Purinergic System and Disease

#### **Chapter 4**

## Graft-Versus-Host Disease: Pathogenesis and Treatment

*Shin Mukai*

#### **Abstract**

Graft-versus-host disease (GVHD) is a disabling complication after allogeneic hematopoietic stem cell transplantation (HSCT) and negatively impacts patients' quality of life. GVHD is classified into 2 forms according to clinical manifestations. Acute GVHD (aGVHD) typically affects the skin, gastrointestinal tract, and liver, whereas chronic GVHD occurs systemically and shows diverse manifestations similar to autoimmune diseases such as eosinophilic fasciitis, scleroderma-like skin disease. GVHD is induced by complicated pathological crosstalk between immune cells of the host and donor and involves various signaling pathways such as purinergic signaling. Although the past several decades have seen significant progress in the understanding of mechanisms of GVHD and several drugs have been approved by FDA for the prevention and treatment of GVHD, there is still vast scope for improvement in the therapy for GVHD. Thus, new drugs for GVHD will need to be developed. Towards this goal, this chapter succinctly summarises the pathogenic process of GVHD and emerging GVHD treatments in order to provide some insights into the mechanisms of GVHD and facilitate the development of novel drugs.

**Keywords:** inflammation, fibrosis, therapeutic targets, drug development

#### **1. Introduction**

Graft-versus-host disease (GVHD) is a debilitating complication that can determine the prognosis of allogeneic hematopoietic stem cell transplantation (HSCT) and subject 40–60% of HSCT recipients to a risk of death and disability [1]. GVHD is composed of acute GVHD (aGVHD) and chronic GVHD (cGVHD). For the classification of the 2 types of GVHD, the classifier should be clinical manifestations instead of time after HSCT [2]. However, in many cases, aGVHD appears within 100 days after HSCT and causes severe inflammation mostly in the skin, gastrointestinal tract, and liver [3]. cGVHD generally occurs systemically 6 months or later after HSCT, and its symptoms are similar to those of autoimmune diseases [4]. Complex interactions between donor and host immune cells are implicated in the pathogenesis of GVHD. It is thought that aGVHD is induced primarily by donor T cells' cytotoxic responses against host tissues through recognition of host polymorphic histocompatibility antigens [5]. On the other hand, the mechanisms of cGVHD are more complicated and still poorly understood [6]. Although the use of corticosteroids alone or in combination with immunosuppressive agents is the recommended first-line strategy

for the treatment of GVHD, its efficacy is not satisfactory [3, 7]. The prevalence of allogeneic HSCT for the treatment of hematologic diseases has increased the need for the development of efficacious second-line therapies which can mitigate symptoms of GVHD without compromising a graft-versus-leukemia effect, where donor T cells eliminate host leukemia cells. To date, various signaling pathways and pathogenic events in the context of GVHD have been intensively investigated. As a result, several FDA-approved drugs for GVHD have recently emerged. This chapter concisely summarises therapeutic targets and newly emerging drugs for the 2 forms of GVHD with the goal to facilitate the development of novel GVHD treatments for human use.

#### **2. Clinical manifestations of GVHD**

aGVHD can occur after the engraftment of donor-derived cells in the transplant recipient [8]. Symptoms of aGVHD can develop within weeks after the transplantation [9]. It has been believed that aGVHD can primarily affect the skin, gastrointestinal (GI) tract, and/or liver [10]. HSCT recipients can manifest rash, increased bilirubin, diarrhea, and vomiting [11]. Most recently, mounting evidence suggests that other organs such as the central nervous system, lungs, ovaries and testis, thymus, bone marrow, and kidney can be susceptible to aGVHD [12].

Clinical manifestations of cGVHD are different from those of aGVHD. The onset of cGVHD can be divided into the following 3 cases: (1) occurring when aGVHD is present, (2) emerging after a period of resolution from aGVHD, and (3) developing de novo [13]. Immune dysregulation and absence of functional tolerance are characteristic of cGVHD, and symptoms of cGVHD are reminiscent of those of autoimmune disorders [13]. Clinical presentations of cGVHD can be as follows: (i) rash, raised or discolored areas, skin thickening or tightening, (ii) dry eye or vision changes, (iii) dry mouth, white patches inside the mouth, (iv) diarrhea and weight loss, (v) shortness of breath due to lung disorders and (vi) abnormal liver function [14]. It was challenging for clinicians to reach an agreement on the diagnosis, the timing of treatment, and how to grade cGVHD [15]. In order to overcome these difficulties, the National Institute of Health (NIH) consensus created diagnostic criteria for cGVHD in 2005 and revised the criteria in 2014 [16, 17]. The authors considered the severity of involvement of the skin, mouth, eyes, gastrointestinal tract, liver, lungs, joint fascia, and genital tract in order to define manifestations of cGVHD in its target organs and establish a scoring system.

Corticosteroids are used with or without immunosuppressive drugs as the firstline therapy for aGVHD and cGVHD in clinical settings [3, 7, 18, 19]. However, approximately 50% of patients who receive steroid therapy will be resistant to it, although mechanisms of steroid resistance remain to be elucidated [3, 7, 18, 19]. In addition, corticosteroid therapies also cause various undesired effects such as diabetes, obesity, osteoporosis, hypertension, glaucoma, and liver damage [20]. Thus, medical settings are in need of effective treatments of steroid-refractory aGVHD and cGVHD [3, 7, 18, 19].

#### **3. General GVHD biology**

GVHD has a complex pathophysiology, which initially begins with damage to host tissues by chemotherapy and radiation therapy before allogeneic HSCT (**Figure 1**) [21].

#### **Figure 1.**

*The overview of aGVHD pathogenesis. The preconditioning regimen causes tissue damage. It generates DAMPs, PAMPs and proinflammatory cytokines such as TNFα, IL-1β and IL-6, which activates host APCs. The activated APCs present antigens to donor T cells, and the activated T cells infiltrate aGVHD target organs and produce an excessive amount of IFNγ and IL-17, leading to abnormal inflammation and tissue damage. This figure is created with BioRender.*

Due to this, damage-associated molecular patterns (DAMPs), pathogen-associated molecular patterns (PAMPs), and inflammatory cytokines are released [22]. These stimuli activate host dendritic cells (DCs), leading to the expression of major histocompatibility complex class I (MHC-I) and class II (MHC-II) on the host DCs [22]. The mature host DCs activate donor-derived T cells in the graft [22]. The activated donor T cells migrate to aGVHD-susceptible organs and promote the excessive production of pro-inflammatory cytokines such as interferon (IFN)-γ and interleukin (IL)-17 [23, 24]. It results in abnormal inflammation and tissue damage [23, 24]. While it is believed that donor-derived CD4+ and CD8+ T cells play a pivotal role in mediating aGVHD [25], several other types of immune cells are reportedly involved in the pathogenic process of aGVHD [26]. Neutrophils contribute to the development of intestinal aGVHD [27]. A previous report suggests that neutrophils in the ileum migrate to mesenteric lymph nodes, presenting antigens on their MHC-II and promoting T cell expansion [28]. Donor monocyte-derived macrophages with potent immunological functions are implicated in the pathophysiology of cutaneous aGVHD by secreting chemokines, stimulating T cells, and mediating direct cytotoxicity [29, 30]. In contrast, regulatory T cells (Tregs) are thought to serve a suppressive role in aGVHD without significantly reducing the graft-versus-leukemia (GVL) effect [31, 32]. Recent reports suggest that donor-derived natural killer (NK) cells can have an inhibitory effect in aGVHD by promoting the depletion of allo-reactive T cells while showing the GVL effect [33]. A recent study indicates that the occurrence and severity of aGVHD could be associated with the disordered reconstitution of CD56high NK cells [34].

While mechanisms of cGVHD are still incompletely understood, recent evidence suggests that there are several observations characteristic of cGVHD (**Figure 2**) [35]. The thymus is damaged due to the conditioning regimen and/or the prior occurrence of aGVHD, leading to impaired negative selection of alloreactive CD4+ T cells [36]. Alloreactive T cells are activated by antigen-presenting cells (APCs), resulting in their expansion and polarization toward type 1, type 2, and type 17 helper T (Th1, Th2, and Th17) cells [35]. These immune deviations lead to the production of proinflammatory and profibrotic inflammatory cytokines such as IFNγ, IL-6, IL-17, IL-4, and transforming growth factor β (TGFβ), which skew macrophages and fibroblasts towards

#### **Figure 2.**

*Overview of cGVHD pathogenesis. The thymus is damaged due to the preconditioning regimen and/or aGVHD. Due to the damage, the negative selection of alloreactive T cell is impaired. Alloreactive T cells are polarised into Th1, Th2 or Th17 cells. Th1 cells produce IFNγ, which drives macrophages to an M1-like phenotype to promote inflammation. IL-4, IL-10 and TGFβ produced by Th2 cells facilitate macrophage differentiation into an M2-like phenotype. Activation and proliferation of tissue fibroblasts are induced by (i) TGFβ from Th2 cells, (ii) PDGFα and TGFβ from M2-like macrophages and (iii) IL-6 and IL-17 from Th17 cells, leading to collagen production and fibrosis. B cells are activated by IL-6 and IL-17 from Th17 cells, and the alloreactivity of B cells is presumably induced by an excessive amount of BAFF. As a result of the above events, systemic inflammation and fibrosis are induced, and autoimmune-like manifestations are observed. This figure is created with BioRender.*

proinflammatory and/or profibrotic phenotypes [35]. Consequently, inflammation and fibrosis are induced in cGVHD target organs [37]. The damaged thymic epithelial cells (required for the generation of Tregs as well as the negative selection) also cause a decrease in the number of Tregs [38]. Furthermore, the dysregulation of B cells causes autoreactive B cells to arise and produce autoreactive antibodies [39]. The emergence and activation of autoreactive B cells presumably stem from B cell exhaustion induced by aberrant levels of B cell-activating factor (BAFF) in the lymphoid microenvironment [40, 41].

#### **4. Therapeutic targets and strategies for GVHD**

#### **4.1 TCR and BCR signaling**

When the T cell receptor (TCR) interacts with an MHC-antigenic peptide complex, it induces molecular and cellular changes in T cells [42]. A wide range of signal transduction pathways in T cells is stimulated due to this interaction, leading to the activation of a variety of genes [43]. Effector enzymes such as kinases, phosphatases, and phospholipases are involved in the TCR signaling pathways, which are integrated by non-enzymatic adaptor proteins acting as a scaffold for interactions between proteins [42]. These intracellular signaling pathways can determine the features of immunity mediated by T cells [44].

The B cell receptor (BCR) complexes on inactivated B cells act as self-inhibiting oligomers [45]. The BCR signaling pathways are initiated, when BCR is bound to an antigen and induces actin-mediated nanoscale recombination of receptor clusters [46]. Due to this event, the BCR oligomers are opened and the ITAM domains are

*Graft-Versus-Host Disease: Pathogenesis and Treatment DOI: http://dx.doi.org/10.5772/intechopen.104450*

revealed, resulting in the transduction of intracellular signals which are crucial for B cell development, activation, proliferation, differentiation, and antibody production in health and disease [47].

In 2017, FDA approved ibrutinib, which targets B cells and T cells, for the treatment of cGVHD. Ibrutinib was the first FDA-approved drug for steroid-refractory cGVHD, and it was a significant milestone for GVHD research [48]. Ibrutinib is reported to modulate the functions of B cells and T cells by potently inhibiting Bruton's Tyrosine Kinase (BTK) and IL-2 Inducible T-cell Kinase (ITK) [49], which are involved in the B cell signaling and T cell signaling pathways, respectively. Treatment of cGVHD-affected recipients with ibrutinib resulted in decreased serumautoantibodies and B-cell proliferation [50]. Data from the clinical trials show that symptoms of cGVHD improved in 67% of patients treated with ibrutinib [48].

#### **4.2 Purinergic signaling**

The Purinergic signaling pathways play a crucial role in a range of physiological systems including the immune system. In the purinergic signaling pathways, extracellular purine nucleosides and nucleotides such as adenosine and adenosine triphosphate (ATP) are used as signaling molecules that mediate the communication between cells through the activation of purinergic receptors [51]. There are four types of P1 (adenosine) receptors (A1, A2A, A2B, and A3). P2 receptors are subdivided into P2X and P2Y [52]. P2X receptors have seven subtypes (P2X1, P2X2, P2X3, P2X4, P2X5, P2X6, and P2X7), and P2Y receptors have 8 subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) [52].

As demonstrated by several studies using mouse models of aGVHD, extracellular ATP is augmented in aGVHD-affected mice, and purinergic signaling is implicated in the pathogenic process of aGVHD (**Figure 3**) [53]. The conditioning regimens prior to allo-HSCT can induce tissue damage, leading to the release of DAMP molecules including ATP, which activates purinergic signaling [53]. The involvement of extracellular ATP is evidenced by the fact that the injection of the soluble ATP diphosphohydrolase (ATPDase) can reduce inflammation in aGVHD target organs and the serum level of IFNγ [53, 54].

Evidence suggests that; (i) P2X7 is a crucial P2X receptor in the development of aGVHD after the release of extracellular ATP, (ii) the expression of the P2X7 receptor is elevated in PBMCs in aGVHD patients, (iii) the liver, spleen, skin, and thymus in aGVHD-affected mice show the increased expression of the P2X7 receptor, (iv) the ATP-induced activation of the P2X7 receptor on host APCs can facilitate the stimulation, proliferation, and survival of donor T cells during aGVHD and (v) the P2X7 activation on host APCs may be associated with the expression of microRNA mir-155 [53, 55–57].

While the host P2X7 receptor is shown to play an integral role in the development of aGVHD, the donor P2X7 receptor is also a contributor to this disease. Evidence suggests that (i) the activation and proliferation of donor CD4+ T cells and (ii) the metabolic fitness of donor CD8+ T cells are also enhanced by the activated donor P2X7 receptor [58, 59]. In addition, the activation of P2X7 on donor Tregs can reduce their suppressive ability and stability of Tregs, promoting their conversion to Th17 cells [60].

Inhibition of the P2X7 receptor is reported to mitigate aGVHD in conventional and humanised mouse models of aGVHD. Treatment of allogeneic HSCT recipient mice with the P2X7 inhibitor pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) can increase the survival rate and the number of Tregs, and reduce the

#### **Figure 3.**

*Link between GVHD and the therapeutically targetable purinergic signaling pathways. In aGVHD, ATP is produced due to tissue damage. Host APCs and donor T cells can be activated by the P2X7 receptor, which results in the progression of aGVHD. The deactivation of donor Tregs can also be induced by the ATP-activated P2X7 receptor, which leads to the reduction of Treg survival and the progression of aGVHD. CD39 and CD73 on donor Tregs can degrade ATP to adenosine. Adenosine can activate the A2A receptor on donor T cells, which culminates in the decrease in the number of CD4+ and CD8+ T cells and the reduction of aGVHD. In cGVHD, ATP is also released because of tissue damage and may promote fibroblast-to-myofibroblast transition through the ATPactivated P2X7 receptor, leading to the augmented collagen production and the progression of tissue fibrosis. In contrast, the ATP-activated P2Y14 receiptor may prevent cellular senescence in macrophages and mitigate cGVHD. This figure is created with BioRender.*

serum level of IFNγ and histological aGVHD [53, 54]. Administration of the P2X7 inhibitor brilliant blue G (BBG) to allogeneic HSCT recipient mice can also prevent weight loss and reduce inflammation in the liver and the production of inflammatory cytokines [56]. Furthermore, a crystal structure of the P2X7 receptor in complex with the inhibitor AZ10606120 has been reported (PDB: 5U1W) [61], and this structural information could be useful for the design and synthesis of novel P2X7 inhibitors which can be used in clinical settings.

The P2Y2 receptor is also reported to contribute to the pathogenesis of aGVHD [22, 57]. Evidence indicates that the number of cells expressing the P2Y2 receptor is increased in the intestinal tract in aGVHD-affected mice and that the increased P2Y2 expression enhances the severity of intestinal aGVHD [62]. Of note, knock-out allogeneic HSCT recipient mice of the P2Y2 receptor show an increased survival rate and decreased cytokine levels [62]. However, in the case where the P2Y2 receptor in donor cells is knocked out, no such improvement is observed [62]. In contrast,

#### *Graft-Versus-Host Disease: Pathogenesis and Treatment DOI: http://dx.doi.org/10.5772/intechopen.104450*

literature precedent suggests that the activation of the P2Y2 receptor can promote the migration of Tregs to sites of inflammation and thereby mitigate aGVHD [63]. Due to the dual functions of the P2Y2 receptor, targeting the P2Y2 receptor for the treatment has been challenging and there have been no reports about systemic injection of P2Y2 inhibitors/activators for the treatment of aGVHD [64].

While ATP is released in damaged tissues in allogeneic HSCT recipients and promotes inflammation, it is also degraded to adenosine by CD39 and CD73 [53]. In particular, a murine study indicates that CD39 and CD73 are highly expressed on CD150high Tregs [65]. As shown by a study using a mouse model of aGVHD, inhibition of CD39 and CD73 with adenosine 5′-(α,β-methylene)diphosphate (APCP) leads to the increase in the number of splenic CD4<sup>+</sup> and CD8+ T cells, the serum levels of IFNγ and IL-6, and the mortality rate [66]. These data suggest that CD39 and CD73 play an alleviatory role in aGVHD. Evidence demonstrates that the production of adenosine by CD39 and CD73 results in the activation of the adenosine A2A receptor [66–68]. The activated A2A receptor can induce the expansion of donor Tregs and thereby mitigate aGVHD-induced inflammation [66–68]. The blockade of A2A with the antagonist SCH58261 exacerbates aGVHD by elevating the levels of TNFα, IFNγ, and IL-6 and the number of CD4+ and CD8+ T cells in sera [66]. In agreement with this report, the A2A agonist ATL-146e reduced weight loss and mortality in aGVHD-affected mice by (i) increasing serum IL-10 and reducing serum IFN-γ and IL-6, (ii) precluding the activation of splenic CD4+ and CD8+ T cells, and the infiltration of T cells into GVHD target organs [67]. Other A2A agonists, ATL-370 and ATL-1223, are reported to exert similar therapeutic effects on aGVHD [68]. Moreover, a crystal structure of the A2A receptor in complex with the activator ZM241385 has been reported (PDB: 5WF5) [69], and this structural information could facilitate the creation of novel A2A activators which can enter the clinic.

Although there are few to no reports about a link between purinergic signaling and cGVHD pathogenesis, activation of the P2X7 receptor is reported to promote fibroblast-to-myofibroblast transformation and contribute to the development of fibrosis [70]. The activation of the P2X7 receptor enhances Ca2+ influx and skews fibroblasts towards a fibrogenic phenotype, leading to augmented collagen production [70]. Considering fibrosis is a significant hallmark of cGVHD, the investigation into a correlation between purinergic signaling and fibroblast activity in cGVHD could open up a new window for the elucidation of mechanisms of cGVHD and the development of novel drugs for cGVHD (**Figure 3**). Furthermore, stress-induced cellular senescence in immune cells is reported to play a detrimental role in the pathogenesis of ocular cGVHD [71, 72], and a murine study indicates that the P2Y14 receptor modulates stress-induced cellular senescence in hematopoietic stem/progenitor cells [73]. Given these findings, the P2Y14 receptor may be a regulator of stress-induced cellular senescence in cGVHD, and development of agonists of the P2Y14 receptor could benefit cGVHD patients.

#### **4.3 JAK/STAT signaling**

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways are regarded as a central communication junction for the immune system [74]. In the JAK/STAT signaling pathways, the cytoplasmatic kinase JAKs interact with the transcription factor STATs, and more than 50 cytokines and growth factors are involved in the JAK/STAT signaling pathways [75]. Mammals have 4 JAKs (JAK1, JAK2, JAK3, JAK4) and 7 STATs (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b,

STAT6) [76], and the dysregulated JAK/STAT signaling pathways contribute to a variety of human diseases, which makes this signaling a promising drug target [77].

In the early phase of aGVHD, tissue damage due to the preconditioning regimen and the disease results in the release of DAMPs, leading to the increased expression of MHC on APCs at the infusion of donor cells [78]. Donor T cells are activated via direct or indirect allorecognition, and the activated donor T cells produce IFNγ to initiate the JAK/STAT signaling pathways through IFNγ receptors [78]. The resultant increase in the expression of the chemokine receptor CXCR3 on T cells enhances their migration to aGVHD target organs, which promotes tissue damage [79].

While clinical manifestations of cGVHD are different from those of aGVHD, they have similarities in some aspects of the pathogenic processes [80]. The JAK/STAT signaling pathways in the context of cGVHD have been intensively investigated [81]. Tregs play a crucial role in the reduction of cGVHD, and JAK1/JAK2 signaling pathways are thought to negatively regulate the development and proliferation of Tregs, as indicated by the fact that JAK2 inhibition can promote Treg proliferation [82, 83]. Tissue fibrosis is highly problematic in cGVHD, and M2-like macrophages producing TGF-β are presumably a key player [84]. IL-10 skews macrophages towards an M2-like phenotype through the IL-10 receptor-JAK1/STAT3 pathway [85]. Given these reports, it would be intriguing to investigate an association between macrophages and the JAK/STAT signaling pathways in the development of cGVHD-induced fibrosis.

Many researchers have focused on the development of inhibitors targeting JAK/ STAT signaling pathways for the treatment of aGVHD and cGVHD [81]. As demonstrated by several preclinical data, inhibition of the JAK/STAT pathways can mitigate GVHD without affecting the GVL effect [81] Most recently, the JAK1/JAK2 inhibitor ruxolitinib has been approved by FDA for aGVHD and cGVHD. In 2019, FDA approved ruxolitinib to treat steroid-refractory aGVHD patients 12 years or older [86]. The clinical trials show that the day-28 overall response rate (ORR) was 100% for Grade 2 aGVHD, 40.7% for Grade 3 aGVHD, and 44.4% for Grade 4 aGVHD [86]. In 2021, FDA approval was also granted to ruxolitinib for the therapy of steroid-resistant cGVHD patients 12 years or older [87]. The clinical trial data demonstrate that the ORR was 70%, and the median durations of response, which were calculated from first response to progression, death, or new systemic therapies for cGVHD, were 4.2 months [87]. A crystal structure of JAK2 in complex with ruxolitinib is provided in the PDB database (PDB: 6VGL) [88], and this structural information could be useful for the design of more potent and selective JAK1/JAK2 inhibitors. Another promising JAK1 inhibitor is itacitinib [89]. Data from a phase 1 clinical trial of itacitinib shows that 70.6% of steroid-refractory cGVHD patients were treated in a satisfactory manner [90]. Furthermore, two clinical trials of itacitinib for cGVHD have recently commenced (ClinicalTrials.gov identifier: NCT04200365, NCT03584516). It is of great medical significance that novel drugs targeting the JAK/STAT signaling will continue to be developed for the treatment of aGVHD and cGVHD.

#### **4.4 NF-κB signaling**

The transcription factor nuclear factor kappa B (NF-κB) controls the expression of various genes important for the induction of inflammatory responses in innate and adaptive immune cells [91]. NF-κB is a family of heterodimers or homodimers generated from different combinations of the following 5 proteins: p65/RelA, RelB, c-Rel, p105/p50 (NF-κB1), and p100/p52 (NF-κB2) [92]. Among them, the p50/p65 complex is thought to be the most abundant form of NF-κB dimer [93]. When NF-κB is inactive,

#### *Graft-Versus-Host Disease: Pathogenesis and Treatment DOI: http://dx.doi.org/10.5772/intechopen.104450*

it is retained in the cytoplasm by the IκB family of inhibitors [94, 95]. In response to a wide range of stimuli such as the proinflammatory cytokines IL-1 and TNF-α, IκB kinase (IKK) is activated to phosphorylate the 2 serine residues of IκBα [96]. The phosphorylation causes the 26S proteasome to induce the ubiquitination and degradation of IKβ. Subsequently, NF-κB is translocated into the nucleus and triggers gene transcription, leading to the production of proteins necessary for immune responses [97]. Thus, NF-κB is regarded as a therapeutic target for the treatment of various inflammatory diseases.

The NF-κB signaling pathways have captured increasing attention from GVHD researchers. It has been reported that the activation of RelB in APCs contributes to the expansion of donor Th1 cells and subsequent alloreactivity, which leads to the development of aGVHD [98]. The NF-kB signaling pathways can be survival and proliferation signals and contribute to B-cell alloantibody deposition and germinal center formation, which play a critical role in the pathogenic process of cGVHD [99, 100].

Bortezomib is an FDA-approved drug for the treatment of multiple myeloma and is known to be an indirect inhibitor of NF-κB [101]. A murine study suggests that aGVHD can be prevented by treatment with bortezomib early after allogeneic HSCT [102, 103]. Bortezomib is undergoing clinical trials for aGVHD (BMT CTN 1203), and the phase1/2 study shows that bortezomib can be used in combination with tacrolimus and methotrexate in a tolerable immunosuppressive regimen after allogeneic HSCT [104]. Bortezomib can also be effective for the treatment of cGVHD. NF-κB inhibition with Bortezomib is suggested to cause apoptosis of germinal center B cells during reconstitution, leading to the decrease in donor-derived B cell numbers and BAFF expression [103]. With these promising data, clinical trials of bortezomib for the treatment of steroid-refractory cGVHD are in progress (NCT01158105). At present, there are no NF-κB inhibitors approved by FDA for aGVHD or cGVHD. Generally, direct inhibitors are superior to indirect ones in terms of selectivity. Thus, novel direct NF-κB inhibitors with high selectivity are greatly anticipated for the treatment of GVHD.

#### **4.5 Hedgehog signaling**

The Hedgehog signaling pathways are involved in the regulation of cell proliferation, survival, and differentiation [105], and its aberrant activation contributes to detrimental events such as the self-renewal and metastasis of cancer stem cells [106]. In the absence of Hedgehog ligand (Hh), the activation of Smoothened (SMO) is inhibited by Patched (PTCH) [107]. Subsequently, the activity of glioma-associated oncogene homolog (Gli) is suppressed by a protein complex mainly composed of a suppressor of fused (SUFU), which phosphorylates Gli and prevents it from entering the nucleus. In the presence of Hh, the binding of Hh to PTCH precludes the SMO inhibition mediated by PTCH [107]. Activated SMO prevents phosphorylation of Gli mediated by the SUFU complex, leading to the migration of Gli to the nucleus and the induction of downstream target gene expression [107].

Fibrosis is a highly problematic feature of cGVHD, and a profibrotic activity of Hedgehog signaling in patients and mouse models of cGVHD has been reported [108]. Overexpression of Hh, which is an inducer of the Hedgehog signaling pathways, is observed in human and murine sclerodermatous cGVHD [108]. The downstream processes of the Hedgehog signaling pathway cause overexpression of Gli-1 and Gli-2, particularly in fibroblasts [109]. The abnormal expression of Gli-1 and Gli-2 may result in the overproduction of collagen and the resultant pathologic fibrosis in cGVHD target organs [109]. Furthermore, the Hedgehog signaling is suggested to contribute to the increase of profibrotic M2-like macrophages in the cGVHD-affected skin [109].

There are several inhibitors of the Hedgehog pathways. Among others, sonidegib, vismodegib, and glasdegib are SMO inhibitors approved by FDA for the treatment of basal cell carcinoma [110]. These 3 SMO inhibitors are currently undergoing clinical trials for cGVHD therapy (NCT02086513, NCT02337517, NCT04111497). According to a report of the Phase-1 trial of sonidegib, where 17 steroid-refractory cGVHD patients participated, protein expression of hedgehog signaling pathway molecules was decreased by treatment with sonidegib as judged by immunohistochemical evaluation of the skin [111]. With respect to the creation of novel SMO inhibitors for the treatment of GVHD, Lacroix et al. found a potential SMO inhibitor by performing structure-based virtual screening of 3.2 million available, lead-like molecules against SMO and subsequent biological validations of the top-ranked compounds [112]. This information could benefit the design and synthesis of more potent and selective inhibitors of SMO.

#### **4.6 Endoplasmic reticulum stress**

While elucidation of mechanisms of cGVHD is still elusive, chronic inflammation is characteristic of cGVHD [113]. Senescent macrophages contribute to ocular cGVHD in mice, and gray eyebrows, skin wrinkles and conjunctival cancer are observed in human cGVHD [71, 114]. These findings suggest that ageing in donor- and recipient-derived cells is induced in cGVHD [71]. Evidence suggests that chronic inflammation and age-related diseases are associated with the elevation of endoplasmic reticulum (ER) stress [115, 116]. Mukai et al found that ER stress was increased in organs affected by cGVHD in mice [117]. Treatment of cGVHD-affected mice with the known ER stress reducer 4-phenylburyric acid (PBA) resulted in mitigation of systemic inflammation and fibrosis induced by cGVHD [117]. Of note, PBA is approved by FDA for the treatment of urea cycle disorders, and its safety was proven [118]. Investigation at the cellular level indicates that ER stress contributes to fibrosis as well as inflammation induced by cGVHD. Elevated ER stress caused (i) the dysregulation of lacrimal-gland-derived fibroblasts and (ii) abnormal production of MCP-1/CCL2, IL-6, and connective tissue growth factor (CTGF) [117]. Suppression of ER stress with PBA reduced their abnormal production of the inflammatory and fibrotic molecules [117]. In addition, ER stress induced by cGVHD skewed splenic macrophages towards an M2-like phenotype, and treatment of them with PBA promoted their differentiation into an M1-like phenotype [117]. Several reports also indicate that the augmentation of M2-like macrophages is implicated in the progression of cGVHD [84, 119, 120]. M2-like macrophages are thought to contribute to the pathogenesis of fibrosis-associated diseases [121], and it seems to be the case with cGVHD. As these analyses were performed in a bulk population, further investigation will be needed. Macrophages and fibroblasts are known to be heterogeneous populations [122–125]. In particular, mounting evidence suggests that macrophage heterogeneity is multidimensional and more complex than M1/M2 classification [126]. Hence, single-cell analyses could greatly facilitate the understanding of a correlation between ER stress and macrophages/fibroblasts in the development of cGVHD and make ER stress a more compelling therapeutic target for cGVHD therapy.

#### **4.7 Aberrant immune cell infiltration**

While aGVHD and cGVHD show different clinical manifestations, one of their common features is abnormal immune cell infiltration, which results in organ damage and severe inflammation and fibrosis. Mukai et al devised a novel therapeutic strategy for both types of GVHD by targeting vascular adhesion protein-1 (VAP-1) [127], which is known to be overexpressed in inflamed organs [128]. VAP-1 is an endothelial surface glycoprotein assisting leucocyte migration from the bloodstream to tissues and possesses the following 2 functional domains: a distal adhesion domain and a catalytic amine oxidase domain [129]. For infiltration into tissues, the amino group in leukocytes undergoes a nucleophilic attack on the carbonyl group in VAP-1 [129]. The subsequent catalytic conversion of the primary amine to the corresponding aldehyde allows immune cells to squeeze into tissues through blood vessels [129, 130]. Pursuant to their study with the use of a mouse model where aGVHD shifts to cGVHD [127], (i) the protein expression of VAP-1 is increased in organs with GVHD, where the number of inflammatory cells is accordingly augmented, (ii) blockade of VAP-1 with a novel inhibitor reduced the number of tissue-infiltrating leukocytes and thereby mitigated GVHD manifestations such as inflammation and fibrosis and (iii) the VAP-1 inhibition caused few to no severe adverse effects. Collectively, inhibition of VAP-1 could be an effective all-in-one approach for the treatment of aGVHD and cGVHD.

#### **4.8 NOTCH signaling**

The Notch signaling pathways are cell-to-cell communication induced by interactions between Notch receptors (NOTCH1, NOTCH2, NOTCH3, and NOTCH4) and NOTCH ligands (Jagged1 (JAG1), JAG2, Delta-like 1 (DLL1), DLL3 and DLL4) [131]. Due to these intercellular interactions, the NOTCH receptor is proteolytically activated by an ADAM family metalloprotease and subsequently by the γ-secretase complex [132]. The sequential cleavages lead to the release of the intracellular NOTCH domain (NICD), which is a transcriptionally active fragment [133]. NICD migrates to the nucleus and binds to the DNA binding CSL/RBP-Jk factor, forming a transcriptional activation complex with a mastermind-like (MAML) family coactivator [133]. This final complex triggers the transcription of target genes which are important for biological processes such as proliferation, differentiation, and survival [134].

A correlation between the Notch signaling pathways and alloimmune responses has gained interest from GVHD researchers. Studies using animal models of aGVHD suggest that; (i) the Notch signaling promotes activation, differentiation, and alloreactivity of T cells [135] and (ii) dendritic cells with high DLL4 expression show an increase in the production of IFN-γ and IL-17 [136]. The Notch signaling is also implicated in the pathogenic process of cGVHD. A murine study shows that NOTCH1 and NOTCH2 as well as DLL1 and DLL4 serve significant functions in regulating proinflammatory cytokine production by T cells [137]. Investigation using *in-vitro* human B-cell assay systems demonstrates that abnormal activation of NOTCH2 is correlated with hyperresponsiveness of BCR on B cells from cGVHD patients [138].

GVHD treatments by targeting the Notch signaling pathway have been reported. A series of experiments using a mouse model of aGVHD reveals; (i) inhibitors of γ-secretase block proteolytic activation of all the NOTCH receptors, but has severe toxicity in the gut epithelium, (ii) NOTCH1 inhibition using an antibody mitigates GVHD but causes serious toxicity and (iii) treatment with a combination

of anti-DLL1 and anti-DLL4 reduces aGVHD without debilitating adverse effects while maintaining a GVL effect of donor T cells [139]. An anti-DLL1 antibody is also effective for the treatment of murine cGVHD in combination with an anti-DLL4 antibody [137]. Treatment with all-trans-retinoic acid (ATRA) prevents NOTCH2 induced BCR hyperresponsiveness, which plays a detrimental role in cGVHD pathogenesis [137]. It appears that NOTCH2 and DLL1/4 are promising drug targets for the treatment of the 2 types of GVHD. Therefore, it is highly anticipated that novel, selective inhibitors of NOTCH2 and DLL1/4 will be developed for use in human GVHD.

#### **4.9 Rho/ROCK signaling**

Rho-associated coiled-coil-containing protein kinases (ROCKs) are serinethreonine-specific protein kinases, and mammals have ROCK1 and ROCK2 [140]. ROCKs are downstream effector proteins of GTPase Rho, and abnormal activation of the Rho/ROCK pathways contributes to the development of various diseases [140]. In particular, ROCK2 is known to regulate (i) the balance of Th17 cells and Tregs and (ii) profibrotic pathways [141]. ROCK2 activation increases Th17 cell-specific transcription factors by promoting STAT3 phosphorylation [142]. In addition, when ROCK2 is activated by profibrotic mediators such as tumor growth factor-β (TGF-β), it causes myocardin-related transcription factors to activate profibrotic genes in fibroblasts [143, 144]. This profibrotic gene activation induces fibroblast-to-myofibroblast differentiation and the resultant increase in collagen production [143, 144].

A study using a cGVHD mouse model shows that treatment with belumosudil, which is a selective ROCK2 inhibitor, can substantially reduce cGVHD-induced fibrosis in the lung [145]. In 2021, belumosudil was approved by FDA for the treatment of cGVHD, and the clinical trial data show that the overall response rate was 75% (6% complete response and 69% partial response) [146].

ROCK1 is also thought to be involved in the development of fibrosis, and pan-ROCK inhibitors targeting ROCK1/2 are thereby expected to show better treatment outcomes for cGVHD [147]. Several pan-ROCK inhibitors have been granted approval for human use [148–151] In particular, netarsudil has been approved by FDA for the treatment of glaucoma [151]. However, due to a lack of overall kinome selectivity of the reported dual ROCK1/2 inhibitors, there is still scope for improvement in pan-ROCK inhibitors [152]. Hu et al. has recently reported the synthesis and *in-vitro* evaluation of a novel series of 5*H*-chromeno[3,4-c]pyridine, 6*H*-isochromeno[3,4-c] pyridine, and 6*H*-isochromeno[4,3-d]pyrimidine derivatives as dual ROCK1/2 inhibitors [152]. Their data show that some of the novel pan-ROCK inhibitors display potent inhibitory activity against ROCK1/2 and possess excellent kinome selectivity [152]. They also provided a crystal structure of ROCK2 in complex with one of the novel dual ROCK1/2 inhibitors (PDB ID: 7JNT). This structural information can be useful in the structure-based design of other new pan-ROCK inhibitors.

#### **5. Conclusion**

While recent decades have seen significant technological and medical advances, aGVHD and cGVHD are still a major hurdle to successful allogeneic HSCT in clinical settings. Systemic corticosteroid therapy, with or without immunosuppressive agents, is the first-line treatment for GVHD, although it can cause severe adverse effects

#### *Graft-Versus-Host Disease: Pathogenesis and Treatment DOI: http://dx.doi.org/10.5772/intechopen.104450*

and approximately 50% of GVHD patients develop steroid-resistant GVHD. Thus, sophisticated treatments of steroid-refractory aGVHD and cGVHD are highly anticipated by medical settings. A great deal of effort has been invested in the elucidation of mechanisms of GVHD and development of safe and efficacious drugs for GVHD. Recently, several drugs have been approved by FDA for the treatment of steroidrefractory aGVHD and cGVHD. Despite this progress, there is still a need to create novel drugs with better efficacy for GVHD therapy. This chapter focused on druggable targets for the treatment of GVHD with an aim to stimulate various GVHD researchers (from medicinal chemists to biologists) to create novel drugs which can enter the clinic. While several signaling pathways have been intensively studied in the context of GVHD, there are underexplored signaling pathways. In particular, the purinergic signaling pathway is one of the understudied signaling pathways in GVHD. The P2X7, A2A, and P2Y14 receptors seem to be compelling drug targets for the treatment of GVHD, and clinical settings could benefit from safe and efficacious (i) inhibitors of the P2X7 receptor and (ii) activators of the A2A and/or P2Y14 receptors. However, the development of new drugs is a costly and time-consuming process. To overcome this setback, the use of AL/ML has captured great interest from many researchers and has been expected to substantially reduce the cost and time of drug development. A combination of AL/ML and molecular design could greatly facilitate the development of novel, effective, safe, and affordable drugs for the treatment of GVHD.

### **Author details**

Shin Mukai Beneras Pharma Inc., Boston, United States of America

\*Address all correspondence to: shin.mukai@beneras-pharma.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 5**

## Involvement of the Purinergic System in Cell Death in Models of Retinopathies

*Douglas Penaforte Cruz, Marinna Garcia Repossi and Lucianne Fragel Madeira*

#### **Abstract**

Literature data demonstrate already that the presence of adenine nucleotides in the extracellular environment induces cell death that leads to several retinopathies. As said, the objective is to carry out a systematized review of the last decade, relating purinergic signaling to the outcome of cell death and retinopathies. It is possible to identify different mechanisms that occur through the activation of purinergic receptors. The exacerbated activation of the P2X7 receptor is mainly involved in the apoptotic death pathway, and this response is due to the dysregulation of some components in the intracellular environment, such as the Ca2+ ion, CD40, MiR-187, and influence of mononuclear macrophages. The A2A receptor is involved in increasing levels of cytokines and promoting inflammatory processes. The data presented can be used as a basis to better understand the mechanisms of death in retinopathies, in addition to proposing therapeutic strategies with the potential to be transposed to several other models.

**Keywords:** P2x7 receptor, A2a receptor, apoptosis, retina

#### **1. Introduction**

The retina is a tissue that is located at the back of the eye and is responsible for converting light stimuli into electrical signals, a process is known as phototransduction, being responsible for the sense of vision [1]. In this tissue, as in many others, cell death is a highly regulated process that is important for maintaining homeostasis, in addition to preserving tissue function by excluding cells whose genome is altered [2]. According to Fricker et al. [3], there are some particularities in cell death in neurons. For example, complexity in nervous system circuitry during development results in programmed cell death of neurons that fail to connect properly. Furthermore, the excitability of neurons causes a high volume of adenosine triphosphate (ATP) and, due to a cytotoxic effect of this molecule in high concentrations, a sensitivity to death in several pathological models [3].

Damage or dysfunction in the retina, through excess cell death, can lead to several pathological conditions that interfere with the normal function of the tissue, compromising the ability to transmit visual stimuli to higher centers. A possible classification of diseases that affect the retina is hereditary retinal dystrophies, a group of pathologies caused by spontaneous genetic alterations that lead to irreversible loss of vision [4]. These diseases have great clinical relevance since they have a high incidence, and in Brazil, the ones that stand out are Non-syndromic Retinitis Pigmentosa, Stargardt's disease, Leber's Congenital Amaurosis, and hereditary retinal syndromic dystrophies [5]. Another known classification is retinopathies, a set of pathologies that affect the retina, which involve damage to the surrounding vasculature [6]. The decrease in the number of blood vessels leads to a process called retinal neovascularization that, occurring in a disorderly way, can deregulate tissue homeostasis or cause the process of retinal detachment [7].

Diseases in which vascular damage occurs are referred to as ischemic retinopathies and include diabetic retinopathy, retinal vein occlusions (RVOs), retinopathy of prematurity (ROP), and sickle-cell retinopathy. Thus, following this definition, other diseases fit this classification as they present stages with vascular damage, such as agerelated macular degeneration (AMD), retinal detachment, and glaucoma [8]. Both in the case of retinopathies and retinal dystrophies, most of them remain without a cure or with inadequate treatment and, therefore, new therapeutic strategies are necessary. In this case, the manipulation of molecular pathways can change the course of cell death, and the participation of the purinergic system is of great interest in these cases.

Cell death in non-neural cells has already been described, in which extracellular ATP triggers cell death by binding to P2X7 receptors, which is the main receptor activated in these cases. Upon activation, P2X7 receptors induce large, non-selective membrane pores, which eventually lead to cell death [9–12]. In addition to the evident relationship between the P2X7 receptor and the cell death process, other purinergic receptors participate in this cellular response. The P2X2 and P2X4 receptors, for example, are upregulated in squirrel ischemia models, and the improvement in cell death with the inhibition of these receptors confirms the participation of purinergic signaling [13].

Purinergic signaling mediated by extracellular ATP and adenosine is involved in the induction and protection of cell death in several models of retinal diseases [14]. One of the mechanisms observed in retinal pathologies is the increased expression of purinergic receptors that contribute to high calcium concentrations. P2X receptors act as direct channels for calcium influx and as indirect activators of voltage-gated calcium channels. Meanwhile, activation of P2Y receptors induces a rapid transient release of calcium from internal stores, followed by an influx of calcium. The increased expression, mainly of the P2X7 receptor, is involved in the pathogenesis of several diseases that affect the retina, in which ATP in high concentrations is capable of inducing apoptosis through this purinergic receptor [15].

Studies show that ATP induces apoptosis in embryonic retinal neurons of chicks in culture through the activation of the P2X7 receptor and ionotropic glutamate receptors [16]. Other research also proves that the direct application of ATP to isolated retinas induces the death of cholinergic amacrine cells that express P2X7 receptors [9, 10].

Although ATP is well known for being toxic in high concentrations, and its receptors are involved in several pathologies as inducers of cell death, adenosine receptors are also worth mentioning. When it comes to cell death, the adenosine A2A receptor plays a role [17]. It has already been seen that blocking the A2A receptor controls microglial reactivity [18], delays excitotoxic death of embryonic motor neurons *in vitro* [19], and is able to prevent cell death in ischemic retinas [20]. This evidence

#### *Involvement of the Purinergic System in Cell Death in Models of Retinopathies DOI: http://dx.doi.org/10.5772/intechopen.103935*

makes it clear that this receptor is of great interest when it comes to therapeutic targets to improve the condition of various diseases in the retina and nervous system.

Considering the close connection between the release of adenine nucleotides in the extracellular environment and cell death through the activation of purinergic receptors, it is of great interest to observe the latest studies carried out on the subject. Therefore, the objective of the present review is to present the knowledge obtained from the studies of the last decade, making clear the participation of purinergic signaling in cell death induced by different models of retinopathies.

#### **2. Age-related macular degeneration (AMD)**

#### **2.1 Participation of calcium pathways**

Age-related macular degeneration (AMD) is the main cause of irreversible vision loss in the elderly in developed countries, being able to affect several cell types in the retina [21, 22]. From the observed damage, numerous studies try to understand the molecular mechanisms involved in this pathology.

The retinal pigment epithelium (RPE) is considered a site of great interest in this pathology, and it is now well known that ATP acts as a key signaling molecule in several cellular processes, including cell death [23]. The P2X7 receptor is also involved in inflammation and oxidative stress in many cell types, and cell death, inflammation, and oxidative stress have been implicated in AMD.

Through the use of apoptotic markers, Yang et al. sought to know whether the presence of ATP, an endogenous P2X receptor agonist, increased the number of cells undergoing apoptosis in human pigment epithelium cell cultures. In cultures treated with ATP, it was possible to observe an increase in the intensity of apoptotic markers when compared to the control. This effect was blocked by the administration of the oxidized P2X7 antagonist (oATP). The selective exogenous P2X7 receptor agonist, 3'-O-(4-benzoyl) benzoyl-ATP (BzATP), was also able to increase apoptosis, but Brillant Blue G (BBG), a P2X7 receptor antagonist, and oATP reverse this effect [24].

Treatment with BAPTA-AM, used to decrease intracellular calcium levels, was able to decrease ATP and BzATP-induced apoptosis, which indicates that Ca2+ is an essential component for signaling the P2X7 receptor pathway and continuation of the apoptotic cascade. In summary, the study by Yang et al. provides the first evidence of the presence of functional P2X7 receptors in human pigment epithelium cell cultures and demonstrates that activation of P2X receptors, especially P2X7 receptors, induces Ca2+ signaling and apoptosis in these cells [24].

#### **2.2 Participation of oxysterols**

Another implication of AMD is the accumulation of drusen, which are extracellular proteolipid deposits, contributing to vision loss in the advanced stages of the disease. These deposits are located between the RPE and Bruch's membrane (inner layer of the choroid) and contain β-amyloid peptide as the main component [25]. Different oxysterols were found in human drusen, which suggests their involvement in AMD. Furthermore, the aggregated form of β-amyloid is well known as an inducer of oxidative stress and cell death [26, 27].

Oliver et al. aimed to highlight the β-amyloid/oxysterols relationship and describe the involvement of the P2X7-pannexin-1 receptor in oxysterol toxicity in human

RPE cell cultures [28]. A link was found between the presence of β-amyloid peptide aggregates and oxysterol levels. Two types of oxysterols, 25-OH and 7-KC seem to play a role in the pathogenesis of AMD through P2X7 activation, but only 25-OH causes pannexin-dependent pore opening in the cell membrane. This pannexinstimulated pore opening is important in the pathological mechanism of the disease, as it promotes the extravasation of ATP to the extracellular environment, and consequent activation of the P2X7 receptor. Thus, the toxicity of this oxysterol occurs in two ways—increased P2X7 receptor activity and oxidative stress-dependent on pannexin-1, and pannexin-1-independent chromatin condensation [28].

The potential relationship between oxysterols and β-amyloid in AMD supports the notion that oxysterols can be considered as biomarkers of retinal degeneration. Considering the fundamental role of P2X7 receptor activation in oxysterol cytotoxicity, this may be an important target for the development of treatments for this disease [28].

#### **2.3 Participation of alu RNA**

Geographic atrophy (GA) is an advanced form of age-related macular degeneration characterized by central loss of vision due to confluent areas of retinal pigment epithelium loss and degeneration of overlying photoreceptors [25]. The DICER1 processing enzyme is specifically reduced in the RPE in eyes with geographic atrophy, as its blockage results in an abundant increase in Alu RNA transcripts (an endogenous retroelement that requires reverse transcriptase for its life cycle), which in turn promotes the cell death of the RPE [29].

More recent studies have identified that the cytotoxicity of Alu RNA in the RPE is mediated by the activation of the NLRP3 inflammasome [30], and it has already been observed that reactive oxygen species (ROS) and the P2X7 receptor are involved in this process in other systems [31, 32]. Therefore, Kerur et al. investigated whether P2X7 signaling was also involved in Alu RNA-induced NLRP3 inflammasome activation, with experiments performed in mouse and human retinal pigment epithelial cell cultures [33].

After transfection of the Alu RNA into the culture media containing the cells of interest, it was seen that NF-kB signaling and P2X7 activation play important roles in Alu RNA-induced inflammasome initiation and activation and RPE degeneration. The authors also suggested, from cell cultures of P2X7 receptor knockout mice, that this receptor is an essential intermediate in the Alu RNA-induced activation of the NLRP3 inflammasome and consequent RPE degeneration. This suggests that manipulating this pathway may be a useful strategy for developing drugs for the treatment of geographic atrophy [33].

In complete agreement with these results, Fowler et al. also investigated the relationship between the P2X7 purinergic receptor and Alu RNA-induced AMD. It was based on what was previously demonstrated, that Alu-derived RNA activates the NLRP3 inflammasome, via the P2X7 receptor, to cause cell death of the retinal pigment epithelium in geographic atrophy [33]. As Alu RNA requires reverse transcriptase for its life cycle, the use of transcriptase inhibitors has been proposed for a definition of other therapeutic alternatives for the disease [34].

After injection of Alu RNA or transfection into human and mouse retinal pigment epithelium cell cultures, Alu RNA was seen to be cytotoxic, as it activates caspase-1 and activates IRAK4 (interleukin-1 receptor-associated kinase 4), whose phosphorylation in these cases leads to degeneration of the pigment epithelium). Alu and LPS,

*Involvement of the Purinergic System in Cell Death in Models of Retinopathies DOI: http://dx.doi.org/10.5772/intechopen.103935*

a bacterial compound known to activate inflammatory pathways, activate the NLRP3 inflammasome via activation of the P2X7 receptor. d4T (reverse transcriptase inhibitor—NRTI) acts in a protective manner, preventing caspase-1 activation and IRAK4 phosphorylation. Several approved and clinically relevant NRTIs, including lamivudine (3TC) and abacavir (ABC), prevented the activation of caspase-1, and the Alu RNA-induced inflammasome effect of NLRP3. NRTIs were effective in mouse models of geographic atrophy, choroidal neovascularization, graft-versus-host disease, and sterile liver inflammation [34].

#### **2.4 Abnormal vascular growth**

In the neovascular form of age-related macular degeneration, visual loss commonly occurs as a result of the invasion of abnormal blood vessels from the choroidal circulation, that is, choroidal neovascularization (CNV), which induces irreversible damage to the overlying retina. CNV mainly occurs due to dysregulation in the production of endothelial growth factors in the retinal vascular network [35].

Photoreceptor degeneration involves the activation of several regulated cell death signaling pathways that may constitute potential therapeutic targets. ATP has already been discovered as an important extracellular messenger that may contribute to lethal signaling [36]. Thus, Notomi et al. hypothesized that ATP acting via the P2X7 receptor is involved in the pathogenesis of photoreceptor loss in subretinal hemorrhage.

The results suggest that ATP levels in the subretinal space could potentially be higher than those detected in the vitreous because extracellular ATP diffuses into the vitreous cavity from the subretinal space. From the analysis of cell death in cultures of primary retinas in vitro and in a subretinal hemorrhage model in vivo, it was observed that a concentration of 1 mM of ATP triggered an apoptotic process in photoreceptor cells through binding to the P2X7 receptor, while the use of a selective inhibitor of the P2X7 receptor (Brilliant Blue G (BBG)) was able to prevent this effect. These results indicate that extracellular ATP can trigger apoptosis of photoreceptor cells via P2X7 receptor-dependent machinery. Thus, it is shown that pharmacological inhibition of the P2X7 receptor with BBG may result in neuroprotection of photoreceptors in cases of subretinal hemorrhage [37].

The study further suggests that similar severe neurodegenerative pathologies, such as subarachnoid hemorrhage or intracerebral hemorrhage, may be related to elevations in extracellular ATP. In this way, P2X7 receptor antagonists including BBG may have a neuroprotective therapeutic effect in retinal diseases as well as in Central Nervous System diseases with excessive extracellular ATP.

#### **2.5 Infiltration and accumulation of mononuclear phagocytes**

Also focusing on damage to photoreceptors in AMD, Hu et al. related this disease to the infiltration and chronic accumulation of mononuclear phagocytes, [38], which in excess lead to neuronal degeneration [39]. It has also been seen that a deficiency in Cx3cr1, a transmembrane chemokine receptor involved in leukocyte adhesion and migration, leads to the accumulation of mononuclear phagocytes, but the mechanism by which this occurs has not yet been well elucidated [38].

From the isolation of bone marrow-derived monocytes that are recruited to the inflammatory site, the expression levels of the P2X7 receptor in these cells were evaluated by flow cytometry. The study confirmed that the accumulation of mononuclear phagocytes in cases of Cx3Cr1 deficiency leads to increased expression of the P2X7 receptor in these cells. The authors observed that, in these situations, P2X7 receptors provoke the opening of pannexin-dependent pores and release ATP to the external environment. This ATP, from the P2X7 receptor, is able to activate inflammasomes which, in turn, are responsible for the maturation and release of interleukin-1 β (IL-1β), responsible for cytotoxicity and increased cell death in photoreceptors. This was confirmed by the ELISA assay, in which IL-1β levels are increased in cases of Cx3Cr1 deficiency [38].

To test whether P2X7 receptor inhibition has a protective effect against death, intravitreal injection of BBG, a selective inhibitor of the P2X7 receptor, was performed. The TUNEL assay showed that the number of apoptotic cells in the photoreceptor layer was reduced after administration of BBG in cases of Cx3Cr1 deficiency. Immunostaining with Iba-1 to quantify inflammation-associated reactive microglia showed that intravitreal injection of BBG was able to protect against inflammation in these cases.

P2X7 receptor inhibitors, therefore, may be a promising therapeutic target to inhibit lesion expansion in cases of Macular Degeneration, as they may prevent RPE cell death, and IL-1β and P2X7 inhibitors may help to prevent RPE cell death. Photoreceptor loss associated with inflammation [38].

#### **3. Diabetic retinopathy**

#### **3.1 Damage to the blood-retinal barrier**

Diabetic retinopathy is a serious complication of diabetes mellitus. Breach of the blood-retinal barrier (BRB) is a hallmark of diabetic retinopathy, as well as other eye diseases [40]. The human retina contains two BRBs, the inner and the outer, including endothelial cells and retinal pigment epithelial cells, respectively [41]. Maintenance of the physiological structure of retinal cells requires complex cell-to-cell interactions. These interactions occur at special contact sites called cell junctions, which include tight junctions (TJs), adherent junctions (AJs), and gap junctions (GJs) [42].

Knowing this, Platania et al. tested the hypothesis that activation of the P2X7 receptor contributes to the degradation of the inner portion of the BRB, also interfering with the integrity of the endothelial barrier, through the disruption of TJs between endothelial cells of human retinas, in an environment with high concentrations of glucose [43]. Using the bioinformatics program GEO2R, used to identify differentially expressed genes between two groups, P2X7 receptor expression was measured in human retinal endothelial cell cultures. The expression of the P2X7 receptor underwent a significant increase, induced by both the high concentration of glucose and the agonist BzATP, when compared to the control. Furthermore, high glucose induced the activation and release of the pro-inflammatory cytokine IL-1β via P2X7 receptor activation in human retinal endothelial cells. Glucose exposure also caused a decrease in endothelial cell viability and damage to the BRB [43].

It was also seen, by performing the transendothelial electrical resistance assay (TEER) to measure cell membrane integrity and cell-to-cell interactions, that blocking the P2X7 receptor with the drug JNJ47965567 was able to protect retinal endothelial cells against damage induced by high glucose concentrations and protected the blood-retinal barrier. In addition, treatment with JNJ47965567 significantly decreased the expression and release of IL-1β, induced by high glucose. These findings suggest that the P2X7 receptor plays an important role in regulating the integrity of the retinal blood barrier, and blocking this receptor was useful to counteract the damage caused by high glucose concentration in retinal endothelial cells. Thus, the use of P2X7 receptor antagonists may be useful in the treatment of diabetic retinopathy [43].

#### **3.2 Participation of P2X receptors in hyperglycemic retinas**

Long-term exposure to high glucose concentration, considered the main factor in the development of diabetic retinopathy, has already been shown to affect extracellular ATP levels in retinal cell cultures [44]. Furthermore, ATP can act as a neurotransmitter in the retina [45, 46], and through activation of plasma membrane receptors, it can increase intracellular calcium concentration. Some of the inflammatory mediators and excitatory neurotransmitters seen in neuronal death in diabetic retinas are released in response to an increase in intracellular Ca2+ concentration. Considering this, Pereira et al. sought to investigate whether the exposure of retinal cells from mice grown under high glucose levels could alter the function of P2X receptors [47].

In this study, through the Western Blot assay, it was seen that cultures of rat retinas exposed to high glucose concentration, the following subunits of the P2X receptor were found—P2X2, P2X3, and P2X7, but these did not undergo any significant change in their content when compared to the control. It is noteworthy that in these retinas the P2X4 receptor was affected by the high concentration of glucose, and its expression was reduced [47].

Through the Fura-2 assay (dye used for labeling intracellular calcium) it was shown that intracellular calcium concentrations triggered by the stimulation of P2 receptors are increased in retinal cells of rats cultured at high glucose concentrations, in a model used to simulate the hyperglycemic conditions seen in diabetes. Also using Fura-2, a difference in the pattern of Ca2+ concentration based on cell type was noted. In retinal neurons, the increase in intracellular Ca2+ concentration was mainly due to the influx of Ca2+ through voltage-sensitive calcium channels. In microglial cells, Ca2+ influx occurred mainly through P2X receptor channels, although there was also a minor component of increased intracellular Ca2+ concentration dependent on calcium release from intracellular stores [47].

These increased calcium responses may be responsible for the increased release of neurotransmitters and/or inflammatory mediators found in diabetic retinas and therefore contribute to retinal neural cell death detected in the early stages of diabetic retinopathy. Since intracellular calcium plays a key role in cell death, inhibition of some purinergic receptors may exert protective effects against retinal neural cell dysfunction or degeneration, and therefore P2 receptors may become a potential therapeutic target for the treatment of early stages of diabetic retinopathy.

#### **3.3 Involvement of the differentiation cluster 40 (CD40)**

Capillary degeneration is a hallmark of early diabetic retinopathy. They are the result of loss of retinal endothelial cells and pericytes (perivascular cells essential in maintaining metabolic, mechanical, and signaling functions in microvessels) [48]. Cluster of differentiation 40 (CD40) is required for retinal capillary degeneration in diabetic mice, a process mediated by the death of retinal endothelial cells [49]. However, binding of CD40 on endothelial cells does not normally induce cell death, likely because CD40 activates PI3K/Akt-mediated pro-survival signals [50, 51]. Thus, Portillo et al. aimed to identify a mechanism by which CD40 triggers programmed

cell death in human retinal endothelial cell cultures and address this apparent contradiction [52].

Administration of CD40 ligand in primary cultures of human retinal endothelial cells did not significantly alter the percentage of apoptotic cells. Given the close connection between these cells and Müller's glia, we sought to determine whether Müller's glia would indirectly influence the triggering of CD40-mediated cell death. The results showed that CD40 does not exert its effects directly on endothelial cells, but on circulating Müller's glia. It was also seen, by measuring cytokines by the ELISA assay, that CD40 also did not provoke the secretion of IL-β or TNF-α. In fact, CD40 stimulated Müller glia releases ATP into the extracellular medium. By performing a qPCR, it was noted that CD40 also upregulated the expression of the P2X7 receptor on the surface of endothelial cells, making them susceptible to the cell death process mediated by ATP/P2X7.

To obtain *in vivo* results, the authors used a model of induced diabetes in mice. By performing a real-time PCR after CD40 activation, they concluded that these animals upregulated P2X7 in the retina in a CD40-dependent manner when compared to control. Finally, inhibition of the P2X7 receptor (with A-438079) caused a decrease in retinal endothelial cell-cell death [52].

In summary, these studies have uncovered a mechanism by which CD40 enhances cell death of retinal endothelial cells and suggest that CD40 signaling on Müller cells may be an important contributor to vascular injury in diabetic retinopathy. The expression of CD40 was responsible for the secretion of ATP to the extracellular medium, favoring a greater activation of the P2X7 receptor. Increased programmed cell death accompanies these disorders and the P2X7 receptor is consistently seen as pathogenic in these diseases [53]. The findings may be relevant to other diseases caused by CD40, such as atherosclerosis and inflammatory bowel disease. Thus, new therapies can be developed to treat these diseases: blocking CD40 or the P2X7 receptor may prove to be effective alternatives in the treatment of diabetic retinopathy [52].

Growing evidence indicates that chronic inflammation is important for the development of diabetic retinopathy [54, 55]. TNF-α and IL-1β are pro-inflammatory molecules upregulated in this disease [56]. In addition to macrophages/microglia, Müller's glia (the main retinal microglia) become dysfunctional in diabetes and contribute to the development of diabetic retinopathy. Since CD40 deficiency impairs this process and prevents diabetic retinopathy [52, 57], Portillo et al. sought to elucidate the mechanisms by which this response occurs [58].

The study carried out showed, through real-time PCR, an increase in CD40 expression by Müller's glia in a mouse model of diabetic retinopathy. Additionally, it was seen that CD40 binding on Müller's glia triggered phospholipase C-dependent release of ATP. This release provoked activation of P2X7 receptors, resulting in the release of TNF-α and IL-1β by macrophages. To better prove the role of the P2X7 receptor in this process, mice that do not express the P2X7 receptor and mice treated with a P2X7 inhibitor were protected from the increase in the concentration of TNFα, IL-1β, ICAM-1, and NOS2 induced by diabetes, thus preventing the inflammatory process and cell death [58].

The observed effects are relevant in vivo because TNF-α is up-regulated in microglia/macrophages of diabetic mice that express CD40 on Müller glia and mice treated with BBG (P2X7 receptor inhibitor) are protected from diabetes-induced upregulation of TNF-α and IL-1β. This protection prevents the inflammatory process that normally accompanies the release of these cytokines, alleviating the pathological effects of diabetic retinopathy. Thus, this study indicates that CD40 in Müller's glia is sufficient to up-regulate retinal inflammatory markers. Furthermore, CD40 appears to promote experimental diabetic retinopathy and Müller's glia orchestrates inflammatory responses in myeloid cells via a CD40-ATP-P2X7 pathway [58].

#### **3.4 Therapeutic potential of the A2A receptor**

Diabetic retinopathy is one of the main complications of diabetes mellitus and one of the main causes of blindness. The pathogenesis of diabetic retinopathy is accompanied by chronic low-grade inflammation. Adenosine is a neuromodulator of the central nervous system that exerts its actions through the activation of its four receptors: A1, A2A, A2B, and A3. Some reports demonstrate that the microglial cell response can be altered by A2A receptor antagonists in the different brain and retinal diseases [59, 60]. Therefore, Aires et al. sought to find out whether blocking the A2A receptor can provide protection to the retina by modulating microglial reactivity [61].

Through the use of specific immunomarkers, it was observed that the number of reactive microglia was increased in the retina of mice in a model of induced diabetes. Intravitreal injection of SCH 58261, the A2A receptor antagonist significantly decreased microglial reactivity in the retinas of diabetic animals. The ELISA assay confirmed that, accompanied by this decrease in microglial activity, treatment with the A2A receptor antagonist was able to decrease the levels of TNF and IL-1β cytokines, also demonstrating the ability to control inflammatory processes [61].

In addition to these results, the injection of SCH 58261 was able to decrease the levels of reactive oxygen species. The TUNEL assay confirmed the neuroprotective potential of this inhibitor, demonstrated in the fall of apoptotic cells in the retina of mice in vivo, and the prevention of cell death preserved the thickness of the retinal tissue. Finally, regarding the vascular damage characteristic of Diabetic Retinopathy, it was seen that the inhibition of the A2A receptor contributes to the preservation of the integrity of the blood-retinal barrier. All these data demonstrate the therapeutic potential of A2A receptor antagonists for the treatment of diabetic retinopathy [61].

#### **4. Photoreceptors' degeneration**

Photoreceptor degeneration involves the activation of several cell death pathways that may constitute potential therapeutic targets, and an alternative for the inhibition of death pathways is to intercept death, such as the activation of caspases. Among the seven mammalian P2X receptors, the P2X7 receptor has the highest affinity for ATP [36]. Thus, extracellular ATP can induce apoptotic and/or necrotic cell death, acting on the P2X7 receptor [62]. Taking into account the therapeutic possibility of P2X7 receptor inhibitors (such as Brillant Blue G) [11, 63], Notomi et al. decided to investigate the pathogenic implications of the P2X7 receptor in the pathological loss of photoreceptors in mice, as well as the therapeutic utility of BBG in this context [64].

By administering ATP or BzATP (selective P2X7 receptor agonist) in primary cultures of mouse retinal cells, it was seen that stimulation of the P2X7 receptor with these ligands could directly mediate the cell death pathway in photoreceptors. Inhibition of the P2X7 receptor with BBG or KN-62 was able to prevent photoreceptor cell death, confirming the role of this purinergic receptor in this process. The pathway followed after activation of the P2X7 receptor was also demonstrated, in which photoreceptor cell death occurred through calcium influx (observed through the use

of the calcium marker Fluo-4 AM) and caspase-8 activation, suggesting a potential mechanism to an extrinsic pathway mediated by the P2X7 receptor [64].

Intravitreal injections of BzATP administered to mice showed that this specific P2X7 receptor agonist has the potential to induce retinal cell death in vivo. Inhibition of the P2X7 receptor proved to be effective in preventing cell death and preserving photoreceptors. Furthermore, blocking the P2X7 receptor indirectly inhibits the caspases of the mitochondrial cell death pathway in retinal cell cultures. Together, these results clarify some of the mechanisms of cell death induced by the binding of ATP to the P2X7 receptor and how antagonists, especially BBG, have clear relevant therapeutic effects that can be transferred to other models of neurodegenerative diseases, having neuroprotective potential that are also relevant. Applies to photoreceptors [64].

#### **5. Glaucoma**

#### **5.1 Ischemia-induced damage**

As a chronic neurodegenerative condition, glaucoma is characterized by the loss of retinal ganglion cells, resulting in progressive optic neuropathy and consequent visual field loss. Reduced blood flow to the optic nerve region and consequent ischemia has been suggested as a mechanism of ganglion cell death in glaucoma [65, 66].

There is currently considerable interest in the P2X7 receptor in mediating neurodegeneration, with increasing evidence indicating its role in chronic disease [67, 68]. Some studies have also provided evidence that the P2X7 receptor may play a role in glaucoma-induced death [69–71]. Taking this into account, Niyadurupola et al. sought to determine whether stimulation of ischemia-induced death in the ganglion cell layer is mediated by P2X7 in the human retina [12].

As a result, it was seen that stimulation of the P2X7 receptor by the selective agonist BzATP induced cell death in ganglion layer cells in organotypic cultures of human retinas, which was inhibited by the P2X7 receptor inhibitor (BBG). In addition, the ischemia caused to cells in culture led to the loss of retinal ganglion cells, and this effect was also inhibited by BBG, which suggests the participation of the P2X7 receptor in the observed degeneration. Finally, it was possible to locate the P2X7 receptor in the outer and inner plexiform layers of the retina, and the ganglion cells also expressed the mRNA encoding the P2X7 receptor protein [12].

All these data confirm the great importance of this purinergic receptor in the retina and its relationship with glaucoma, since the stimulation of the P2X7 receptor can mediate the death of retinal ganglion cells and that this mechanism plays a role in ischemia-induced neurodegeneration in the human retina. In addition, the therapeutic potential of P2X7 receptor inhibitors is clear, with the aim of preventing cell death [12].

#### **5.2 NMDA-induced damage**

It is known that glutamate receptor stimulation by excess glutamate during hypoxia [72] and ischemia-reperfusion [73] is toxic to neurons. Activation of the N-methyl-D-aspartic acid (NMDA) receptor, a subtype of glutamate receptor, is followed by a large influx of Ca2+. This excess of intracellular Ca2+ is predominantly involved in neuronal excitotoxicity processes and is considered one of the

#### *Involvement of the Purinergic System in Cell Death in Models of Retinopathies DOI: http://dx.doi.org/10.5772/intechopen.103935*

mechanisms of glaucoma-induced neuronal cell death [71]. Furthermore, some studies have also suggested that the P2X7 receptor plays a role in retinal ganglion cell death induced by high ocular pressure [69, 74, 75]. So, Sakamoto et al. sought to examine the role of P2X7 receptors in NMDA-induced retinal damage in mice in vivo [76].

The results obtained in the study demonstrate that, as expected, the intravitreal injection of the P2X7 receptor agonist (BzATP) induces cell death in the rat retina, an effect that was prevented by the administration of receptor antagonists (A438079 and Brillant Blue G). After this confirmation, it was evaluated whether the NMDA receptor produces its toxic effects through this receptor. Injections of the P2X receptor inhibitors, A438079 and BBG, were able to reduce the deleterious effects of NMDA, decreasing the number of apoptotic cells in cases of glaucoma induced through the NMDA receptor, confirming the neuroprotective effect of these drugs on the retina against toxicity of the drug. Glutamate. Finally, the immunohistochemistry technique was performed to determine the distribution pattern of the P2X7 receptor in mouse retinas. The results indicated that P2X7 receptors were expressed in the somatic region of RGCs that had DAPI-labeled nuclei in the ganglion cell layer and in the inner and outer plexiform layers [76].

These results then showed for the first time that P2X7 receptor activation is, at least in part, involved in NMDA-induced retinal damage. In summary, the study authors demonstrate the possibility that P2X7 receptor antagonists are effective in preventing retinal diseases caused by glutamate excitotoxicity [76].

#### **5.3 Participation of miR-187**

MicroRNAs (miRNAs) are a class of non-coding RNAs that regulate transcription and translation of target genes by interacting with the 3′-untranslated region of the target gene (3'-UTR), thus mediating the pathogenesis of multiple human diseases [77]. Previous studies confirmed that miR-187 promoted retinal ganglion cell survival and decreased apoptosis of these cells in human ganglion cell culture incubated with TGF-β, suggesting a protective role of miR-187 against glaucoma [78]. Given the role of miR-187 and the P2X7 receptor in glaucoma, Zhang et al. sought to know whether there is a functional correlation between miR-187 and the P2X7 receptor in apoptosis in a mouse retinal ganglion cell culture-induced model of glaucoma [79].

The results showed that high pressure-induced oxidative stress in retinal ganglion cells was accompanied by a decrease in miR-187 expression and an increase in P2X7 receptor expression. It was also found that miR-187 down-regulated P2X7 receptor expression in ganglion cells, and this inhibition was able to inhibit oxidative stress and apoptosis in these cells [80]. These data demonstrated that miR-187/P2X7 signaling is involved in retinal cell apoptosis, at least in part, through oxidative stress activation. In vitro experiments showed that both miR-187 and the P2X7 receptor were upregulated in the retina of mouse models of chronic ocular hypertension [79]. Thus, miR-187 and the P2X7 receptor promise to be a new target for the prevention and treatment of ophthalmic neurodegenerative diseases.

#### **6. Retinopathy of prematurity (ROP)**

Retinopathy of prematurity (ROP) is a disease that can cause blindness in very low birth weight babies and remains a leading cause of childhood blindness in many parts of the world [81, 82]. As a disease that mainly affects the retinal vasculature, existing

treatments for this disease, such as anti-VEGF therapy, can have adverse effects, compromising the development of blood vessels and leading to peripheral blindness [80]. The therapeutic potential provided by the antagonism of the purinergic A2A receptor has already been verified, and it may represent a new and promising pharmacological strategy to control pathological retinal angiogenesis under ROP conditions. This strategy avoids the onset of negative effects observed in the anti-VEGF strategy, as it alters molecular mechanisms without compromising the maintenance of the vasculature or the formation of new blood vessels [83]. That said, Zhou et al. sought to extend this potential of A2A receptor inhibition, using it as a therapeutic strategy to selectively control pathological retinal neovascularization, in a model of oxygeninduced retinopathy leading to ROP [84].

To verify whether the use of the A2A receptor inhibitor would alter the retinal vasculature under physiological conditions, KW6002 (A2A receptor inhibitor) was administered intraperitoneally in mice of the C57BL/6 strain. Repeated exposure to KW6002 did not alter the mice's normal retinal vasculature, showing that the treatment has no unwanted effects. After that, immunohistochemistry and immunofluorescence assays showed that, in a model of induced RDP in mice, the administration of KW6002 reduced avascular areas and neovascularization, with apoptosis and cell proliferation also reduced, and astrocyte functions increased. Thus, KW6002 treatment increased astrocyte participation and reduced cell proliferation and apoptosis to confer protection against pathological angiogenesis in ROP [84].

#### **7. Retina detachment**

Retinal detachment involves the separation of the sensorineural retina (responsible for receiving and conducting light stimuli to the higher centers of vision) from the retinal pigment epithelium. Direct contact between the retina and the pigment epithelium is essential for its normal function, and detachment can lead to irreversible vision loss [85]. A2A adenosine receptor signaling has been shown to be neuroprotective in some models of retinal damage, but its role in neuronal survival during retinal detachment is unclear. Therefore, Gao et al. sought to modulate the A2A receptor-dependent signaling cascade and observe whether there would be changes in the rate of photoreceptor apoptosis [86].

In a mouse model of retinal detachment, A2A receptor expression was determined from real-time PCR and Western blot assays. It was found that A2A receptor protein was detected in the ganglion cell layer, the inner and outer plexiform layers, and the inner nuclear layer after the retinal detachment protocol. It is worth noting that this receptor was predominantly expressed in microglia and in Müller's glia [86].

The role of the A2A receptor in different models of pathologies is quite controversial, and the cellular response followed may favor cell death or neuroprotection. However, the effect caused by this receptor in cases of retinal detachment had not been well elucidated so far. Thus, in this study, intravitreal injection of the drug ZM241385, a selective antagonist of the A2A receptor, was performed. Through immunofluorescence assays using specific markers, it was seen that blockade of the A2A receptor inhibited microglia reactivity after the triggering of retinal detachment, accompanied by a reduction of microglial proliferation. The drug

#### *Involvement of the Purinergic System in Cell Death in Models of Retinopathies DOI: http://dx.doi.org/10.5772/intechopen.103935*

also decreased the expression of GFAP (reactive gliosis marker) and decreased the expression of the inflammatory cytokine IL-1β. Furthermore, by performing a specific measurement assay for oxidative stress, it was seen that inhibition of the A2A receptor reduced the amount and production of reactive oxygen species in detached retinas [86].

Finally, through the TUNEL assay, the rate of apoptotic cells in the retina after the induction of retinal detachment was evaluated. The administration of ZM241385 was able to prevent the loss of photoreceptors caused by the high concentration of reactive oxygen species triggered after retinal detachment, further reinforcing the role of A2A receptor inhibition in the control of neuroinflammation. Thus, the involvement of the purinergic A2A receptor in the pathogenesis of retinal detachment was confirmed, making it a promising therapeutic target in the treatment of the pathology [86].

#### **8. Transitory retina ischemia**

Transient retinal ischemia refers to a pathological condition that involves loss of blood supply to the retina, resulting in cell damage and death from lack of oxygen supply [87]. It has already been seen that a mechanism for triggering this pathological condition is microglia-mediated neuroinflammation, raising the hypothesis that the control of microglial reactivity may provide neuroprotection. Furthermore, it has already been seen that inhibition of the A2A receptor led to neuroprotection from microglial control in cases of ischemia [20]. Taking this into account, Boia et al. aimed to investigate the therapeutic potential of oral administration of the A2A receptor antagonist and the effects of caffeine ingestion (adenosine receptor antagonist) against neuroinflammation and cell death in a model of ischemia caused by intraocular pressure. in mice [60].

Knockout mice for the A2A receptor were used to evaluate the effects of the absence of this receptor in relation to the control in cases of ischemia. From the quantification of TNF and IL-1β levels by the ELISA assay, it was seen that IL-1β levels were not altered, but TNF levels were significantly reduced in A2A knockout retinas when compared to Wild-Type animals. Likewise, the use of the A2A receptor antagonist (KW6002) caused a reduction in TNF levels. In addition, labeling of reactive microglial activity was also found to be decreased when the A2A receptor was inhibited by the drug KW6002. The TUNEL assay confirmed the neuroprotection caused by the use of the A2A receptor inhibitor since the number of apoptotic cells was found to be reduced in relation to the control group [60].

Focusing on caffeine, which is an adenosine receptor antagonist, we quantified the expression of the same cytokines seen previously through qPCR and ELISA assay. In ischemic retinas, the acute administration of the substance was not able to change the levels of TNF or IL-1β; however, the transcriptional levels of the two cytokines were found to be elevated after 24 hours of administration. Regarding microglial activity, caffeine showed dichotomous results: after 24 hours, caffeine increased microglial reactivity, inflammatory response, and ischemia-induced cell death compared to the control group. However, at 7 days of reperfusion, caffeine administration decreased microglia reactivity and reduced levels of pro-inflammatory cytokines and cell death. This indicates that prolonged treatment with caffeine induces the beneficial effects presented [60].

#### **9. Conclusion**

Purinergic signaling has already been shown to be important for several cellular processes in different organs and systems. The present study showed the relevance of purinergic receptors in signaling cell death pathways in the retina, and the cytotoxic effects can be applied in the various retinopathies addressed (**Figure 1**). When it comes to cell death, the participation of the P2X7 receptor in cytotoxic and inflammatory processes is clear. Thus, despite the search to encompass the entire purinergic system, P2X7 and A2A receptors were the most found when it comes to cell death in the retina.

In the studies analyzed in this review, it is possible to recognize different cellular mechanisms that occur through the activation of the P2X7 receptor. Dependence on the influx of Ca2+ ions after receptor activation was present, since the lack of these ions prevents the apoptotic cascade from occurring, and this pattern was present in models of Macular Degeneration and Diabetic Retinopathy. In AMD models, other mechanisms by which the activation of the P2X7 receptor can act can also be observed, such as the increase in the levels of oxysterols, Alu RNA and the infiltration of mononuclear macrophages. All these factors contribute to the onset of cytotoxic effects and the initiation of cell death.

Cell death induced by purinergic signaling also extends to other pathologies: in Diabetic Retinopathy, it is noted that, in addition to calcium-dependent signaling, the P2X7 receptor is also involved in damage to the blood-retinal barrier (BRB), and damage mediated by the differentiation cluster 40 (CD40); in cases of glaucoma, the ischemic injury induced by NMDA or involving the MicroRNA MiR-187 has its toxic effects also mediated by this same receptor.

We can also see that the adenosine A2A receptor plays an important role in triggering several diseases, mainly in alterations of vascular integrity. This receptor has been

#### **Figure 1.**

*Synthesis of the involvement of the P2X7 receptor in the presented retinopathies. In the studies analyzed, it was possible to recognize different cellular mechanisms that occur through activation of the P2X7 and A2A receptors, each of them leading to the final outcome, which is cell death.*

#### *Involvement of the Purinergic System in Cell Death in Models of Retinopathies DOI: http://dx.doi.org/10.5772/intechopen.103935*

seen to be involved in increasing cytokine levels in pathological conditions such as TGF and IL-1B; also in triggering microglial reactivity and promoting inflammatory processes.

With the results obtained from this analysis, it is clear that more and more studies claim that the exacerbated activation of the P2X7 receptor by extracellular ATP is highly involved mainly in the apoptotic pathway of cell death. When it comes to possible therapeutic targets for the diseases addressed, it is interesting to consider P2X7 receptor inhibitors for this purpose, as some of the studies show the effectiveness of these inhibitors in improving the effects observed in each model. Thus, the data presented here can be taken as a basis to better understand the mechanisms of death in various retinopathies, in addition to proposing therapeutic strategies with the potential to be transposed to several other models.

### **Author details**

Douglas Penaforte Cruz, Marinna Garcia Repossi and Lucianne Fragel Madeira\* Department of Neurobiology at the Fluminense Federal University, Institute of Biology, Niterói, Rio de Janeiro, Brazil

\*Address all correspondence to: lfragel@id.uff.br

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 6**

## Leukaemia: The Purinergic System and Small Extracellular Vesicles

*Arinzechukwu Ude and Kelechi Okeke*

#### **Abstract**

Haematopoiesis is a tightly regulated process, by intrinsic and extrinsic factors, to produce lifelong blood cell lineages within the bone marrow. In the bone marrow microenvironment, mesenchymal stem cells and haematopoietic stem cells play important roles to ensure that haematopoiesis is maintained. These cells contain purines and pyrimidines that control intercellular process such as energy transport. However, in some cases, this process may be misregulated thus leading to the production of various diseases, including leukaemia. As a result, bone marrow cells may be stimulated via stress or induced hypoxia, and this leads to the release of purine and pyrimidine nucleotides and nucleosides into the extracellular space, and activation of autocrine/paracrine feedback loops. These extracellular nucleotides and nucleosides, and their respective cell surface receptors are involved in purinergic signaling that control different physiologic functions in cells including proliferation, differentiation, and cell death. These extracellular nucleotides and nucleosides include ATP, UTP, adenosine diphosphate (ADP), UDP and adenosine however the most important players are ATP and its metabolite adenosine. ATP is degraded via a sequential activity of ectonucleotidases. ATP, adenosine and these ectonucleotidases play very important roles in the tumour microenvironment crucial to disease development, progression, and aggressiveness by modulating immune response to leukaemia treatment and increasing homing of leukaemic cells.

**Keywords:** cancer, communication, vesicles, transplantation

#### **1. Introduction**

Leukaemia is a malignant disorder involving early blood-forming cells of the bone marrow in which there is lack of control in the haematopoietic process. As a result, abnormal or immature blood cells accumulate in the bone marrow, bloodstream or lymphatic system thus causing debilitating effects in the patient [1, 2]. Leukaemia is a heterogenous disease hence its diagnosis is based on a complete blood workup (full blood count) and bone marrow studies (aspiration and biopsy) incorporating clinical, morphological, immunophenotypical, cytogenetic and genetic data.

Leukaemia is classified into different types depending on the blood cell type that the cancer originates. Leukaemia could be myeloid (myelogenous) if it involves the myeloid lineage such as red blood cells (RBC), platelets and white blood cells

(WBCs) or lymphoid (lymphocytic/lymphoblastic) if it involves cells originating from the lymphoid lineage such as lymphocytes [1, 3]. Leukaemia is also classified into acute and chronic based on the progression of the disease. Acute leukaemia is often abrupt and fast-growing due to the accumulation of immature cells in the bone marrow whilst chronic leukaemia is rather slow growing [1, 3]. Therefore, leukaemia is categorized into four main groups: acute myeloid leukemia (AML), acute lymphocytic leukaemia (ALL), chronic myeloid leukaemia (CML) and chronic lymphocytic leukaemia (CLL).

#### **2. Epidemiology and aetiology of leukaemia**

Leukaemia is one of the most common cancers in the world and accounts for about 2.6% of all cancer cases worldwide [4]. In 2020, leukaemia was reported to be the 13th most diagnosed cancer case and 10th leading cause of cancer death [5]. Caucasians especially the men are more predisposed to the disease compared to other ethnic groups. The disease progresses with age hence adults are more susceptible than children. However, the outlook for patients is much better than three decades ago, with better cure rates and longer-term disease-free survival. Chemotherapy and radiotherapy are the mainstay treatment for leukaemia however the overall survival rate has improved remarkably in recent years with almost half of the population diagnosed with leukaemia surviving for at least five years or more [3, 6]. This is due to the advent of different therapies including stem cell transplantation, targeted therapy, combined therapy, immune cell engineering such as chimeric antigen receptor (CAR)-T cell therapy, innovative clinical trials and immunotherapies, and patient's improved access to these therapies [6, 7].

Though an increase in age is a well-known factor, the aetiology of leukaemia remains unclear. Exposure to certain viruses (for example, human T-cell leukaemia virus; HTLV-1), ionizing radiation, chemicals such as benzene or formaldehyde, smoking and other environmental cues have all been reported to be risk factors for leukaemia [8–10]. These environmental cues induce genetic and chromosomal aberrations such as chromosomal deletions or translocations, point mutations and epigenetic factors, that cause maturation arrest of the normal haematopoietic stem cells (HSCs). This leads to development of leukaemia due to the proliferation and clonal expansion of leukaemic stem cells (LSC). Leukaemia could also arise following exposure to chemotherapy, radiotherapy and/or stem cell transplantation [6, 7].

#### **3. Leukaemia and the purinergic system**

Haematopoiesis is a tightly regulated process that leads to lifelong production of a sustainable pool of functional blood cells within a compartmentalized bone marrow. The bone marrow consists of osteocytes, osteoblasts, osteoclasts, the bone matrix, perivascular cells, immune cells, sinusoidal endothelium as well as regenerative cells; mesenchymal stem cells (MSC) and HSC that inhabit a unique hypoxic microenvironment [11, 12]. These cells are responsible for the provision of cell signals required for the support and regulation of haematopoiesis as well as maintenance of homeostasis within the bone marrow microenvironment [12, 13].

Additionally, there is prevalence of purines and pyrimidines in these bone marrow cells thus modulating intracellular processes such as energy transport [14, 15]. These

cells can also release the purine and pyrimidine nucleotides and nucleosides into the extracellular space under normal circumstance in the absence of any stimulus. However, the stimulation of these bone marrow cells via stress or induced hypoxia leads to the release of purine and pyrimidine nucleotides and nucleosides, and activation of autocrine/paracrine feedback loops [12–15]. These extracellular nucleotides and nucleosides, and their respective cell surface receptors control a form of cellto-cell communication called purinergic signaling. This complex network controls various physiologic functions in the body, including cell proliferation, differentiation, and cell death. As a result, any aberration to this network will lead to the development of diseases such as leukaemia [11, 16–19].

These extracellular nucleotides and nucleosides include ATP, UTP, adenosine diphosphate (ADP), UDP and adenosine however the most important players are ATP and its metabolite adenosine [11, 19, 20]. ATP is degraded via a sequential activity of four ectonucleotide enzyme subtypes (ectonucleotide pyrophosphate, ectonucleotide triphosphate diphosphohydrolase, alkaline phosphatase and ecto5-nucleotidase) leading to adenosine, which binds P1 receptors, as end-product [2, 11, 17, 18, 21–24]. Thus, extracellular ATP is the primary source of adenosine, and adenosine is degraded by adenosine deaminase (ADA). HSCs express several receptors for nucleotides and nucleosides, which belong to two different purinergic receptor families, P1 and P2. The P2 family includes eight receptors that have been identified so far (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2Y14), which are G protein-coupled receptors that respond to stimulation by ATP, ADP, UTP or UDP, depending on the receptor subtype and seven ionotropic receptors (P2X1, P2X2, P2X3, P2X4, P2X5, P2X6, P2X7), which are sensitive to ATP [15, 18, 25]. The P1 receptor family consists of four G proteincoupled receptor subtypes, A1, A2A, A2B, and A3, which are activated by adenosine.

Extracellular ATP is a crucial component of the tumour microenvironment. The balance between extracellular ATP and adenosine is pivotal to cancer progression, promoting proliferation of HSCs and thus, cancer cell proliferation [19]. Extracellular ATP also mediate proinflammatory activity on specific P2Y (P2Y1, P2Y2, P2Y4, P2Y6 and P2Y12) and P2X (P2X4 and P2X7) receptors. In addition, extracellular ATP exerts angiogenic effects on P2Y1, P2Y2 and P2X7 receptors and immunosuppressive effects on P2Y11 receptors thereby affecting interaction with the immune system via trafficking of granulocytes and monocytes and inhibiting proliferation and migration of leukemic cells [2, 16, 18, 23, 24, 26, 27]. Cellular stress or damage to stromal host cells promotes and controls inflammatory response resulting in ATP release and subsequent accumulation of ATP in extracellular space, which can be beneficial or harmful to the host depending on its concentration.

Notably, the concentration gradient of ATP is maintained by two important enzymes, CD39 (ectonucleotide triphosphate diphosphohydrolase-1) and CD73 (ecto5-nucleotidase) that are found in abundance in immune cells, endothelial cells, and tumour cells [18, 19, 28]. This enzymatic chain is also responsible for adenosine production, which can accumulate in the tumour environment and stroma where it acts as a potent immunosuppressant that exerts its effects mainly at A2A receptors. In addition, adenosine also modulates cell growth when acting on A3 receptors. Multiple mechanisms have been implicated in adenosine effects and these include deregulation of mononuclear phagocyte cell differentiation and maturation, suppression of effector T cells, inhibition of T helper 1 cell cytokine production, and generation of an angiogenic and matrix remodeling environment [17, 21, 23]. Extracellular adenosine also modulates and protects cells and tissues from excessive inflammatory and immune responses, which favor angiogenesis and thus, carcinogenesis [2, 18, 19, 29].

Adenosine acts on the A2A, A2B and A3 receptors to induce macrophages to promote the release of anti-inflammatory cytokine such as tumour necrosis factor (TNF), nitric oxide (NO), macrophage inflammatory protein (MIP), interleukin (IL)-6, IL-10, and IL-12. Therefore, ATP exerts dual mechanism in cancer, facilitating proand anti-tumoral immune response whilst adenosine is a known immunosuppressive mediator facilitating tumour immune evasion [11, 18, 19, 23]. An immunosuppressed microenvironment and inflammation enhance the development and metastasis of cancer via release of a wide variety of cytokines and other proinflammatory markers.

In various cancers, including leukaemia, it is widely acknowledged that purinergic receptors of the P2 family (P2Rs) are required for anti-cancer immune response induced by chemotherapy. In leukaemia, chemotherapeutic agents such as doxorubicin and daunorubicin induce intracellular ATP release to create an immunosuppressed tumour microenvironment, which leads to cell death [2, 19, 20]. Once released, the extracellular ATP can bind to P2 receptors to regulate cell invasion and migration. Treatment with ATP inhibitors or ATP analogues has a strong cytotoxic effect on several tumours, including leukaemia [23, 24]. This leads to a decrease in the intracellular concentration of ATP until it falls to undetectable levels when cells enter secondary necrosis. Low ATP doses have a growth-promoting effect and depending on the P2 receptor subtypes expressed, tumour cells may be more sensitive to the death inducing or the trophic effect of ATP [21].

Recent developments have shown that the purinergic system is potentially beneficial in leukaemia [2, 16, 29]. ATP/P2X7 axis is very vital in regulating the proliferation and homing of leukaemic initiating cells. Purinergic receptors, P2XRs, particularly P2X7R, are elevated in patients with acute and chronic forms of leukaemia [2, 11, 15, 16]. Studies show that patients with acute and chronic forms of leukaemia express higher levels of P2X7 than the healthy bone marrow controls, with the P2X7-loss-offunction polymorphism linked to increased capacity to evade apoptosis and therefore, to progression of chronic leukaemia [2, 11, 15, 16]. Knockdown studies have shown that ATP in the leukaemia microenvironment decreases upon knockdown of P2X7 thus leading to the lysis of leukaemic cells [16, 18, 19, 29]. P2X7 and P2Y11 were also identified in leukaemic cell lines, HL-60 and NB-4 cell lines [11, 29]. Leukaemic cell lines also express elevated levels of ecto-enzymes CD39 and CD73 [2, 19, 22]. In another study, the levels of these enzymes that are concerned with purine degradation, CD73, ADA and purine nucleoside phosphorylase were varied in patients with different forms of chronic leukaemia [19, 21, 22]. This infers that these enzymes may be beneficial to the survival of leukemic cells and promote metastatic spread.

Since promotion of inflammatory response is a hallmark of cancer, and ATP and adenosine exert pro-inflammatory and anti-inflammatory activities, the development of innovative agonists and antagonists that target these specific receptors will be a relevant therapeutic mechanism in leukaemia. Adenosine analogues have been proposed as a possible differentiation-inducing agent against AML. Adenosine exerts cytotoxic effects on leukaemic cells after ectoenzymic breakdown of ATP, with ATP increasing the cation permeability of acute and chronic leukaemia lymphocytes and ADP increasing the calcium mobilization of myeloid leukaemia cells [23, 24]. A voltagegated potassium channel blocker, 4-aminopyridine, also induced apoptosis in human AML cells via increasing the calcium ions through P2X7 receptor pathways. Activation of the P2X7 receptor induces the formation of reactive oxygen species in erythroleukemia cells whilst P2X7 receptor agonists mediate cation uptake into human myeloid leukaemic cells [2, 23, 25, 29]. Evidence suggest that P2XR expression and activity may be relative to disease severity and depends on the level of activation as shown in

#### *Leukaemia: The Purinergic System and Small Extracellular Vesicles DOI: http://dx.doi.org/10.5772/intechopen.104326*

lymphocytes of patients with varying forms of B-cell CLL [16, 23, 25]. Remission in patients with CML has also been associated with a frameshift polymorphism of the P2X5 receptor that elicits an allogeneic cytotoxic T lymphocyte response. Low levels of ATP triggered anti-proliferative effects in AML cells and except for P2X2, P2X3 and P2X6, AML cells are known to express all subtypes of P2Rs thereby suggesting targeting ATP is a promising alternative therapy in AML with favourable outcome in patients [2, 19, 21, 22]. Taken together, ATP/P2R axis demonstrate sustainable leukaemia-initiating cell signaling activities thus suggesting targeting and inhibiting ATP/P2XR signaling may completely hinder leukemogenesis.

#### **4. The role of ectonucleotidases in leukaemia**

Metabolic stress due to hypoxia, ischemia, and pro-inflammatory signals lead to abundant release of ATP in the extracellular space within the tumour microenvironment. CD39 and CD73 are ectonucleotidases that catabolize high extracellular levels of ATP to produce molecules that modulate intracellular calcium levels and activate the purinergic receptors [22, 28]. Under normal conditions, these ectonucleotidases potentially stimulate immune cells to fight against the tumour. However, these ectonucleotidases are also capable of modulating immune response to favour tumour growth.

CD39 converts extracellular ATP or ADP to adenosine monophosphate (AMP) whilst CD73 converts AMP to adenosine [22, 28]. Adenosine then accumulates in the tumour microenvironment thereby supporting tumour growth by skewing immune cells towards tolerance. Therefore, these enzymes have significant effects in pathogenesis and progression of leukaemia by enhancing chemoresistance and homing of leukaemic cells [22]. In support of this, high expression of ectonucleotidases increase homing of leukaemic cells to protected niches, survival, and proliferation of leukaemic cells and modulation of immune cells towards tolerance.

Since these enzymes are deeply involved in the pathogenesis of leukaemia, their expression in leukaemia may be of prognostic value in leukaemia, marking disease progression and aggressiveness, and immunosuppression. They may act as reliable markers to monitor and distinguish specific cellular populations or subset of patients characterized by a different prognosis. CD39 and CD73 are expressed in leukaemia of lymphoid and myeloid lineage such as AML, B-ALL and CLL [2, 19, 22]. In these subtypes of leukaemia, there's a significant crosstalk between these leukaemic cells and the bystander non-leukaemic cells. Therefore, the levels of these ectonucleotidases, both on the leukaemic and bystander non-leukaemic cells, may be used as disease markers or prognostic factors.

In ALL, CD73 was identified as a differentially expressed molecule in a genomewide analysis, which compared 270 newly diagnosed ALL patients to normal B cell progenitors [30]. Following chemotherapy, flow cytometry validation revealed that these patients had upregulated expression of these enzymes thereby suggesting that CD73 could serve as a useful marker for minimal residual disease (MRD) and predicting ALL relapse following leukaemia treatment.

In AML, a more permissive immune environment is associated to high expression of CD39 and CD73, which favour leukaemia progression and aggressiveness [2, 19, 22]. AML cells release ATP following exposure to chemotherapy and upregulate CD39 expression on immune cells, and through this they modulate immune cells and skew them towards tolerance. In contrast, increased expression of CD73 on immune cells elicits a reawakening of the immune response. However, CD73 can also influence

leukaemic cell proliferation and aggressiveness via CAAT enhancer-binding protein alpha (CEBPA) gene [19, 22].

In CLL, an increase in expression level of CD39 in patients is linked to an increase in circulating leukaemic cells as this contributes to disease progression and aggressiveness [2, 19, 22]. Thus, expression of CD39 on these immune cells can be used as a marker for advanced disease stage and a predictive factor of treatment requirement. An increased expression of CD73 is significant in CLL cells and is associated with a higher cellular turnover, an increased recirculation to and from the lymphoid niche, a more aggressive clinical behavior and associated with time to disease progression after chemotherapy [2, 22]. Therefore, CLL cells are well equipped with CD39/CD73 that protect these cells from drug-induced toxicity thereby causing MRD, a reservoir that fuels disease progression. In addition, adenosine, which is produced by these enzymes, further acts on immune cells and polarize them towards tolerance thus sustaining leukaemic cell expansion. Upregulation of CD73 and adenosine is mediated by hypoxia thus influencing the metabolic adaptation of immune cells surrounding the tumour. This induces a switch towards glycolysis, with upregulation of glucose and lactate transporters and of lactate dehydrogenase and pyruvate kinase [18]. This further links hypoxia and CD73/adenosine in a common axis in which the tumour microenvironment is reshaped with tumour-supportive and immunosuppressive features.

#### **5. Leukaemia and small extracellular vesicles**

Extracellular vesicles (EVs) are nanosized lipid bilayered membrane vesicles that are virtually released by all cells and secreted at higher numbers in cancer cells. They can also be found in varied body fluids such as semen, blood, follicular fluid, and urine. Based on their size, intercellular origin and release mechanisms, these vesicles are grouped into microvesicles (≥200 nm and ≤1000 nm), exosomes (≤200 nm) and apoptotic bodies [31, 32]. These vesicles originate from plasma membrane (microvesicles) and endosomes (exosomes) from inward budding of the plasma membrane into the cytoplasm to form multivesicular bodies (MVBs). These vesicles are then released into the extracellular milieu to be transported to neighbouring or distant cells to induce phenotypic and functional changes in the recipient cells [31, 33]. There are also other types of small EV that have been mentioned in recent literature such as exomeres, large oncosomes and enveloped viruses [34, 35]. Nevertheless, the detection and classification of these vesicles remain an uphill task due to this heterogeneity.

The recipient cells internalize their vesicles via different routes in different ways such as clathrin-dependent and clathrin-independent pathways like phagocytosis, macropinocytosis, caveolin-mediated uptake or lipid raft-mediated internalization [36, 37]. However, the mechanism of vesicular uptake and internalization remains a conundrum, but it is postulated that it depends on some factors found on the surface membrane of the recipient cells and the vesicles such as surface proteins, glycoproteins and glycolipoproteins. Therefore, it can be inferred that these surface membrane factors promote adhesion of the vesicles to the recipient cells thus facilitating their internalization.

Upon uptake, the function and fate of these vesicles differ depending on the physiological or pathological state of origin cells releasing and receiving these vesicles [36, 37]. These vesicles often carry a cargo of bioactive molecules, including proteins, lipids, and nucleic acids, which they can transfer to the recipient cells to modulate and reprogram the bone marrow microenvironment to promote their survival and

#### *Leukaemia: The Purinergic System and Small Extracellular Vesicles DOI: http://dx.doi.org/10.5772/intechopen.104326*

induce biological effects in these cells [33, 38]. These vesicles play important roles in the regulation of immune stimulation or suppression that can drive inflammatory, autoimmune, and infectious disease pathology. EV could also alter the fate of their target cells by regulating gene expression through epigenetic changes in the recipient cells [36–38]. EV produced by cancer cells are in abundance in the tumour microenvironment and can enhance malignancy by transferring regulatory factors to normal cells within the tumour microenvironment. They also enhance anti-tumour immune response by inducing immunity to antigens that are carried by tumour EV.

In leukaemia, EV have a key role in the early stages of leukemogenesis as predominant EV population changes during leukaemia progression. Leukaemic cells utilize EV to transfer functional information to either MSC, HSC or myeloid progenitor cells in the BM microenvironment in amounts sufficient to induce phenotypic and functional changes in these cells, which are necessary for the development of leukaemia [33, 39]. These vesicles bridge the gap between leukaemic cells and the stromal cells that reside in the BM microenvironment thus initiating a crosstalk between these cells. This crosstalk between the leukaemic cells and stromal cells is crucial in remodelling and transforming the BM microenvironment into a leukaemia-permissive space, where leukaemic cells could proliferate, grow, and survive [29, 40, 41]. Leukaemia-derived EV transform and potentiate the phenotypic change of healthy MSC into cancer associated fibroblasts, which then proliferate, release inflammatory cytokines, and increase angiogenesis. As a result, leukemic cells survive and protect against apoptotic stimuli, including cytotoxic chemotherapy. AML-derived EV also alter the differentiation of stromal cells upon uptake thus leading to a decrease in the development of osteoblasts [42, 43].

The main mechanism through which EV promote the development and progression of leukaemia is not yet fully elucidated but much evidence supports through the delivery of microRNAs (miRNAs) into HSC within the BM microenvironment to change its characterization for developing into leukaemic cells [43–45]. These miRNAs are short non-coding RNAs that can mediate RNA silencing and regulate genes at the post-transcriptional level thus influencing the translocation of genes and signaling cascades. As thus, these small RNAs play vital roles in different cellular processes, including cell cycle, proliferation, angiogenesis, inflammation, immune reaction, and cell death [45, 46]. These small RNAs are either oncogenic or tumour suppressive, and once transferred from leukaemic cells to bone marrow microenvironment induce epigenetic changes that will support leukemogenesis. For example, in ALL, leukaemic cells release EV that carries miR-43a-5p to the bone marrow microenvironment and upon internalization, targets *Wnt* signaling pathway [45, 47]. This signaling pathway is vital for regulating haematopoiesis and induces the inhibition of osteogenesis in the bone marrow. Once suppressed, malignant HSC can then transform to leukaemic cells. MSC in the bone marrow microenvironment also secrete EV containing miR-21, which are then delivered into HSCs to enhance the development of B-ALL cells [40, 43]. These vesicle-derived miR-21 also interacts with transforming growth beta (TGF-β) to suppress the anti-tumour immune responses in the BM microenvironment.

In AML, the levels of serum-derived EV containing miR-10b, which is crucial in abrogating granulocytic/monocytic differentiation in HSCs, are elevated in patients compared to healthy individuals [12, 40, 43]. This suggests that miR-10b may play a vital role in inducing AML by enhancing the proliferative capacity of immature myeloid progenitors thus leading to the development of AML. Another miRNA, miR-4532 that targets the signal transducer and activator of transcription (*STAT-3*) signaling pathway, could also be transferred to HSCs from AML-derived EV to suppress the expression of leucine zipper downregulated in cancer 1 (*LDOC*), a well-known inhibitor of the *STAT-3* signaling pathway [47]. This leads to manipulation of the proliferative capacity of the cells in the BM microenvironment via this pathway. AML-derived EV also induce early leukemogenesis in myeloid progenitors through the transfer of miR-155 that inhibits *c-Myb* [31, 40, 47, 48]. *c-Myb* is a differentiating transcription factor in myeloid cells that induce differentiation arrest, which is critical in the development of AML. EV containing miR-155 and miR3-375 could also be transferred from MSC to AML cells to confer drug resistant phenotype against tyrosine kinase inhibitors [40]. Other miRNAs, such as miR-17-92 family, also induce chemoresistance in AML cells through activation of TGF-β and PI3K/Akt signalling axis. In CLL, EV released by leukaemic cells express a cell differentiating miRNA, miR-202-3p, which is taken up by stromal cells to influence the stroma cell transcriptome thereby resulting in altered growth characteristics [40].

However, miRNAs are not the only RNAs that have been implicated in the development and progression of leukaemia. Oncogenic messenger RNA (mRNA) derived from EV can be transferred to myeloid progenitors to encode nucleophosmin (*NPM1*) and FMS-like tyrosine kinase 3 *(FLT3*) gene with internal tandem duplication (ITD) (*FLT3-ITD*) thereby leading to the development of leukaemia [40]. Insulin-like growth factor 1 receptor (*IGF-1R*) and epidermal growth factor receptor (*EGFR*) mRNA enriched in AML vesicles are also transferred to stromal cells, leading to alterations in cell proliferation, secretion of growth factors and induction of downstream gene expression [31, 40, 47, 48]. AML-derived EV could also silence the expression of haematopoiesis-related growth factors such as *IGF-1,* C-X-C motif chemokine ligand 12 (*CXCL12), KIT* ligand and *IL-7*, and osteogenesis (osteocalcin; *OCN,* collagen type 1 alpha 1 chain; *Col1A1, IGF1*) whilst increasing the expression of genes that support AML growth such as Dickkop *wnt* signaling pathway inhibitor 1 (*DKK1*)*, IL-6* and C-C motif chemokine ligand 3 (*CCL3*) [31, 40, 47, 48]*.*

As thus, these enforce HSC committed to the myeloid progenitors. Anti-apoptotic proteins such as myeloid leukaemia 1 (*MCL-1*)*,* B-cell lymphoma 2 (*BCL-2*) and B-cell lymphoma extra-large (*BCL-XL*) could also be transferred to the immature myeloid blasts to guarantee their survival in the BM microenvironment [46, 48]. By modulating the promoting loss of apoptosis or cell death, EV play a vital role in drug resistance. Drug-resistant AML cells also transfer p-glycoprotein to induce chemoresistance in drug-sensitive leukaemia cells via their nucleic acid and multidrug resistance protein 1 (*MRP1*) cargo [40].

In ALL, leukemic vesicles reduce mitochondrial respiration and cause metabolic switch, from oxidative phosphorylation to anaerobic glycolysis, in MSC thereby changing their ability to respond to metabolic changes [46]. This could provide the desired energy for ALL development in the BM microenvironment. In CLL, these vesicles also mediate the activation of *AKT* signaling pathway, which in turn induces the production of vascular endothelial growth factor (*VEGF*) to enhance progression of CLL [40]. Furthermore, EV from MSC of CLL patients could rescue leukaemic cells from drug-induced apoptosis and enhance their migratory capacity [31, 40, 49]. Lastly, CML-EV also promote the growth and maintenance of leukemic cells via internalization of their own EV or activation of *EGFR* signaling following delivery of amphiregulin protein [40, 49, 50]. Amphiregulin is a ligand of EGFR and stimulates cell growth, survival, and migration via juxtacrine, autocrine and paracrine signaling. EV from CML also contain *BCR-ABL* transcripts that induce increased proliferation in MSC upon uptake [31, 40].

#### *Leukaemia: The Purinergic System and Small Extracellular Vesicles DOI: http://dx.doi.org/10.5772/intechopen.104326*

Since leukaemic EV are enriched in tumour signature molecules and cargo antigens and immunological molecules associated with leukaemia cells, the role of EV within the leukaemic microenvironment may provide insight for therapeutic advances. Drug resistance is a substantial impediment to successful treatment in leukaemia. Leukaemia EV could act as circulating biomarkers for diagnosis and detection of leukaemia [31, 40, 46]. Their evaluation in body fluids especially peripheral blood and urine could provide relevant information for early and highly sensitive method for detection and monitoring of leukaemia progression in patients. EV could also be harnessed for gene delivery and personalized therapy in leukaemia [31, 50]. EV offer beneficial characteristics that synthetic vectors cannot such as high physiochemical stability, long distance communication, inherent cell signaling, cell-to-cell communication and bioactive delivery whilst protecting the interior cargo from stress-induced necrosis and the environment. EV can cross the blood brain barrier and target neuronal cells; this may be useful in treating central nervous system (CNS)-associated leukaemia, which has poor prognosis [46]. EV can also be synthetically modified by attaching cell-specific targeting ligands to the EV surface to cargo chemotherapeutic drugs directly to leukaemia cells thereby enhancing their functionality, specificity of cell targeting and decrease adverse immune response. Chemotherapeutic drugs such as imatinib and paclitaxel have been incorporated into EV and delivered to target IL-3 receptor on the CML blasts [40]. However, it is important to mention that the study of EV as biomarkers in clinical medicine is still a new field. No standard methods have been established yet for proper enrichment and isolation of these circulating vesicles. Varied methods such as differential ultracentrifugation, density gradient centrifugation, polymer-facilitated precipitation, immune-affinity isolation, and size exclusion chromatography are currently employed for isolation of EV. This heterogeneity in isolation techniques causes variability in the results thus affecting the purity of EV and subsequent precise molecular characterization of EV.

#### **Author details**

Arinzechukwu Ude1 \* and Kelechi Okeke2

1 University of the West of England Bristol, Bristol, UK

2 London South Bank University, London, UK

\*Address all correspondence to: arinze\_ude@yahoo.co.uk

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 3
