### signal transduction pathways for angiogenesis process

Napione Lucia* ^{∗∗}*, Manini Daniele

*, Cordero Francesca*

^{∗}*, Horvath Andras*

^{∗∗∗}*, Picco Andrea*

^{∗}*, De Pierro Massimiliano*

^{∗∗}*, Pavan Simona*

^{∗}*, Sereno Matteo*

^{∗∗}*, Veglio*

^{∗}Andrea* ^{∗∗}*, Bussolino Federico

*and Balbo Gianfranco*

^{∗∗}*(*) Dipartimento di Informatica, Universit`a di Torino, Torino, Italy;*

^{∗}(**) Institute for Cancer Research and Treatment, Candiolo (TO), Italy;

(***) Molecular Biotechnology Center, Torino, Italy

Building and analyzing models of complex biological systems is a major challenge in modern computational biology that can greatly benefit from the application of model- ing frameworks developed within the field of computer science. In this paper we present a methodology for qualitative and quantitative analysis of a selected portion of signal transduction events involved in angiogenesis process. To overcome the problem that derives from the lack of experimental data from wet biology, we suggest ways of sim- plifying a detailed representation of the system that reduce the number of parameters of the model. Starting from a detailed Stochastic Petri net (SPN) model that accounts for all the reactions of the signal transduction cascade considered, using structural prop- erties combined with the knowledge of the biological phenomena, we propose a set of model reductions. The result is a simplified SPN (e.g., with a smaller number of param- eters) which is qualitatively validated through the preservation of most of the relevant properties computed from the original (detailed) model. To support our methodology from a quantitative point of view too, the temporal evolutions of a few relevant biolog- ical products represented in the models are computed, showing that for all of them a satisfactory agreement exists among the indications provided by the different models evaluated using coherent input parameters.

### 1 Introduction

Formal modeling is a central theme in systems biology in which mathematical mod- eling and simulation can play in important role. The Petri nets (PN) formalism [13] is a framework that allows the construction of a formal and clear representation of bio- logical systems based on solid mathematical foundation. This formalism permits the study of qualitative properties related to the structure of the model (e.g., the structure of a biological pathway). The variant of the PN, called Stochastic Petri Nets (SPN) [12][11][1] and characterized by the addition of timing and/or stochastic information, can be used for quantitative analysis (e.g., analysis that involve the rates in biochemical reactions). PN have been first proposed for the representation of biological pathways by Reddy et al [15]. Since their introduction, many other researcher constructed PN models of biological pathways [6] with the aim of using their representation to obtain qualitative information on the behavior of these systems, mostly via simulation [7, 4].

The interaction of qualitative and quantitative analysis is necessary to check a model for

consistency and correctness; following this idea, Heiner et al [5] proposed a methodol- ogy to develop and analyze larger biological model in a step-wise manner.

In this paper we present our experience in modeling signal transduction pathways for angiogenesis process using SPN. The general goal is to provide an approach that al- lows to analyze temporal dynamics of the biological phenomenon under study. The key contribution of this work is the simplification process that is essential to make the model feasible for the analysis. Besides that, this process represents the basis of the development of arguments useful for identifying both critical complexes and interac- tions that play a crucial role in the biochemical system under study. With respect to the framework proposed by Gilbert et al [2], where the main idea is to illustrate the com- plementarity among the three different ways of modeling biochemical network , i.e.

qualitative, stochastic and continuous, here we focus our attention on the definition of
the simplification process to limit the complexity of the model. The paper is organized
as follows. Section 2 provides an overview of PN, SPN and of their use in biochemical
systems. Section 3.1 describes the angiogenesis case study with special emphasis on
*two signal modules induced by the activation of the tyrosine kinase receptor KDR.*

Section 3.2 presents the approach we followed to build the SPN and Section 3.3 shows the formal and biological rules used in the simplification process as well as the resulting SPN. The quantitative analysis performed in order to verify the mathematical robust- ness of the simplified model is proposed in Section 3.4. We conclude with a discussion and an outlook of future works in Section 4.

### 2 Modeling formalism and solution techniques

The descriptions commonly applied in biology, where the relations among components are described by biochemical reactions, or by interactions of genes as well as by cell population interactions, are easy to transform into PN in which places correspond to genes/proteins/compounds (substrates) and transitions to their interactions.

2.1 Petri net representation for biochemical entities interactions

PN are a graphical language for the formal description of distributed systems with con- currency and synchronization. PN are bipartite graphs with two types of nodes, namely places and transitions, connected by directed arcs (see Definition 1). The state of the system is given by the quantities of the entities represented by the distribution of tokens over the places of the net. The dynamics of the model (starting from an initial marking) is captured by state changes due to firing of transitions and by the consequent movement of tokens over the places.

*Definition 1. [PN - syntax ]A Petri Net graph is a tuple (P, T, W, m*0), where
*– P is a finite set of places;*

*– T is a finite set of transitions;*

*– P and T are such that P ∩ T = ∅;*

*– W : (P × T ) ∪ (T × P ) → IN defines the arcs of the net and assigns to each of*
them a multiplicity;

– m0*is the initial marking which associates with each place a number of tokens.*

When applied in a system biology, places represent biochemical entities (enzymes,
compounds, etc.) and transitions represent the interactions among entities [15]. The
biological system we consider consists of biochemical reactions similar to that reported
in Fig. 1-a showing the PN representation scheme we adopted to describe all reactions
*of this type. Fig. 1-b represents the state evolution due to firing of transition k*53.

P ip3

P ten

P ip3: P ten

P ip3

P ten

P ip3: P ten

(a) (b)

k53 k53

k_{54} k_{54}

*Fig. 1. PN representation of reactions P ip3 + P ten** ^{k}*À

^{53}

*k*_{54}

*P ip3 : P ten.*

2.2 Analysis techniques based on structural properties

One main objective in system biology is to model and analyze temporal dynamics of the phenomenon under study. By using the SPN as the formalism for the construction of the model, the analysis is performed in two phases: the first provides qualitative infor- mation on the structure of the model and the second investigates quantitative properties including statistical indices describing the temporal behavior of the system.

The PN graph inspection can provide several functional properties of the model,
whose validity is true independently of the initial state of the system: such properties
are, for instance, the boundedness and the existence of structural deadlocks and traps
[13][1]. In deriving such kind of information an important role is played by the so called
*net’s invariants. There exist two kinds of invariants: place invariants (P-invariants) and*
transition invariants (T-invariants) [13]. In this paper we deal with P-invariants only.

A P-invariant is a weighted sum of tokens contained in a subset of places of the net
that remains constant through the entire evolution of the model starting from an initial
marking. The subset of places used for the computation of the P-invariant is the support
(i.e. the set of nonzero components) of a P-semiflow f which is a vector of nonnegative
weights assigned to all the places of the net and that is an integer and nonnegative
*solution of the matrix equation f W = 0.*

The interpretation of an invariant in a biological context, where tokens represent compounds, enzymes etc., is relatively simple: the set of places that support the semi- flow f represents a logical path in the PN graph along which a given kind of correlated matter is conserved.

2.3 Quantitative temporal analysis

One main objective in systems biology is to model and analyze temporal dynamics of the phenomenon under study. It is natural hence to apply an extension of PN that allows to introduce in the model temporal specifications. The most common timed extension of PN is SPN in which exponentially distributed random delays (interpreted as dura- tions of certain activities) are associated with the firings of the transitions. SPN are

qualitatively equivalent to PN, meaning that for their structural analysis it is sufficient to disregard their time specifications. The temporal stochastic behaviour of an SPN is isomorphic to that of a continuous time Markov chain (CTMC). This CTMC can be built automatically from the description of the SPN and corresponds to the behaviour of the biological system described by the Master Chemical Equations [3]. This “stochastic approach” based on SPN adopts a discrete view of the quantity of the entities and sees their temporal behaviour as a random process.

Another possibility is to adopt a “deterministic approach” with which the temporal behaviour of the entities is seen as a continuous and completely predictable process. In our contest we make use also of the deterministic approach because it allows for faster and simpler evaluation of the simplification process we propose for SPN.

The deterministic approach translates the interactions into a set of coupled, first- order, ordinary differential equations (ODE) with one equation per entity. These equa- tions describe how the quantity of the entities changes based on the speed and the struc- ture of the interactions and taking into account the quantity of the reactants as well.

Let us illustrate the procedure by a simple example. The ODEs corresponding to the
*reactions A + B* *→ C and B + C*^{k}^{1} *→ A are*^{k}^{2}

*dX**A**(t)*

*dt* *= −k*1*A(t)B(t) + k*2*B(t)C(t),* *dX**B**(t)*

*dt* *= −k*1*A(t)B(t) − k*2*B(t)C(t),*
*dX**C**(t)*

*dt* *= k*1*A(t)B(t) − k*2*B(t)C(t)*

*where X**i**(t) denotes the quantity of reactant i at time t. Having the ODEs and the initial*
quantities of the entities, numerical integration of the ODEs is applied to calculate the
quantities at a given time instant.

### 3 A Stochastic Petri Nets based approach applied to signal transduction pathways for angiogenesis process

The approach we present is based on the construction of a SPN model for the analysis of a biological system on the angiogenesis task.

3.1 Biological case study definition

Angiogenesis, defined as the formation of new vessels from the existing ones, is a topic of great interest in all areas of human biology, particularly to scientists studying vascular development, wound healing, vascular malformation and cancer biology. Angiogenesis is a complex process involving the activities of many growth factors and relative recep- tors, which trigger several signaling pathways resulting in different cellular responses.

The ability of pro-angiogenic growth factor/receptor axis to induce multiple intracel- lular signal events makes them interesting subjects for modeling studies in the context of Systems Biology. The Vascular Endothelial Growth Factor (VEGF) family proteins are widely regarded as the most important growth factors involved in angiogenesis.

VEGF-A, a member of VEGF family, has been most carefully studied and is thought to
*be of singular importance. VEGF receptor-2 (KDR in humans) is thought to mediate*

most of VEGF-A’s angiogenic functions, including cell proliferation, survival, and mi-
*gration. The main pathway through which VEGF-A induces cell proliferation involves*
*the activation of P LCγ [14]. Activation of P LCγ promotes phosphatidylinositol 4,5-*
*bisphosphate (P ip*2*) hydrolysis giving rise to 1,2-diacylglycerol (DAG). VEGF-A-*
*induced cell survival is dependent on the activity of P I3K [8]. P I3K phosphory-*
*lates P ip*2 generating the second messenger phosphatidylinositol-3,4,5-trisphosphate
*(P ip*3*). This recruits Akt to the membrane where it is activated through phosphoryla-*
*tion. Activated Akt induces cell survival.*

Although the core components of the main VEGF-A-induced pathways have been
identified, the molecular mechanisms involved need to be characterized in fine details
in order to better understand the flow of information. Indeed, a strong body of evidences
indicates the presence of common adaptor/effector proteins involved in different signal
*modules induced by VEGF-A/KDR axis, pointing out the difficulty to isolate a spe-*
cific pathway and suggesting the presence of common nodes which contribute to create
*an intricate signaling network. In particular, considering the KDR-proximal signaling*
events triggering the survival and proliferation pathways, a possible molecular can-
*didate able to form common nodes could be recognized in the adaptor protein Gab1*
[10][9]. Taking into account these notions, we wrote and analysed a system of bio-
chemical reactions based on the available biological information together with further
supposed mechanisms which could contribute to underline the presence of additional
molecular nodes in the context of VEGF-A-induced proliferation and survival path-
ways.

3.2 Model construction

In this section we discuss the approach we followed to represent the signal transduction
cascade using SPN. Consider the detailed biological model depicted in Fig. 2. These
*reactions describe KDR-proximal signaling events in the context of the survival and*
proliferation signal modules induced by receptor activation. In particular, reactions are
split into four blocks. The First Block represents the earliest signaling events which
*include KDR*^{∗}*(we use the star to denote that proteins are active), Gab1, and P ip*3. The
*Second Block concerns the regeneration of P ip*2, a common substrate for the two signal
modules that we are considering. The Third Block includes the reactions describing
the survival pathway, whereas the Fourth Block represents the proliferation pathway.

We analyzed the biochemical reactions in order to identify possible pathways and sub- pathways that describe embedded behaviors of the complete model. We denoted the reactions by means of their kinetic constants.

*In the model P ip*_{3} *and DAG have been considered as the end points of the sur-*
vival and proliferation pathways, respectively. Moreover, in the context of the survival
module it has also been included a signal step downstream the end point through which
*P ip*3 *is used to allow Akt activation, an index of cell survival. To analyze the signal*
transduction cascade, we focused our attention on the reactions that lead to the above-
mentioned end points. Following these sub-paths backward, we identified two reac-
*tions that produce P ip*3 *using P ip*2 *(i.e. k*21*and k*27*). The reactions involving P ip*2

*are k*19*k*20 *and k*25*k*26 *where P ip*2 *is a substrate of P i3k** ^{∗}*. The reactions that acti-

*vate P i3k are k*18

*and k*24. Hence we identified two different sub-pathways that lead to

Pip2 Production (Second Block)

P ip3 + P ten

k53

⇄

k54

P ip3:P ten
P ip3:P ten^{k}→ P ip2 + P ten^{55}

—————- P ten + P ip2

k_{56}

⇄

k57

P ten:P ip2

P ten:P ip2 + P ip3

k58

⇄

k_{59}

P ten:P ip2:P ip3
P ten:P ip2:P ip3^{k}→ P ten:P ip2 + P ip2^{60}

—————- Dag + E

k61

⇄

k62

Dag:E
Dag:E^{k}→ P ip2 + E^{63}

Survival (Third Block)

Gab1^{∗}:P ip3 + P i3k

k12

⇄

k_{13}

Gab1^{∗}:P ip3:P i3k

Gab1^{∗}:P ip3:P i3k + Kdr^{∗}

k14

⇄

k15

Kdr^{∗}:Gab1^{∗}:P ip3:P i3k

—————-
Kdr^{∗}:Gab1^{∗}+P i3k

k16

⇄

k17

Kdr^{∗}:Gab1^{∗}:P i3k
Kdr^{∗}:Gab1^{∗}:P i3k^{k}→ Kdr^{18} ^{∗}:Gab1^{∗}:P i3k^{∗}
Kdr^{∗}:Gab1^{∗}:P i3k^{∗}+P ip2

k19

⇄

k20

Kdr^{∗}:Gab1^{∗}:P i3k^{∗}:P ip2
Kdr^{∗}:Gab1^{∗}:P i3k^{∗}:P ip2^{k}→ Kdr^{21} ^{∗}:Gab1^{∗}:P i3k + P ip3

—————-
Kdr^{∗}:Gab1^{∗}:P ip3 + P i3k

k22

⇄

k23

Kdr^{∗}:Gab1^{∗}:P ip3:P i3k
Kdr^{∗}:Gab1^{∗}:P ip3:P i3k^{k}→ Kdr^{24} ^{∗}:Gab1^{∗}:P ip3:P i3k^{∗}
Kdr^{∗}:Gab1^{∗}:P ip3:P i3k^{∗}+P ip2

k25

⇄

k26

Kdr^{∗}:Gab1^{∗}:P ip3:P i3k^{∗}:P ip2
Kdr^{∗}:Gab1^{∗}:P ip3:P i3k^{∗}:P ip2^{k}→ Kdr^{27} ^{∗}:Gab1^{∗}:P ip3:P i3k + P ip3

—————- P ip3 + Akt

k_{28}

⇄

k29

P ip3:Akt
P ip3:Akt^{k}→ P ip3 + Akt^{30} ^{∗}
KDR-Receptor (First Block)

Kdr^{∗}+Gab1

k0

⇄

k_{1}

Kdr^{∗}:Gab1
Kdr^{∗}:Gab1^{k}→ Kdr^{2} ^{∗}:Gab1^{∗}
Gab1 + P ip3

k3

⇄

k_{4}

Gab1:P ip3

Kdr^{∗}+Gab1:P ip3

k_{5}

⇄

k6

Kdr^{∗}:Gab1:P ip3
Kdr^{∗}:Gab1:P ip3^{k}→ Kdr^{7} ^{∗}:Gab1^{∗}:P ip3
Kdr^{∗}:Gab1^{∗}:P ip3

k_{8}

⇄

k9

Gab1^{∗}:P ip3 + Kdr^{∗}

Kdr^{∗}:Gab1^{∗}+P ip3

k10

⇄

k11

Kdr^{∗}:Gab1^{∗}:P ip3

Proliferation (Fourth Block)

Kdr^{∗}+P lcγ
k31

⇄

k32

Kdr^{∗}:P lcγ

Kdr^{∗}:P lcγ
k33

→ Kdr^{∗}:P lc^{∗}γ

Kdr^{∗}:P lc^{∗}γ+P ip2

k34

⇄

k35

Kdr^{∗}:P lc^{∗}γ:P ip2
Kdr^{∗}:P lc^{∗}_{γ}:P ip2^{k}→ Kdr^{36} ^{∗}:P lcγ+Dag

—————-
Kdr^{∗}:Gab1^{∗}+P lcγ

k37

⇄

k38

Kdr^{∗}:Gab1^{∗}:P lcγ

Kdr^{∗}:Gab1^{∗}:P lcγ
k39

→ Kdr^{∗}:Gab1^{∗}:P lc^{∗}γ

Kdr^{∗}:Gab1^{∗}:P lc^{∗}γ+P ip2

k40

⇄

k41

Kdr^{∗}:Gab1^{∗}:P lc^{∗}γ:P ip2
Kdr^{∗}:Gab1^{∗}:P lc^{∗}γ:P ip2^{k}→ Kdr^{42} ^{∗}:Gab1^{∗}:P lcγ+Dag

—————-
Kdr^{∗}:Gab1^{∗}:P ip3 + P lcγ

k43

⇄

k44

Kdr^{∗}:Gab1^{∗}:P ip3:P lcγ

Kdr^{∗}:Gab1^{∗}:P ip3:P lcγ
k45

→ Kdr^{∗}:Gab1^{∗}:P ip3:P lc^{∗}γ

Kdr^{∗}:Gab1^{∗}:P ip3:P lc^{∗}_{γ}+P ip2

k46

⇄

k47

Kdr^{∗}:Gab1^{∗}:P ip3:P lc^{∗}_{γ}:P ip2
Kdr^{∗}:Gab1^{∗}:P ip3:P lc^{∗}γ:P ip2^{k}→ Kdr^{48} ^{∗}:Gab1^{∗}:P ip3:P lcγ+Dag

—————-
Gab1^{∗}:P ip3 + P lcγ

k49

⇄

k50

Gab1^{∗}:P ip3:P lcγ

Gab1^{∗}:P ip3 : P lcγ+Kdr^{∗}

k51

⇄

k52

Kdr^{∗}:Gab1^{∗}:P ip3:P lc^{∗}γ

**Pip2 Regeneration (Second Block)**

Fig. 2. Reactions of the detailed model.

survival effect. The distinguishing elements of these sub-pathways are the complexes
*KDR*^{∗}*:Gab1*^{∗}*and KDR*^{∗}*:Gab1*^{∗}*:P ip*3that belong to the First Block. Actually, the
*sub-pathways that determine the survival behavior are three since Gab1*^{∗}*:P ip*3leads to
*the complex KDR*^{∗}*:Gab1*^{∗}*:P i3k:P ip*3already involved in one of the identified sub-
pathways. Summarizing there are three sub-pathway that lead to survival effect starting
*from: KDR*^{∗}*:Gab1*^{∗}*, KDR*^{∗}*:Gab1*^{∗}*:P ip*3*and Gab1*^{∗}*:P ip*3.

Using the same approach of analysis we identified four different sub-pathways that
*lead to DAG production. Again the sub-pathways are distinguished by the compounds*
*belonging to the First Block, i.e.: KDR*^{∗}*, KDR*^{∗}*:Gab1*^{∗}*, KDR*^{∗}*:Gab1*^{∗}*:P ip*3and
*Gab1*^{∗}*: P ip*3. In this case the sub-pathways that lead to proliferation are four since
*there is a further sub-pathway starting from KDR** ^{∗}*. Notice that this last module has

*one additional sub-pathway that starts from KDR*

*. Using all these observations, we have been able to identify the sub-components of the detailed model that we exploited to build the SPN. The resulting net is illustrated in Fig. 3.*

^{∗}Note that the time evolution of this SPN is intuitively portrayed by a top-down view.

*On the top is depicted the place KDR** ^{∗}* that represents the starting point of the sig-

*nal cascade induced by its ligand. From the KDR*

*cascade start all the sub-pathways that characterize the proliferation and survival pathways. The places describing the end*

^{∗}*points of the respective pathways (i.e. DAG and P ip*3) are aligned on the bottom of the net. It is interesting to note that the sub-pathways we identified in the detailed model are represented in the SPN with structures such as the one outlined by a dashed box in Fig. 3, that corresponds to the reaction groups separated by continuous lines in Fig. 2.

We denote these Sub-Components by SC. Each SC involves:

– the binding between an enzyme and other species present in the cascade (i.e. tran-
*sitions k*31*k*32);

*– the enzyme activation (i.e. transition k*33);

*– the recruitment of the P ip*2*(i.e. transitions k*34*k*35);

– the production of the molecules representing the pathway end point and the enzyme
*deactivation (i.e. transition k*36).

The SPN defined in this section is a representation of the detailed biological model describing the signal transduction pathways under investigation. The aim of this study is to provide a tool that allows to analyze temporal dynamics of this network.

3.3 Model simplification and accuracy

The SPN we obtained requires a simplification process to limit the complexity of the
model in order to make it feasible for the analysis that helps to face the biological
problems we pointed out in Section 3.1. The presence of repeated structures in the SPN
model suggests that the biological model is characterized by the presence of several
similar reaction groups, and this observation can be used to identifying simplification
steps to be applied to the detailed model. In particular, the SCs shown in the previous
section are enzymatic kinetics reaction groups. Each of these groups can be written as
*the reactions set 1 where the enzyme E binds reversibly to the compound C.*

*E + C À EC → E*^{∗}*C* (1)

*E*^{∗}*C + S À E*^{∗}*CS*
*E*^{∗}*CS → EC + P*

**KDR***

**Gab1Pip3** **Plc**
**Gab1**

**Pi3k**

**KDR*Gab1Pip3**
**KDR*Gab1**

**KDR*Gab1***

**KDR*Gab1*Pip3**

**Gab1*Pip3**

**G*PgP3**

**Kd*G*PgP3**

**Kd*G*Pg** **Kd*Pg**

**Kd*Pg***

**Kd*Pg*P2**
**Kd*G*Pg*P2**

**Kd*G*Pg*P3P2**

**DAG**
**E**
**DAGE**
**Pip2**

**Kd*G*Pg*P3** **Kd*G*Pg***

**Akt***

**AktP3**

**Akt** **Pip3**

**PtP3P2**
**PtP3**

**Pten**

**PtP2**
**G*P3kP3**
**Kd*G*P3kP3**

**Kd*G*P3k**

**Kd*G*P3k***

**Kd*G*P3k*P2**

**Kd*G*P3k*P3P2**
**Kd*G*P3k*P3**

*K4*
*K3*

*K0* *K6* *K5*

*K1*

*K2* *K7*

*K11* *K10*

*K9* *K8*

*K16* *K17*

*K22* *K23* *K12*

*K13*

*K50* *K49* *K44* *K43* *K38* *K37* *K32* *K31*

*K52*
*K51*

*K45* *K39* *K33*

*K34*

*K36*
*K35*
*K40*

*K42*
*K41*
*K46*

*K48*
*K47*

*K61*
*K62*
*K63*

*K18* *K24*

*K15*
*K14*

*K21* *K27*

*K26*
*K25*
*K20*
*K19*

*K30*
*K28*
*K29*

*K60*
*K59* *K58*
*K57* *K56*

*K54*

*K53*

n_{1}

n_{2}

n3

n_{4}

n_{5}

n6 n7

n8

*K55*

γ

*Fig. 3. SPN representing the detailed model. Compound symbols: KDR ≡ Kd, Gab1 ≡ G,*
*P i3k ≡ P 3k, P lc**γ**≡ P g, P ip3 ≡ P 3, P ip2 ≡ P 2, P ten ≡ P t.*

*This complex EC become in irreversibly way E*^{∗}*C that means the enzyme is activated.*

*E*^{∗}*C binds reversibly to the substrate S (forming E*^{∗}*CS), before converting it into a*
*product P and releasing the complex EC. In order to simplify the detailed model we*
can represent each SC by the following couple of “merging” pseudo-reactions:

*E + S → E*^{∗}*S* (2)

*E*^{∗}*S + S*^{0}*À E + S + P*

Survival (Third Block)

Gab1^{∗}:P ip3 + Kdr^{∗}+P i3k^{k}→ Kdr^{12} ^{∗}:Gab1^{∗}:P ip3:P i3k^{∗}
Kdr^{∗}:Gab1^{∗}:P ip3:P i3k^{∗}+P ip2^{k}→ Gab1^{13} ^{∗}:P ip3 + Kdr^{∗}+P i3k + P ip3

—————-

Kdr^{∗}:Gab1^{∗}+P i3k^{k}→ Kdr^{14} ^{∗}:Gab1^{∗}:P i3k^{∗}
Kdr^{∗}:Gab1^{∗}:P i3k^{∗}+P ip2^{k}→ Kdr^{15} ^{∗}:Gab1^{∗}+P i3k + P ip3

—————-

Kdr^{∗}:Gab1^{∗}:P ip3 + P i3k^{k}→ Kdr^{16} ^{∗}:Gab1^{∗}:P ip3:P i3k^{∗}
Kdr^{∗}:Gab1^{∗}:P ip3:P i3k^{∗}+P ip2^{k}→ Kdr^{17} ^{∗}:Gab1^{∗}:P ip3 + P i3k + P ip3

—————- P ip3 + Akt

k18

⇄

k19

P ip3:Akt
P ip3:Akt^{k}→ P ip3 + Akt^{20} ^{∗}
KDR-Receptor (First Block)

Kdr^{∗}+Gab1

k0

⇄

k1

Kdr^{∗}:Gab1
Kdr^{∗}:Gab1→ Kdr^{k}^{2} ^{∗}:Gab1^{∗}
Gab1 + P ip3

k3

⇄

k4

Gab1:P ip3
Kdr^{∗}+Gab1:P ip3

k5

⇄k6

Kdr^{∗}:Gab1:P ip3
Kdr^{∗}:Gab1:P ip3→ Kdr^{k}^{7} ^{∗}:Gab1^{∗}:P ip3
Kdr^{∗}:Gab1^{∗}:P ip3

k8

⇄

k9

Gab1^{∗}:P ip3 + Kdr^{∗}

Kdr^{∗}:Gab1^{∗}+P ip3

k10

⇄

k11

Kdr^{∗}:Gab1^{∗}:P ip3

Pip2 Production (Second Block)

P ip3 + P ten

k29

⇄

k30

P ip3:P ten
P ip3:P ten^{k}→ P ip2 + P ten^{31}

—————- P ten + P ip2

k_{32}

⇄

k33

P ten:P ip2

P ten:P ip2 + P ip3

k34

⇄

k35

P ten:P ip2:P ip3
P ten:P ip2:P ip3^{k}→ P ten:P ip2 + P ip2^{36}

—————- Dag + E

k_{37}
k⇄38

Dag:E
Dag:E^{k}→ P ip2 + E^{39}

Proliferation (Fourth Block)

Kdr^{∗}+P lcγ
k21

→ Kdr^{∗}:P lc^{∗}γ

Kdr^{∗}:P lc^{∗}γ+P ip2^{k}→ Kdr^{22} ^{∗}+P lc^{∗}γ+DAG

—————-
Kdr^{∗}:Gab1^{∗}+P lcγ

k23

→ Kdr^{∗}:Gab1^{∗}:P lc^{∗}γ

Kdr^{∗}:Gab1^{∗}:P lc^{∗}γ+P ip2^{k}→ Kdr^{24} ^{∗}:Gab1^{∗}+P lcγ+DAG

—————-
Kdr^{∗}:Gab1^{∗}:P ip3 + P lcγ

k25

→ Kdr^{∗}:Gab1^{∗}:P ip3:P lc^{∗}γ

Kdr^{∗}:Gab1^{∗}:P ip3:P lc^{∗}γ+P ip2^{k}→ Kdr^{26} ^{∗}:Gab1^{∗}:P ip3 + P lcγ+DAG

—————-
Gab1^{∗}:P ip3 + Kdr^{∗}+P lcγ

k27

→ Kdr^{∗}:Gab1^{∗}:P ip3:P lc^{∗}γ

Kdr^{∗}:Gab1^{∗}:P ip3:P lc^{∗}γ+P ip2^{k}→ Gab1^{28} ^{∗}:P ip3 + Kdr^{∗}+P lcγ+DAG
**Pip2 Regeneration (Second Block)**

Fig. 4. Reactions after first step of simplification.

By exploiting this representation, we rewrite the reactions of the Third and the Fourth Blocks as shown in Fig. 4, and we use them for simplifying the SPN obtaining the net depicted in Fig. 5. Note that in this SPN any SC is now represented by a sequence of transition-place-transition, such as the one outlined by the dashed box, again. These SCs are defined by reaction groups separated by continuous line in Fig. 4. A further step in the simplification process is based on the representation provided by reaction 3:

*E + S + S*^{0}*→ E + S + P* (3)

The application of this observation to reactions of the Third and Fourth Blocks provides

n_{1}

n2

n3

n_{4}

n5

n6

n7

n8

**Gab1Pip3**
**Gab1**

**KDR*Gab1Pip3**
**KDR*Gab1**

**KDR*Gab1***

**KDR*Gab1*Pip3**

**Kd*G*P3Pg***

**Kd*G*Pg***

**Kd*G*P3k***

**Kd*G*P3k*P3** **Kd*G*P3k*P3-alt** **Kd*G*P3Pg*-alt**
*K4*

*K3*

*K0* *K6* *K5*

*K1*

*K2* *K7*

*K11* *K10*

*K9* *K8*

*K14*

*K16* *K12*

*K13*
*K15*

*K17* *K28* *K26* *K24* *K22*

*K23*
*K25*

*K27* *K21*

*K20* *K19*

*K35* *K34*
*K29*

*K30* *K31*

*K33* *K32* *K39*

*K37*
*K38*

*K36*
*K18*

**Kd*Pg***

**KDR***

**Plc**
**Pi3k**

**Gab1*Pip3**

**DAG**

**E**
**DAGE**
**Pip2**

**Akt*** **AktP3**

**Akt**
**Pip3**

**PtP2P3**
**PtP3**
**Pten**

**PtP2**

γ

Fig. 5. SPN obtained after the first step of simplification (Simplification1).

the reactions illustrated in Fig. 6, utilized to define the new simplified net illustrated in
Fig. 7. Note that in this SPN any SC is now represented by a singular transition such as
*the one oulined again by the dashed box. Moreover reactions k*29*k*30*, k*31*and k*37*k*38,
*k*39*of the Second Block are simplified following the scheme: E + S → E + P .*
The definition of structural criteria that we used to guide the process of simplification
of the model helped us to verify that the reduced models maintain the biological sig-
nificance of the original one and represent a good approximation of its behavior. The
conservation laws represented by the P-invariants can be used as a criterion to justify
formally the simplification process. We compute the P-invariants for the SPN describ-
ing the detailed model (Fig. 3) and we verified its boundedness. Then, we performed
the same analysis on the SPN corresponding to the simplified models (Figures 5 and 7)
and we verified that they were still bounded.

These structural properties, that from a biological point of view represents the law of conservation of mass through the equilibrium aspect, point out that the amount of the compounds described by places in any SPNs (original and simplified) cannot increase indefinitely. We found the same eight invariants in all the three nets. They are:

*– one P-invariant including KDR** ^{∗}*and the complexes containing it that are present in
the sub-pathways (both in proliferation and in survival) that bring to the recruiting

*of substrate P ip*2;

*– one P-invariant including Gab1 and the complexes containing it that are present in*
the sub-pathways (both in proliferation and in survival) that bring to the recruiting
*of substrate P ip*2;

*– one P-invariant including Akt and the complexes that lead to its activation;*

Pip2 Production (Second Block)

P ip3 + P ten^{k}→ P ten + P ip2^{22}

—————- P ten + P ip2

k_{23}

⇄

k_{24}

P ten:P ip2 P ten:P ip2 + P ip3

k_{25}

⇄

k_{26}

P ten:P ip2:P ip3
P ten:P ip2:P ip3^{k}→ P ten:P ip2 + P ip2^{27}

—————-
Dag + E^{k}→ P ip2 + E^{28}

Proliferation (Fourth Block)

Kdr^{∗}+P lcγ+P ip2^{k}→ Kdr^{18} ^{∗}+P lcγ+DAG

—————-

Kdr^{∗}:Gab1^{∗}+P lcγ+P ip2^{k}→ Kdr^{19} ^{∗}:Gab1^{∗}+P lcγ+DAG

—————-

Kdr^{∗}:Gab1^{∗}:P ip3 + P lc_{γ}+P ip2^{k}→ Kdr^{20} ^{∗}:Gab1^{∗}:P ip3 + P lc_{γ}+DAG

—————-

Gab1^{∗}:P ip3 + Kdr^{∗}+P lcγ+P ip2^{k}→ Gab1^{21} ^{∗}:P ip3 + Kdr^{∗}+P lcγ+DAG
KDR-Receptor (First Block)

Kdr^{∗}+Gab1

k_{0}

⇄

k_{1}

Kdr^{∗}:Gab1
Kdr^{∗}:Gab1→ Kdr^{k}^{2} ^{∗}:Gab1^{∗}
Gab1 + P ip3

k_{3}

⇄

k_{4}

Gab1:P ip3
Kdr^{∗}+Gab1:P ip3

k_{5}

⇄

k_{6}

Kdr^{∗}:Gab1:P ip3
Kdr^{∗}:Gab1:P ip3→ Kdr^{k}^{7} ^{∗}:Gab1^{∗}:P ip3
Kdr^{∗}:Gab1^{∗}:P ip3

k_{8}

⇄

k_{9}

Gab1^{∗}:P ip3 + Kdr^{∗}
Kdr^{∗}:Gab1^{∗}+P ip3

k_{10}

⇄

k_{11}

Kdr^{∗}:Gab1^{∗}:P ip3

Survival (Third Block)

Gab1^{∗}:P ip3 + Kdr^{∗}+P i3k + P ip2^{k}→ Gab1^{12} ^{∗}:P ip3 + Kdr^{∗}+P i3k + P ip3

—————-

Kdr^{∗}:Gab1^{∗}:P ip3 + P i3k + P ip2^{k}→ Kdr^{13} ^{∗}:Gab1^{∗}:P ip3 + P i3k + P ip3

—————-

Kdr^{∗}:Gab1^{∗}+P i3k + P ip2^{k}→ Kdr^{14} ^{∗}:Gab1^{∗}+P i3k + P ip3

—————- P ip3 + Akt

k_{15}

⇄

k_{16}

P ip3:Akt
P ip3:Akt^{k}→ P ip3 + Akt^{17} ^{∗}

**Pip2 Regeneration (Second Block)**

Fig. 6. Reactions after second step of simplification.

*– one P-invariant including the two pathways end points, i.e. P ip*3 *and Dag, and*
*P ip*2that is the common substrate in both pathways. In this invariant are also in-
*cluded the cascade complexes containing P ip*3;

*– each enzyme present in the model (P i3k, P lcγ, P ten, E) has an invariant includ-*
ing the complexes containing it.

Due to the simplification process, the P-invariants of the simplified SPNs corre- spond to P-semiflows with smaller supports. The analysis we performed above allowed us to consider the simplified models valid from a qualitative point of view.

3.4 Model analysis for validation

The simplification process proposed in Section 3.3 results in SPNs which maintain the qualitative properties of the original, detailed SPN. In this section we report in silico experiments that were performed in order to validate the robustness of the simplifica- tions from the point of view of quantitative properties. The validation was performed applying the “deterministic approach” described in Section 2.3. We compared the tem- poral behaviour of the detailed SPN with respect to those of the simplified SPNs in case of several different initial markings and several sets of transition rate parameters.

Throughout the comparisons we concentrated on three important entities, in particu-
*lar, the end point compounds, P ip*3 *and Dag, and the substrate P ip*2 which is com-
mon to both the survival and the proliferation pathways. Hereinafter we report on two
cases which illustrate the obtained results. The initial condition is identical for the two

**KDR***

**Plc**
**Gab1Pip3**
**Gab1**

**Pi3k**

**KDR*Gab1Pip3**
**KDR*Gab1**

**KDR*Gab1***

**KDR*Gab1*Pip3**

**Gab1*Pip3**

**DAG**

**E**

**PtP2P3**
**Pip2**

**Akt***

**AktP3**
**Akt**

**Pip3**

**Pten**
*K4*
*K3*

*K0* *K6* *K5*

*K1*

*K2* *K7*

*K11* *K10*

*K9* *K8*

*K12*
*K13*

*K14* *K19* *K18*

*K16* *K15*

*K17*

*K26*
*K23*

*K21*
*K20*

*K27*
*K25*
*K24*
n_{2}

n3

n_{4}

n5

n6 n7

n8

n_{1}

*K22*

**PtP2**
*K28*

γ

Fig. 7. SPN obtained after the second step of simplification (Simplification2).

*cases: for all three SPNs (see Figures 3, 5 and 7) we use the initial marking n*1 = 2,
*n*2 *= n*3 *= n*4 *= 1, n*5 *= 20, n*6 *= n*7 *= n*8 = 1. In the first case the transition
rates are such that the transitions along the survival pathway are ten times faster than all
the others. Figure 8A depicts the temporal behaviour for the detailed model. With these
*parameters the concentration of P ip*3*increases, the concentration of P ip*2 decreases
*and the concentration of DAG remains low. The temporal behaviour of the simplified*
models, depicted in Figures 8B-C, shows the same major characteristics. In the second
case the transitions along the proliferation pathway are ten times faster than all the oth-
ers. Figures 8E-F-G depict the temporal behaviour for the three models. Also in this
*case, the major characteristic, i.e. the fact that the concentration of DAG prevails over*
*the concentration of P ip*2*and P ip*3, is maintained. The general agreement among the
results of all these models was also tested by computing the steady state distribution of
*tokens in the places corresponding to the end-point compounds P ip*3and Dag and to
*the substrate P ip*2 obtained from the solutions of the Continuous Markov Chains cor-
responding to all the SPN models we have constructed (that we do not report in detail
in this paper due to space constraints).

In the experiments reported by now, the rate of a given transitions of the simplified
SPNs was simply set equal to the rate of the corresponding transition of the detailed
SPN. As a result, even if the overall characteristics are maintained, the shape of the
curves can be rather different and the dynamics take place on different time scales. Fo-
cussing our attention on the curves corresponding to the first set of parameters (Figures
*8A-B-C), we can notice that the crossing of the two concentration curves for P ip*2,

*which decreases, and P ip*3that grows takes place at time instants that are of the same
order of magnitude in all the three cases. A more important difference, however, is ob-
*served when we concentrate on the dynamics of DAG that shows a small initial growth*
followed by a descent to a value next to zero. For this case, the most simplified model
predicts an important initial climb that makes the shape of the curve quite different from
the others (see Figure 8C). Turning our attention to the curves corresponding to the sec-
ond set of parameters ( Figures 8E-F-G), we can notice that all the models predict a
*crossing between the concentration of P ip*2which decreases and the concentration of
*DAG that instead grows. With these parameters the concentration of P ip*3 remains
always extremely small. In this case the predictions of the more compact model are
quantitatively quite different since the crossing is reported to happen quite sooner and
the shapes of the curves are very different.

In order to mimic better the temporal behaviour of the detailed SPN, we applied an optimization technique to determine the transition rates for the simplified SPNs.

We used the nonlinear optimization function of MatLab to find these transition rates with which the curves resulting from the simplified SPNs became closer to the curves resulting from the detailed SPN. The results are illustrated for the SPN obtained after the second step of simplification (Simplification2) in Figure 8D and 8H. It can be seen that by properly setting the transition rates, even the more simplified SPN (Simplification2) can mimic quite precisely the behaviour of the detailed model (compare the diagrams A with D and E with H of Figure 8).

### 4 Discussion

The formal description of biological systems often leads to the definition of complex models. These models are difficult to deal with both because they have a large number of parameters that must be determined and because their state space can be enormous. It is necessary, hence, to simplify the original, detailed models in order to obtain compact representations that can still be used for a reliable analysis of the dynamic behaviour of the biological system. Consequently, both the model construction and the analysis process must be supported by appropriate tools that allow for structured handling of the detailed models, for finding the proper simplification scheme, and for validating the performed simplifications.

In this paper we showed how to use structural properties of SPNs and biochemi- cal properties of the system in guiding the simplification process. The procedure was presented through a case study, namely, the model of signaling transduction pathways involved in the angiogenesis process. We showed that the procedure results in simplified SPNs that are able to mimic precisely the temporal behavior of the detailed SPN.

One non trivial step is to determine the transition rates in the simplified SPNs in such a way that the resulting temporal behavior is a good approximation of that of the detailed SPN. In this work we faced this problem by applying optimization.

In the future, on the basis of the simplification schemes presented in this report, we plan to work on generalizations of these reduction steps which will allow to operate on other portions of the detailed model and on the identification of rules concerning the relations existing among the corresponding rates of the detailed and simplified models.

0 2 4 6 8 10 12 14 16 18 20 0

2 4 6 8 10 12 14 16 18 20

DAG Pip3 PiP2

*time*

*concentration*

0 5 10 15

0 2 4 6 8 10 12 14 16 18 20

DAG Pip3 PiP2

*time*

*concentration*

**A** **E**

0 5 10 15

0 2 4 6 8 10 12 14 16 18 20

DAG Pip3 PiP2

*time*

*concentration*

0 0.5 1 1.5 2 2.5

0 2 4 6 8 10 12 14 16 18 20

DAG Pip3 PiP2

*time*

*concentration*

**B** **F**

0 1 2 3 4 5 6 7

0 2 4 6 8 10 12 14 16 18 20

DAG Pip3 PiP2

*time*

*concentration*

0 1 2 3 4 5 6 7 8

0 2 4 6 8 10 12 14 16 18 20

DAG Pip3 PiP2

*time*

*concentration*

**G**
**C**

**Detailed Model**

**Simplified Model 1**

**Simplified Model 2**

**Detailed Model**

**Simplified Model 1**

**Simplified Model 2**

**Optimized Model**

0 2 4 6 8 10 12 14 16 18 20

0 2 4 6 8 10 12 14 16 18 20

DAG Pip3 PiP2

*time*

*concentration*

0 5 10 15

0 2 4 6 8 10 12 14 16 18 20

DAG Pip3 PiP2

*time*

*concentration*

**Optimized Model**

**D** **H**

Fig. 8. The left (right) column shows the ODE results related to the first (second) set of parame- ters.