angle glaucoma.

#### **2.2. The role of fibronectin in pathogenesis of primary open-angle glaucoma**

growth factor. The authors conclude that IGF-1 produced in epitheliocytes regulates N-

There is an opinion that IGF-1 and IGF-2 regulate the processes of proliferation and apoptosis in corneal epitheliocytes [*Yanai R. et al., 2006*]. According to K. Izumi and co-workers (2006), TGFβ produces an influence to corneal fibroblasts differentiation into myofibroblasts. More‐ over, IGF-1 is engaged in this mechanism. Thus, treatment with TGFβ-2 caused expression of IGF-1, mRNA, IGF ВР-3 and IGF ВR-3 protein in human corneal. According to N. Yamada et al. (2005), IGF-1, alongside with fibronectin, IL-6 and substance P, actively participate in

The analysis of above mentioned scientific publications signifies to the important role of *in situ* produced TGFβ-2 and IGF-1 in mechanisms of fibroblastic processes formation and their cellular metaplasia in specific eye membranes: in post-barrier eye membranes in ACAID mechanisms and withdrawal. One cannot exclude that locally produced cytokines possess short-distant range of activity; moreover, their realization might occur according to either the principles of intercellular interaction, i.e. through the paracrine mechanism, or on the basis of intercellular autocrine regulation. Apparently, both mechanisms have an important part in

As known, prostaglandins play an important role in integrative activity of the mammalian organism, in particular, in regulation of immunogenesis, hemostasis, non-specific resistance

However, until present the probability of prostaglandins synthesis in post-barrier membranes

There are only sporadic communications related to the mentioned aspect; an attempt was made to reveal Е1, Е2 and F2α prostaglandins in post-barrier membranes of an eye and in the aqueous

It is considered established that prostaglandins increase intraocular pressure and infringe the function of hematoophthalmic barrier [*Podos S.M. et al., 1972 a; b; Podos S.M., 1976a ; b; c*]. Moreover, the drainage function of an eye is simultaneously realized by prostaglandins in the

According to C.B. Toris and associates, numerous prostaglandin-dependent effects in postbarrier membranes of an eye are realized according to the receptor mechanism associated at the level of mRNAs [*Toris C.B. et al., 2008*]. Similar receptors were revealed in the trabecular

Prostaglandin-dependent receptors in post-barrier eye membranes were revealed not only in humans, but also in rats, mice, rabbits, pigs and monkeys. Considering the vasoactive properties of prostaglandins, as well as their role in sustaining the drainage function of an eye, the sysnthetic analogs of prostaglandins are widely applied in ophthalmological practice for

Therefore, it is no excluded that Е2 prostaglandins might participate in maintenance of the drainage function and, appropriately, the intraocular pressure as well; similar mechanisms of

treatment of glaucoma [*Bucci F.A., Waterbury L.D., 2008; Toris C.B. et al., 2008*].

infringement of drainage function of an eye at different types of glaucoma.

cadherin positive expression in corneal fibroblasts.

78 Glaucoma - Basic and Clinical Aspects

stimulation of fibroblastic processes in cornea.

at the organism level [*Kuznik B. et al., 1989*].

of an eye seems still disputable.

meshwork, celiar muscle, and sclera.

humour.

aqueous humour.

Fibronectins are a group of cold-insoluble glycoproteids with the molecular mass 400.000– 450.000 D localized both on the surface of connective tissue cells and in the extracellular matrix. The following functionally active domains were revealed in fibronectin structure: NH2 terminal domain includes sites of fibrin binding; then collagen and heparin binding domains and domain ensuring cells adhesion are localized; at COOOH-cone there is one more heparin binding domain [*Kuznik B. et al., 1989*]. Fibronectins have an important part in cells prolifera‐ tion and differentiation, morphogenesis and embryogenesis of tissues. In particular, tissue fibronectin of fibroblastic genesis actively participates in collagen formation both at norm (at the stage-by-stage process of the connective tissue maturation), and in pathology state (different distrophic and inflammatory processes occurring at the site of the connective tissue. Thus, in particular, an important role is assigned to fibronectin in reparative processes, influence on cells migration, growth and proliferation. Fibronectin produced by cells of the connective tissue (fibroblasts, endotheliocytes, smooth-muscle cells of arterioles, etc.) in its turn actively participates in formation of the extra-cellular matrix, especially at early stages of the connective tissue formation. The following cells of different genesis serve as the main source of fibronectin synthesis: endothelium, hepatocytes, fibroblasts, smooth myocytes, Schwann's cells, alveolar and peritoneal macrophages, epitheliocytes, and thrombocytes [*Kuznik B. et al., 1989*].

Currently the sources of fibronectin synthesis in the post-barrier membranes of an eye are disputable as well. According to H.B. Hindman et al. (2010), corneal keratocytes might be considered as probable sources of fibronectin synthesis. This fact was revealed under cultiva‐ tion of keratocytes localized in the anterior and posterior parts of cornea. Furthermore, in the process of cultivating corneal cells of fibroblastic line the authors established TGFβ-1-dependent activation of keratocytes, in which a marked activation of fibronectin synthesis occurred. Simultaneously, Thy-1 secretion increases in the same keratocytes. According to D. Karami‐ chos et al. (2010), *in situ* (in post-barrier membranes of an eye) produced TGFβ-2, under conditions of pathology might serve as a provoking factor binging forth fibrosis of the cornea. Therefore, we cannot exclude that realization of this TGFβ-2 related effect is mediated due to the activation of fibronectin in the same keratocytes.

C. Stefan et al. (2008) use the immune enzyme assay to determine TGFβ-2 and fibronectin concentration in aqueous humour of patients with anterior open-angle glaucoma. The authors revealed a significant increase of TGFβ-2 concentration in this cohort of patients and draw a conclusion that TGFβ-2 produced in post-barrier membranes of the eye should be considered as a "special" cytokine that increases fibronectin concentration in the trabecular meshwork; moreover, it might be considered as a local pro-fibrotic factor.

Sporadic, though rather informative, evidences are available according to which the trabecular meshwork localized in the angle of an anterior chamber of the eye serves as the possible source of fibronectin synthesis. In particular, R.J. Wordinger et al. (2007) studied the probable mechanisms of synthesis of the biologically active substances by trabecular meshwork cells. As known, the cells of trabecular meshwork synthesize and excrete "bone morphogenic protein" – BMP-4. The authors, under conditions of trabecular cells cultivation studied the synthetic potencies thereof at addition of BMP-4 and TGFβ-2 to the culture media. The study results demonstrated that TGFβ-2 treated cells of the trabecular meshwork launched an intense synthesisn of fibronectin, while BMP-4, if additionally introduced to TGFβ-2 containing media, blocked this induction of fibronectin.

pressure might be "leveled" by TGFβ-2 -mediated receptors type 1 through prevention of

Modern Aspects of Glaucoma Pathogenesis Local Factors for Development of Primary Open-Angle Glaucoma…

http://dx.doi.org/10.5772/55037

81

According to mentioned authors, understanding these inter-mediatory and receptor interac‐ tions, which occur at the site of trabecular meshwork of an angle of anterior chamber of the eye, would then allow to develop new efficient approaches for treatment of glaucoma.

There is an opinion [*Gonzales J.M. et al., 1998*], according to which it is merely domain of heparin II (Hep II) in the structure of fibronectin that regulates the ability outflow (excretory system) in cultured anterior segments through the effects produced to the cytoskeleton in transformed cells of the trabecular meshwork of the angle of the anterior chamber of an eye. The mentioned authors cultivated cells of the trabecular meshwork under conditions of Hep II domain and revealed an active site of this domain that regulates the ability of aqueous humor efflux. According to researchers, precisely this site of a domain is responsible in case of disorders in

Fibronectin concentration in aqueous (intraocular) humour of patients with cataracts and glaucomas, according to K.S. Kim et al. (1992), widely varies from 5 *ng/ml* to 100 *ng/ml* (data of immune enzyme assay – ELISA). Authors separated the aqueous humor by aspiration from the eyes of patients with cataract and glaucoma using a special puncture needle introduced through the limbal zone before the limbal incision in the anterior chamber of the eye, that is before the surgical intervention. Due to the performed immune enzyme assay the researchers managed to establish that at glaucomas the level of fibronectin significantly increases com‐ pared to its level in aqueous humour of patients with cataracts. At the same time, fibronectin levels in aqueous humor patients with cataract and glaucoma had no dependence on either

The aspects related to fibronectin sources in post-barrier membranes of the eye are also discussed. An assumption was made that at primary glaucomas relatively high concentrations of fibronectin accumulate in the anterior chamber of an eye, as it cannot escape the drainage pathways. There are quite opposite data, according to which in patients with the open-angle glaucoma fibrinogen concentration in the aqueous humor significantly did not differ from that of aqueous humor of patients with cataracts [*Vesaluoma M. et al., 1998*]. At the same time, upon comparison of obtained results of immune enzyme analysis for fibrinogen content on the one hand, in aqueous humor of patients with cataracts and primary glaucomas, and, on the other hand, in patients with exfoliative glaucoma, the level of fibronectin in aqueous humor significantly increased in the latter case. The authors consider that significantly higher concentration of fibronectin in patients with the pseudoexfoliative glaucoma might result from infringement of the hematoophthalmic barrier. There is evidence [*Tripathi B.J. et al., 2004*] that the growth factor (TGFβ-2) under conditions *in vivo* modulates fibronectin and stromelysin-1 (MMP-3) in trabecular cells of the anterior chamber of an eye. Mentioned authors studied expression of RNA and fibronectin protein at presence of growth factors in primary and secondary humour of the anterior chamber (taken in pre- and post-operative period, appro‐ priately). In particular, under conditions of incubation of trabecular cells of the anterior chamber of the eye, growth factor containing aqueous humors taken from patients with glaucoma prior to and post the surgery were added to the culture medium. Compare to control,

TGFβ-2 stimulating effect to cells of the extracellular matrix.

actinic cytoskeleton of the trabecular meshwork at glaucomas.

age or gender of patients under preoperative study.

Mentioned authors studied the expression of BMP-4 family gens in normal and glaucomatous cells of the trabecular meshwork. Under the influence of these receptors the levels of TGFβ-2 and BMP anatagonist, protein gremlin, significantly increased. The authors succeeded to establish that gremlin blocked the negative impact of BMP-4 towards TGFβ-2 induction of fibronectin.

Another result obtained by the same authors is of no less importance: gremlin introduced in the medium *ex vivo*, caused the prototype of increased intraocular pressure glaucoma. Research findings of these authors reflect the main statements of the hypothesis according to which in case of the anterior open-angle glaucoma the enhanced expression of gremlin by trabecular meshwork cells inhibits BMP-4 antagonism to TGFβ-2, eventually, might bring forth the increase in deposition of the extracellular matrix and intraocular pressure.

The analysis of rather informative data obtained by mentioned authors allows to draw a conclusion, according to which the trabecular meshwork of an angle of anterior chamber of the eyes should not considered as an object that passively ensures the drainage function thus sustaining optimally stable levels of the intraocular pressure.

To our mind, the drainage function of trabecular meshwork is an active process and the leading role here belongs to "secretory" cells of the meshwork predominantly functioning according to the autocrine mechanism. At dysfunctions of trabecular meshwork cells, especially in case of open-angle glaucoma, the synchronous activity of these cells is infringed; this latter might enhance their specific medatory function – in view of the increased synthesis of fibronectin. Precisely, fibronectin depositions and the subsequent intensification of fibroblastic processes *in situ* might bring to disorders in drainage function of the trabecular meshwork of the angle of anterior chamber of the eye and, finally to the stable increase of intraocular pressure.

According to D. Fleenor et al. (2006), TGFβ-2 treatment of segments of trabecular meshwork cells of the angle of anterior chamber of the eye resulted in modulation of multiple gens regulating the structure of extracellular matrix. In the trabecular meshwork cells TGFβ-2 brings forth an increased secretion of fibronectin. TGFβ-2 action to cells of the trabecular meshwork was blocked by inhibitors of receptor type 1 TGFβ. In perfusion anterior segments of human eyes TGFβ-2 treatment increased the intraocular pressure and elution of fibronectin. In our opinion the authors come to the rather reasonable conclusion: TGFβ-2 influence on intraocular pressure might be "leveled" by TGFβ-2 -mediated receptors type 1 through prevention of TGFβ-2 stimulating effect to cells of the extracellular matrix.

Sporadic, though rather informative, evidences are available according to which the trabecular meshwork localized in the angle of an anterior chamber of the eye serves as the possible source of fibronectin synthesis. In particular, R.J. Wordinger et al. (2007) studied the probable mechanisms of synthesis of the biologically active substances by trabecular meshwork cells. As known, the cells of trabecular meshwork synthesize and excrete "bone morphogenic protein" – BMP-4. The authors, under conditions of trabecular cells cultivation studied the synthetic potencies thereof at addition of BMP-4 and TGFβ-2 to the culture media. The study results demonstrated that TGFβ-2 treated cells of the trabecular meshwork launched an intense synthesisn of fibronectin, while BMP-4, if additionally introduced to TGFβ-2 containing media,

Mentioned authors studied the expression of BMP-4 family gens in normal and glaucomatous cells of the trabecular meshwork. Under the influence of these receptors the levels of TGFβ-2 and BMP anatagonist, protein gremlin, significantly increased. The authors succeeded to establish that gremlin blocked the negative impact of BMP-4 towards TGFβ-2 induction of

Another result obtained by the same authors is of no less importance: gremlin introduced in the medium *ex vivo*, caused the prototype of increased intraocular pressure glaucoma. Research findings of these authors reflect the main statements of the hypothesis according to which in case of the anterior open-angle glaucoma the enhanced expression of gremlin by trabecular meshwork cells inhibits BMP-4 antagonism to TGFβ-2, eventually, might bring forth the

The analysis of rather informative data obtained by mentioned authors allows to draw a conclusion, according to which the trabecular meshwork of an angle of anterior chamber of the eyes should not considered as an object that passively ensures the drainage function thus

To our mind, the drainage function of trabecular meshwork is an active process and the leading role here belongs to "secretory" cells of the meshwork predominantly functioning according to the autocrine mechanism. At dysfunctions of trabecular meshwork cells, especially in case of open-angle glaucoma, the synchronous activity of these cells is infringed; this latter might enhance their specific medatory function – in view of the increased synthesis of fibronectin. Precisely, fibronectin depositions and the subsequent intensification of fibroblastic processes *in situ* might bring to disorders in drainage function of the trabecular meshwork of the angle of anterior chamber of the eye and, finally to the stable increase of intraocular pressure.

According to D. Fleenor et al. (2006), TGFβ-2 treatment of segments of trabecular meshwork cells of the angle of anterior chamber of the eye resulted in modulation of multiple gens regulating the structure of extracellular matrix. In the trabecular meshwork cells TGFβ-2 brings forth an increased secretion of fibronectin. TGFβ-2 action to cells of the trabecular meshwork was blocked by inhibitors of receptor type 1 TGFβ. In perfusion anterior segments of human eyes TGFβ-2 treatment increased the intraocular pressure and elution of fibronectin. In our opinion the authors come to the rather reasonable conclusion: TGFβ-2 influence on intraocular

increase in deposition of the extracellular matrix and intraocular pressure.

sustaining optimally stable levels of the intraocular pressure.

blocked this induction of fibronectin.

80 Glaucoma - Basic and Clinical Aspects

fibronectin.

According to mentioned authors, understanding these inter-mediatory and receptor interac‐ tions, which occur at the site of trabecular meshwork of an angle of anterior chamber of the eye, would then allow to develop new efficient approaches for treatment of glaucoma.

There is an opinion [*Gonzales J.M. et al., 1998*], according to which it is merely domain of heparin II (Hep II) in the structure of fibronectin that regulates the ability outflow (excretory system) in cultured anterior segments through the effects produced to the cytoskeleton in transformed cells of the trabecular meshwork of the angle of the anterior chamber of an eye. The mentioned authors cultivated cells of the trabecular meshwork under conditions of Hep II domain and revealed an active site of this domain that regulates the ability of aqueous humor efflux. According to researchers, precisely this site of a domain is responsible in case of disorders in actinic cytoskeleton of the trabecular meshwork at glaucomas.

Fibronectin concentration in aqueous (intraocular) humour of patients with cataracts and glaucomas, according to K.S. Kim et al. (1992), widely varies from 5 *ng/ml* to 100 *ng/ml* (data of immune enzyme assay – ELISA). Authors separated the aqueous humor by aspiration from the eyes of patients with cataract and glaucoma using a special puncture needle introduced through the limbal zone before the limbal incision in the anterior chamber of the eye, that is before the surgical intervention. Due to the performed immune enzyme assay the researchers managed to establish that at glaucomas the level of fibronectin significantly increases com‐ pared to its level in aqueous humour of patients with cataracts. At the same time, fibronectin levels in aqueous humor patients with cataract and glaucoma had no dependence on either age or gender of patients under preoperative study.

The aspects related to fibronectin sources in post-barrier membranes of the eye are also discussed. An assumption was made that at primary glaucomas relatively high concentrations of fibronectin accumulate in the anterior chamber of an eye, as it cannot escape the drainage pathways. There are quite opposite data, according to which in patients with the open-angle glaucoma fibrinogen concentration in the aqueous humor significantly did not differ from that of aqueous humor of patients with cataracts [*Vesaluoma M. et al., 1998*]. At the same time, upon comparison of obtained results of immune enzyme analysis for fibrinogen content on the one hand, in aqueous humor of patients with cataracts and primary glaucomas, and, on the other hand, in patients with exfoliative glaucoma, the level of fibronectin in aqueous humor significantly increased in the latter case. The authors consider that significantly higher concentration of fibronectin in patients with the pseudoexfoliative glaucoma might result from infringement of the hematoophthalmic barrier. There is evidence [*Tripathi B.J. et al., 2004*] that the growth factor (TGFβ-2) under conditions *in vivo* modulates fibronectin and stromelysin-1 (MMP-3) in trabecular cells of the anterior chamber of an eye. Mentioned authors studied expression of RNA and fibronectin protein at presence of growth factors in primary and secondary humour of the anterior chamber (taken in pre- and post-operative period, appro‐ priately). In particular, under conditions of incubation of trabecular cells of the anterior chamber of the eye, growth factor containing aqueous humors taken from patients with glaucoma prior to and post the surgery were added to the culture medium. Compare to control, fibronectin mRNA expression by trabecular cells increased by 50 and 100% after incubation in primary samples of aqueous humor during 48 hours or 7 days, as well as by 50 and 160% after incubation in secondary samples of the aqueous humor. MMP-1 mRNA expression decreased by 25 and 50% after incubation in samples of primary aqueous humor during 48 hours or 7 days, as well as by 80 and 85% after incubation during 48 hours or 7 days in secondary samples of aqueous humor. The level of fibronectin increased 3.5 times and 6-fold after incubation during 48 hours with primary and secondary samples of aqueous humor.

terone, estradiol, triiodothyronine and thyroxine, the results of immune enzyme assay were negative even despite their high solubility and relatively small size of their molecules. According to A. Steiger (2003), the role of somatotropin, somatostatin and adrenocorticotropic hormone (ACTH) in the genesis of a wide range of eye diseases with both inflammatory and

Modern Aspects of Glaucoma Pathogenesis Local Factors for Development of Primary Open-Angle Glaucoma…

http://dx.doi.org/10.5772/55037

83

The results obtained by mentioned authors testify in favour of the local synthesis of certain

It should be specially noted that regional neuroendocrine mechanisms underlying the induction of primary open-angle glaucoma have not been sufficiently studied yet. In this aspect the role of *in situ* produced cortisol in mechanisms of impaired ion exchange is exceptionally connected with disbalance of sodium ions transport between the cells and liquid media of an

As known, in peripheral "epithelial" tissues sodium and water transport are regulated by corticosteroids, 11-β-hydroxysteroid-dehydrogenase (11-β-HSD), its isoform (11-β-HSD1), due to which there occurs formation of cortisol molecule from cortisone. Considering this latter, some researchers [*Rauz S. et al., 2003*] determined levels of cortisol, cortisone, 11-β-HSD and 11-β-HSD1 in ciliary body of actually healthy volunteers. The study was aimed to reveal the role of cortisol and 11-β-HSD in regulation of intraocular pressure that is sustained due to balance of aqueous humour (intraocular liquid) depending on the sodium transport through the ciliated epithelium and drainage via the trabecular meshwork. In both study groups cortisol concentrations were higher than cortisone levels. In both groups oral application of carbenoxolone, 11-β-HSD inhibitor, was accompanied by a marked decrease of intraocular pressure. To our mind, data obtained by mentioned authors, on the one hand, signify in favour of the above-mentioned cascade of reactions for maintenance of intraocular pressure, on the

other hand, in favour of cortisol local synthesis in post-barrier membranes of the eye.

There is an opinion, according to which merely 11-β-HSD1 ensures receptor Nf-dependent mechanisms through the ciliated epithelium, thus regulating the level of intraocular pressure [*Rauz S. et al., 2001*]. Mentioned authors revealed the fine mechanisms, which provide the level of glucocorticoids mediated intraocular pressure. However, the potentiating role of cortico‐ steroids in regulation of intraocular pressure was revealed much earlier [*Jacob E. et al., 1996*]. As known, the rate of aqueous humour production is stimulated by adrenalin. The authors studied the joint and isolated effects of adrenalin and hydrocortisone to the rate of aqueous humor production in 20 volunteers. As demonstrated by study results, joint oral application of adrenaline and hydrocortisone significantly (by 42%) enhanced production of aqueous humour compared to placebo. The authors consider that both factors simultaneously function within the post-barrier membranes of the eye (ciliary body), thus ensuring the rate of aqueous

Molecular mechanisms underlying the biological action of glucocorticosteroids in eye mem‐ branes were also studied. Specifically, in the experiment, under conditions of cornea trans‐ plantation the influence of glucocorticosteroids (SEGRA) was studied to the labeled synthesis of anti- and pro-inflammatory cytokines. The application of glucocorticosteroids brought forth

degenerative genesis is also disputable.

hormones in eye membranes and tissues.

eye and the impaired catecholamines exchange.

humour production.

Study results obtained by the abovementioned authors allow to draw a conclusion that induction of MMP-3 in the trabecular meshwork of glaucomatous eyes might decrease fibronectin formation in aqueous humor excretion pathways, thus decreasing the resistance of liquid outflow into the anterior chamber of an eye.

The analysis of publications relevant to the role of *in situ* produced fibronectin in post-barrier membrane of an eye allows to come the following conclusions.

*Firstly,* the role of *in situ* produced fibronectin in mechanisms on sustaining the local homeo‐ stasis remains debatable.

*Secondly*, the available scientific literature indicates to the fact that under conditions of norm fibronectin produced by cells of the trabecular meshwork performs the drainage function in outflow of the aqueous humor.

*Thirdly*, at some eye diseases and especially at primary open-angle glaucoma and pseudoex‐ foliative syndrome, the excessive synthesis of fibronectin by cells of the trabecular meshwork might bring forth a disorder of the drainage function that eventually in its turn is fraught with the increase of intraocular pressure.

*Fourthly*, it is not excluded that in post-barrier membranes of the eye there are engaged fibronectin-dependent mechanisms, which function according to both principles of intercellular interactions and the autocrine mechanism.

#### **2.3. The role of cortisol in pathogenesis of anterior open-angle glaucoma**

At present, aspects related to studies on "endocrine homeostasis" in post-barrier membrane of an eye at both norm and pathology are the subject of a wide discussion in ophthalmology. The available publications are not numerous; furthermore, they are of a rather statement-ofthe-fact character [*Southren A. et al., 1976; Floman N., Zor U., 1977; Kasavina B. et al., 1977; Weinstein B. et al., 1983; Stone R., Wilson C., 1984; Stojek A. et al., 1991; Chiquet C., Denis P., 2004; Burch J. et al., 2005; Pleyer U. et al., 2005; Schwartz B. et al., 2005; Vessey K. et al., 2005*]. In particular, there are reports discussing the possibility of cortisol local synthesis in eye mem‐ branes.

The autopsy material (vitreous body and blood serum of healthy subjects with fatal injury) was subject to immune enzyme assay for determination of progesterone, estradiol, thyroxine, triiodothyronine, thyrotropic hormone, luteinizing hormone, follitropin, cortisol and prolactin [*Chong A., Aw S., 1986*]. The thyroid-stimulating hormone, luteinizing hormone, follitropin, cortisol and prolactin were revealed in the vitreous humour. As to other hormones, proges‐ terone, estradiol, triiodothyronine and thyroxine, the results of immune enzyme assay were negative even despite their high solubility and relatively small size of their molecules. According to A. Steiger (2003), the role of somatotropin, somatostatin and adrenocorticotropic hormone (ACTH) in the genesis of a wide range of eye diseases with both inflammatory and degenerative genesis is also disputable.

fibronectin mRNA expression by trabecular cells increased by 50 and 100% after incubation in primary samples of aqueous humor during 48 hours or 7 days, as well as by 50 and 160% after incubation in secondary samples of the aqueous humor. MMP-1 mRNA expression decreased by 25 and 50% after incubation in samples of primary aqueous humor during 48 hours or 7 days, as well as by 80 and 85% after incubation during 48 hours or 7 days in secondary samples of aqueous humor. The level of fibronectin increased 3.5 times and 6-fold after incubation

Study results obtained by the abovementioned authors allow to draw a conclusion that induction of MMP-3 in the trabecular meshwork of glaucomatous eyes might decrease fibronectin formation in aqueous humor excretion pathways, thus decreasing the resistance of

The analysis of publications relevant to the role of *in situ* produced fibronectin in post-barrier

*Firstly,* the role of *in situ* produced fibronectin in mechanisms on sustaining the local homeo‐

*Secondly*, the available scientific literature indicates to the fact that under conditions of norm fibronectin produced by cells of the trabecular meshwork performs the drainage function in

*Thirdly*, at some eye diseases and especially at primary open-angle glaucoma and pseudoex‐ foliative syndrome, the excessive synthesis of fibronectin by cells of the trabecular meshwork might bring forth a disorder of the drainage function that eventually in its turn is fraught with

*Fourthly*, it is not excluded that in post-barrier membranes of the eye there are engaged fibronectin-dependent mechanisms, which function according to both principles of inter-

At present, aspects related to studies on "endocrine homeostasis" in post-barrier membrane of an eye at both norm and pathology are the subject of a wide discussion in ophthalmology. The available publications are not numerous; furthermore, they are of a rather statement-ofthe-fact character [*Southren A. et al., 1976; Floman N., Zor U., 1977; Kasavina B. et al., 1977; Weinstein B. et al., 1983; Stone R., Wilson C., 1984; Stojek A. et al., 1991; Chiquet C., Denis P., 2004; Burch J. et al., 2005; Pleyer U. et al., 2005; Schwartz B. et al., 2005; Vessey K. et al., 2005*]. In particular, there are reports discussing the possibility of cortisol local synthesis in eye mem‐

The autopsy material (vitreous body and blood serum of healthy subjects with fatal injury) was subject to immune enzyme assay for determination of progesterone, estradiol, thyroxine, triiodothyronine, thyrotropic hormone, luteinizing hormone, follitropin, cortisol and prolactin [*Chong A., Aw S., 1986*]. The thyroid-stimulating hormone, luteinizing hormone, follitropin, cortisol and prolactin were revealed in the vitreous humour. As to other hormones, proges‐

**2.3. The role of cortisol in pathogenesis of anterior open-angle glaucoma**

during 48 hours with primary and secondary samples of aqueous humor.

liquid outflow into the anterior chamber of an eye.

stasis remains debatable.

82 Glaucoma - Basic and Clinical Aspects

branes.

outflow of the aqueous humor.

the increase of intraocular pressure.

cellular interactions and the autocrine mechanism.

membrane of an eye allows to come the following conclusions.

The results obtained by mentioned authors testify in favour of the local synthesis of certain hormones in eye membranes and tissues.

It should be specially noted that regional neuroendocrine mechanisms underlying the induction of primary open-angle glaucoma have not been sufficiently studied yet. In this aspect the role of *in situ* produced cortisol in mechanisms of impaired ion exchange is exceptionally connected with disbalance of sodium ions transport between the cells and liquid media of an eye and the impaired catecholamines exchange.

As known, in peripheral "epithelial" tissues sodium and water transport are regulated by corticosteroids, 11-β-hydroxysteroid-dehydrogenase (11-β-HSD), its isoform (11-β-HSD1), due to which there occurs formation of cortisol molecule from cortisone. Considering this latter, some researchers [*Rauz S. et al., 2003*] determined levels of cortisol, cortisone, 11-β-HSD and 11-β-HSD1 in ciliary body of actually healthy volunteers. The study was aimed to reveal the role of cortisol and 11-β-HSD in regulation of intraocular pressure that is sustained due to balance of aqueous humour (intraocular liquid) depending on the sodium transport through the ciliated epithelium and drainage via the trabecular meshwork. In both study groups cortisol concentrations were higher than cortisone levels. In both groups oral application of carbenoxolone, 11-β-HSD inhibitor, was accompanied by a marked decrease of intraocular pressure. To our mind, data obtained by mentioned authors, on the one hand, signify in favour of the above-mentioned cascade of reactions for maintenance of intraocular pressure, on the other hand, in favour of cortisol local synthesis in post-barrier membranes of the eye.

There is an opinion, according to which merely 11-β-HSD1 ensures receptor Nf-dependent mechanisms through the ciliated epithelium, thus regulating the level of intraocular pressure [*Rauz S. et al., 2001*]. Mentioned authors revealed the fine mechanisms, which provide the level of glucocorticoids mediated intraocular pressure. However, the potentiating role of cortico‐ steroids in regulation of intraocular pressure was revealed much earlier [*Jacob E. et al., 1996*]. As known, the rate of aqueous humour production is stimulated by adrenalin. The authors studied the joint and isolated effects of adrenalin and hydrocortisone to the rate of aqueous humor production in 20 volunteers. As demonstrated by study results, joint oral application of adrenaline and hydrocortisone significantly (by 42%) enhanced production of aqueous humour compared to placebo. The authors consider that both factors simultaneously function within the post-barrier membranes of the eye (ciliary body), thus ensuring the rate of aqueous humour production.

Molecular mechanisms underlying the biological action of glucocorticosteroids in eye mem‐ branes were also studied. Specifically, in the experiment, under conditions of cornea trans‐ plantation the influence of glucocorticosteroids (SEGRA) was studied to the labeled synthesis of anti- and pro-inflammatory cytokines. The application of glucocorticosteroids brought forth more efficient engraftment. Moreover, the terms of engraftment correlated with the low expression of cytokines, especially IL-I [*Pleyer U. et al., 2005*].

chamber associated immune deviation (ACAID) [*Wilbanks G., Streilein J., 1990; Streilein J. et al.,*

Modern Aspects of Glaucoma Pathogenesis Local Factors for Development of Primary Open-Angle Glaucoma…

http://dx.doi.org/10.5772/55037

85

In pathogenesis of the primary open-angle glaucoma the specific gravity of regional immu‐ nepathological disorders, which are pathognomonic for cataracts, are open for a special discussion, because data of available scientific publications are scarce, fragmentary, sometimes

At the same time, to our mind, it is rather expedient to perform studies at which in case of complicated cataracts associated with glaucoma and pseudoexfoliative syndrome the subject matter would be the entire specter of biologically active substances produced in eye mem‐ branes, which were earlier considered by us as pathogenetic factors of open-angle glaucoma. Such scientific and methodical approach is rather substantiated, as it will allow to answer the question: to what extent the processes of impaired synthesis of fibronectin, IGF-1, PgE2 and cortisol in eye membranes are engaged in mechanisms of primary open-angle glaucoma, namely: in pathogenesis of impaired drainage function and increase of intraocular pressure.

Under our observation there were 960 patients with the senile and complicated cataracts operated at "Shengavit" Medical Center within a period of 2008-2012. The degree of lens opacity was assessed according to Emery colorimetric classification and generally accepted classification of cataracts proposed by Buratto. Undoubtedly, the state of lens capsule, folding, presence of elements of fibrous filaments, pseudoexfoliative deposits on the anterior surface of the capsule, lens subluxation to some degree, were taken into consideration together with classification of phakodonesis suggested by Pashtaev. Actual expressiveness of the pseudoex‐ foliative syndrome was considered based on the classification proposed by Yeroshevskaya.

The studied groups of patients involved civil contingent: residents of Yerevan and different

The first group included patients with senile cataract. The second group was made up of patients with the complicated cataract on the background of existing anterior open-angle glaucoma, with initial and developed stages of the glaucomatous process. The third group involved patients with complicated cataract on the background of existing pseudoexfoliative

The analyses were performed using the main clinical laboratory methods accepted in oph‐

Irrespective of the cataract degree and stage, all patients underwent microaxial Phacoemulsi‐ fication ‒ Microincision Cataract Surgery (MICS) through 2.2 *mm* incision with implantation of posterior chamber intraocular lens. Intra-chamber administration of antibiotics was not

The methodical procedure of extracting aqueous humour was used intra-operatively under conditions of sterility. The corneocentis was done by insulin syringe through the limb; 0.1-0.2 *ml* aqueous humour was extracted. The fluid remained in a syringe until laboratory research

*1992; Abrahamian A. et al., 1995; Muhaya M. et al., 1999; Fleenor D. et al., 2006*].

contradictory and inconsistent.

All operated patents were divided into three groups.

glaucoma and pseudoexfoliative syndrome.

thalmology.

applied in these groups.

provinces (marzes) of Armenia; age range was from 40 to 82 years.

A. Southren et al. (1979) performed experiments in rabbits and revealed endoplasmatic reception of glucocorticoids in corneal cells and the ciliary body. Translocation of cortisol from the surface of the cell nucleus occurred within 30 minutes after injection. As to authors, this mechanism is a stereotype for glucocorticoids towards other sensitive tissues.

It is important to note the following phenomenon as well. Similar translocation was not observed when experimental animals were administered testosterone, estradiol and proges‐ terone. At the same time, different membranes and liquid media of the eye possess different ability of affinity to glucocorticoids and their realization (accumulation and excretion).

In 1977, B. Kasavina et al. (1977) studied cortisol distribution in sclera, ciliary body, cornea, iris, lens capsule, vitreous body and the aqueous humour. Radionuclide methods of investi‐ gation allowed to reveal that tissues and media of the eye have different intensity of cortisol absorption and excretion. According to authors, the sclera, ciliary body, and lens capsule served as target tissues for cortisol.
