Solutions to (4.23) for which the real part of *σ* is positive will grow; this is the classic Turing instability problem (Turing, 1952). For the Turing stability problem, one considers the case for which Λ has only negative eigenvalues and **M***s e*, the symmetric part of **M***e*, has only positive eigenvalues. (This latter condition will hold if and only if *χ*(I*e*, C*e*) < 2 √*μIμC*, and this inequality follows from the conditions *C*∇*u* > 0 and *C*∇*w* > 0 appearing in theorem 4.1.)

For any domain Ω, the first eigenpair is *λ*0 = 0 and *φ*0 = *constant*, and it is well known that there is an enumerable set of positive eigenvalues 0 < *λ*1 < *λ*2 < ··· and corresponding eigenfunctions {*φλn* } that form an orthonormal basis for *L*2(Ω). In the case that Λ has only negative eigenvalues, an instability must come from one of the larger eigenvalues. Unfortunately, finding these eigenvalues explicitly for a general domain is not possible. For an annulus (**R**2), or an annular cylinder (**R**3), they can be found by separation of variables. The corresponding eigenmodes in these cases are nonaxisymmetric suggesting that lesion initiation should also be nonaxisymmetric—this is consistent with clinical observations.

For the present case with no transport of any species across the endothelial layer, we can study the effect of the antioxidant level on the stability of the healthy state. If *Mφ*0 in equation (4.1) is replaced with *Mφ*0C (to make the source explicitly dependent on the chemoattractant), or if *Mφ*0 = 0, then the equilibrium solution is (I*e*, D*e*, C*e*,L*e*,L*oxe*, R*e*)=(0, 0, 0, L*e*,L*oxe*, R*e*).

modeling process. With this in mind, adaptations are easily included as our understanding of this complex medical process increases. We believe that mathematical modeling provides a useful tool to meet the goals of medical research on atherogenesis—identifying vulnerability to disease, development of treatments, and promotion of preventative interventions. Computer simulation (*in silico* analysis) requires a model consistent with and able to capture

Illuminating Atherogenesis Through Mathematical Modeling 67

Here, we have also studied the model by performing stability analyses under two different assumptions regarding the supply of inflammatory components—macrophages, chemotactic chemokines, and LDL. Taking the vasa vasorum as the sole source of these species, we arrive at a distinct set of inequality conditions on the system parameters that will guarantee that perturbations off of the healthy equilibrium state will decay. Bio-medically, the perturbations are interpreted as the start of inflammation, and the starting equilibrium as a disease-free state. A stable equilibrium is then seen as representing a cellular configuration that is robust—where a lesion is unlikely to develop in the short term. An unstable one suggests that (bio-chemically) the location is vulnerable to atherogenesis and the potential for development of a fibrofatty lesion or latter fibrous plaque. In addition to the positive stability criteria obtained using the energy estimate, we offer a negative result in the form of construction of an instability example. This latter condition highlights the inflammation mitigating effects of antioxidant presence and the significant interplay between chemotaxis and diffusion when

We also raise this same stability question under the assumption that the supply of inflammatory components is from influx from the blood flow via the endothelial interface. We again produce several inequalities that when satisfied by the system parameters ensure that the equilibrium solution is linearly asymptotically stable to small perturbations. Of particular interest in this latter case is the stabilizing effect of reverse transport of native LDL from the intima back to the blood stream. That reverse transport of LDL is stabilizing is not surprising given the corruptive nature of oxidized LDL on macrophage function. Our finding further supports the development of treatment modalities aimed at not only reducing serum LDL levels but at facilitating reverse transport of cholesterol (Superko, 2006). Although the conditions are numerous, clinical values of the various parameters can be easily compared in

The availability of clinical values for several parameters is lacking. Moreover, the parameters appearing in table 1 need not be constant, and determination of appropriate functional forms is an important and difficult task. This will require a process of "fine tuning" through

Acosta G. & Durán, R. G. (2003). An optimal Poincaré inequality in *L*1 for convex domains,

Baek, S., Gleason, R. L., Rajagopal, K. R., & Humphrey, J. D. (2007). Theory of small on large:

Boullier, A., Bird, D. A., Chang, M., Dennis, E. A., Friedman, P., Gillotte-Taylor, K., Hörkkö, S.,

Potential utility in computations of fluidâA ¸ ˘ Ssolid interactions in arteries *Comput.*

Palinski, W., Quehenberger, O., Shaw, P., Steinberg, D., Terpstra, V., & Witztum, J. L. (2001). Scavenger receptors, oxidized LDL, and atherosclerosis, *Ann. N. Y. Acad. Sci.*

light of the various inequalities derived and presented in theorems 4.1 and 4.2.

collaboration with clinicians and experimental scientists.

*Proc. Am. Math. Soc.* 132(1): 195–202.

*Methods Appl. Mech. Engrg.* 196(31-32): 3070–3078

**6. References**

947: 214–223

the characteristics of disease as observed *in vivo*.

the antioxidant level becomes negligible.

We can, after some lengthy calculations, show that in the limiting cases as *Aox* → 0+ and *Aox* → ∞ (Ibragimov et al., 2010b)

$$
\mathcal{L}\_{\rm oxx} \propto A\_{\rm ox}^{-1} \quad \text{and} \quad \mathcal{R}\_{\varepsilon} \propto p\_{\rm R} \quad \text{as} \quad A\_{\rm ox} \to 0^+
$$

and

$$
\mathcal{L}\_{\alpha \varkappa \varepsilon} \propto A\_{\alpha \chi}^{-2} \quad \text{and} \quad \mathcal{R}\_{\varepsilon} \propto A\_{\alpha \chi}^{-1} \quad \text{as} \quad A\_{\alpha \chi} \to \infty.
$$

The latter result demonstrates that the antioxidants strongly control free radical production and LDL oxidation. When studying the spectrum of Λ − *λ***M***e*, this asymptotic result shows that for *Aox* sufficiently large, the lower 3 × 3 block corresponding to the lipid chemistry produces no eigenvalues with positive real part. The question of most interest is what conditions are required for stability (or produce an instability) for the full system in the case that the lipid chemistry alone is stable. If (L*e*,L*oxe*, R*e*) is a stable equilibrium for the lipid equations in isolation, what effect does the antioxidant level have on the stability of the equilibrium (0, 0, 0,L*e*,L*oxe*, R*e*)? In the limit as the antioxidant level vanishes, the equilibrium will be unstable whenever

$$(\lambda \mu\_D + d\_2)(\lambda \mu\_\mathbb{C} + d\_3) < (\lambda \chi(0,0) - M\phi\_0) \left(\frac{\varepsilon p}{c}\right). \tag{4.24}$$

The critical and competing roles of diffusion and chemotaxis are prominent in this criterion providing an unstable equilibrium. For any positive eigenvalue *λ* and diffusive capacity of the chemoattractant *μC*, if the chemotactic sensitivity *χ*(0, 0) is large enough, the perturbation will grow away from the healthy equilibrium to some other state.

An analysis like the above can be employed with any variation of the system (3.1)–(3.8) provided the boundary conditions considered are completely homogeneous of Neumann type. For example, in (Ibragimov et al., 2008) the present authors consider a system characterized by two distinct macrophage phenotypes each subject to diffusion, chemotaxis, the potential to change phenotypes, but for which only one subspecies was subject to foam cell formation. We showed that the stability result provided therein—that analogous to theorem 4.1 here—was strongly dependent on the dominance of diffusion over chemotaxis. As is seen here, for any set of other parameter values, the chemotactic sensitivity coefficient can always be taken sufficiently large to produce an unstable equilibrium.

The question of unstable equilibria for the case with boundary transport can likewise be considered. Not surprisingly this presents a far more complicated situation mathematically. Even if we only consider constant equilibria, the special approach based on the ansatz (4.21) and the spectral analysis above does not yield any instability examples. Moreover, the coupled boundary conditions provide a Laplacian that is not self-adjoint and does not allow us the option to expand all of the perturbation variables using any single family of eigenfunctions. Nevertheless, a careful construction within the appropriate mathematical framework will provide conditions for which an unstable equilibrium exists. The effect of antioxidant concentration on stability can be analyzed. The reader is encouraged to see (Ibragimov et al., 2010b) for a construction in this case.

#### **5. Conclusion**

The purpose of this work is twofold. We have formulated a mathematical model of the inflammatory process that characterizes atherogenesis. This model given in equations (3.1)–(3.8) is presented in general terms to provide a framework for the ongoing 18 Will-be-set-by-IN-TECH

We can, after some lengthy calculations, show that in the limiting cases as *Aox* → 0+ and

The latter result demonstrates that the antioxidants strongly control free radical production and LDL oxidation. When studying the spectrum of Λ − *λ***M***e*, this asymptotic result shows that for *Aox* sufficiently large, the lower 3 × 3 block corresponding to the lipid chemistry produces no eigenvalues with positive real part. The question of most interest is what conditions are required for stability (or produce an instability) for the full system in the case that the lipid chemistry alone is stable. If (L*e*,L*oxe*, R*e*) is a stable equilibrium for the lipid equations in isolation, what effect does the antioxidant level have on the stability of the equilibrium (0, 0, 0,L*e*,L*oxe*, R*e*)? In the limit as the antioxidant level vanishes, the

*ox* , and R*e* ∝ *A*−1

(*λμD* + *d*2)(*λμC* + *d*3) < (*λχ*(0, 0) − *Mφ*0)

will grow away from the healthy equilibrium to some other state.

be taken sufficiently large to produce an unstable equilibrium.

The critical and competing roles of diffusion and chemotaxis are prominent in this criterion providing an unstable equilibrium. For any positive eigenvalue *λ* and diffusive capacity of the chemoattractant *μC*, if the chemotactic sensitivity *χ*(0, 0) is large enough, the perturbation

An analysis like the above can be employed with any variation of the system (3.1)–(3.8) provided the boundary conditions considered are completely homogeneous of Neumann type. For example, in (Ibragimov et al., 2008) the present authors consider a system characterized by two distinct macrophage phenotypes each subject to diffusion, chemotaxis, the potential to change phenotypes, but for which only one subspecies was subject to foam cell formation. We showed that the stability result provided therein—that analogous to theorem 4.1 here—was strongly dependent on the dominance of diffusion over chemotaxis. As is seen here, for any set of other parameter values, the chemotactic sensitivity coefficient can always

The question of unstable equilibria for the case with boundary transport can likewise be considered. Not surprisingly this presents a far more complicated situation mathematically. Even if we only consider constant equilibria, the special approach based on the ansatz (4.21) and the spectral analysis above does not yield any instability examples. Moreover, the coupled boundary conditions provide a Laplacian that is not self-adjoint and does not allow us the option to expand all of the perturbation variables using any single family of eigenfunctions. Nevertheless, a careful construction within the appropriate mathematical framework will provide conditions for which an unstable equilibrium exists. The effect of antioxidant concentration on stability can be analyzed. The reader is encouraged to see (Ibragimov et

The purpose of this work is twofold. We have formulated a mathematical model of the inflammatory process that characterizes atherogenesis. This model given in equations (3.1)–(3.8) is presented in general terms to provide a framework for the ongoing

*ox* , and R*e* ∝ *pR*, as *Aox* → 0+

*ox* , as *Aox* → ∞.

 *cp*ˆ *c*

. (4.24)

*Aox* → ∞ (Ibragimov et al., 2010b)

equilibrium will be unstable whenever

al., 2010b) for a construction in this case.

**5. Conclusion**

and

L*oxe* ∝ *A*−1

L*oxe* ∝ *A*−2

modeling process. With this in mind, adaptations are easily included as our understanding of this complex medical process increases. We believe that mathematical modeling provides a useful tool to meet the goals of medical research on atherogenesis—identifying vulnerability to disease, development of treatments, and promotion of preventative interventions. Computer simulation (*in silico* analysis) requires a model consistent with and able to capture the characteristics of disease as observed *in vivo*.

Here, we have also studied the model by performing stability analyses under two different assumptions regarding the supply of inflammatory components—macrophages, chemotactic chemokines, and LDL. Taking the vasa vasorum as the sole source of these species, we arrive at a distinct set of inequality conditions on the system parameters that will guarantee that perturbations off of the healthy equilibrium state will decay. Bio-medically, the perturbations are interpreted as the start of inflammation, and the starting equilibrium as a disease-free state. A stable equilibrium is then seen as representing a cellular configuration that is robust—where a lesion is unlikely to develop in the short term. An unstable one suggests that (bio-chemically) the location is vulnerable to atherogenesis and the potential for development of a fibrofatty lesion or latter fibrous plaque. In addition to the positive stability criteria obtained using the energy estimate, we offer a negative result in the form of construction of an instability example. This latter condition highlights the inflammation mitigating effects of antioxidant presence and the significant interplay between chemotaxis and diffusion when the antioxidant level becomes negligible.

We also raise this same stability question under the assumption that the supply of inflammatory components is from influx from the blood flow via the endothelial interface. We again produce several inequalities that when satisfied by the system parameters ensure that the equilibrium solution is linearly asymptotically stable to small perturbations. Of particular interest in this latter case is the stabilizing effect of reverse transport of native LDL from the intima back to the blood stream. That reverse transport of LDL is stabilizing is not surprising given the corruptive nature of oxidized LDL on macrophage function. Our finding further supports the development of treatment modalities aimed at not only reducing serum LDL levels but at facilitating reverse transport of cholesterol (Superko, 2006). Although the conditions are numerous, clinical values of the various parameters can be easily compared in light of the various inequalities derived and presented in theorems 4.1 and 4.2.

The availability of clinical values for several parameters is lacking. Moreover, the parameters appearing in table 1 need not be constant, and determination of appropriate functional forms is an important and difficult task. This will require a process of "fine tuning" through collaboration with clinicians and experimental scientists.

#### **6. References**

Human Macrophages with Oxidized Low-Density Lipoprotein Is Associated with

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**4**

*Malta*

Lawrence M. Agius

**Parametric Determination of Hypoxic**

*Department of Pathology, Mater Dei Hospital, Tal-Qroqq,*

*University Of Malta Medical School, Msida,*

**Ischemia in Evolution of Atherogenesis**

Atherosclerosis constitutes a primarily destructive phenomenon inherently arising from dynamics of pathobiologic effect within the intima of elastic and muscular arteries. It is significant to view the development of elevated intimal lesions within dimensions of

Considerable interactivity evolves within plaques in consequence to neovascularization in particular. The outline evolution of individual atherosclerotic plaques would considerably modulate the dynamics of migration of smooth muscle cells to the intima and as a consequence of various agonists such as hypoxia, growth factors and coagulationanticoagulation-fibrinolysis systems. Also, insulin appears to exert toxicity on the vascular

System pathways constitute a representative sequence of events that depend on dysfunctional activation of endothelial cells. It is within scope of parameters of permeability and loss of endothelial cells that a full plethora of forms of injury converge as intimal cell proliferation and as deposition of protein matrix proteoglycans. Oxidative stress and chronic inflammation

The individual roles played by various agonist actions in the definition of the atherosclerotic plaque would evolve within the specificity of focal injury to the intima in particular. The convergence of such injuries appears a constitutive attribute of the variable expression of

Developmental parameters of modelling include the delineation of individual pathogenic events in terms that integrally reconstitute the modified anatomy of the individual atherosclerotic plaque. In this regard, Ghrelin improves endothelial function, lowers blood

Significance in terms of complicated plaques as constitutive pathways in modelling of plaques includes the essential interactivity of endothelium with smooth muscle cell trophic effect. Macrophages in particular implicate a series of converging events that sequentially re-define in repetitive form the dynamics of atherogenesis. Dyslipidemmia increases lipid

**1. Introduction**

ongoing further injury to the endothelium.

**2. System pathways of injury**

sequence prototypes in lesion demarcation.

pressure and regulates atherosclerosis (Zhang et al., 2011).

wall and possibly promote atherogenesis (Nandish et al., 2011).

promote diabetes, hypertension and atherosclerosis (Sewon et al., 2011).

content in foam cells found in atherosclerotic plaques (Wong et al., 2011).

### **Parametric Determination of Hypoxic Ischemia in Evolution of Atherogenesis**

Lawrence M. Agius

*Department of Pathology, Mater Dei Hospital, Tal-Qroqq, University Of Malta Medical School, Msida, Malta*

#### **1. Introduction**

22 Will-be-set-by-IN-TECH

70 Atherogenesis

Tabas, I. (2004). Apoptosis and plaque destabilization in atherosclerosis: the role of

Tall, A. R. (1998). An overview of reverse cholesterol transport, *Euro. Heart J.* 19(A): A31–A35 Taylor, C. A. & Humphrey, J. D. (2009). Open problems in computational vascular

Turing, A. (1952). The chemical basis of morphogenesis, *Phil. Trans. Royal Soc. Lond.* B237:

Veneziani, A. (1998). Mathematical and numerical modeling of blood flow problems, Ph.D.

Watanabe, A., Noguchi, N., Takahashi, M. & Niki, E. (1999). Rate constants for hydrogen

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Zhang, Y. & Wahl, L. M. (2006). Synergistic enhancement of cytokine-induced human

S12–S16

37–72

*Mech. Engrg.* 198(45-46): 3514–3523

Thesis, Politecnico di Milano, Italy

acid, *Chem. Lett.* 7: 613–614

E2, *J. Leukoc. Bio.* 79: 105–113

Medicine, Inc. 2 edition

*Trans.* 29: 358–362

macrophage apoptosis induced by cholesterol, *Cell Death and Differentiation* 11:

biomechanics: Hemodynamics and arterial wall mechanics, *Comput. Methods Appl.*

atom abstraction by *α*-tocopherol radical from lipid, hydroperoxide and ascorbic

monocyte matrix metalloproteinase-1 by C-reactive protein and oxidized LDL through differential regulation of monocyte chemotactic protein-1 and prostaglandin Atherosclerosis constitutes a primarily destructive phenomenon inherently arising from dynamics of pathobiologic effect within the intima of elastic and muscular arteries. It is significant to view the development of elevated intimal lesions within dimensions of ongoing further injury to the endothelium.

Considerable interactivity evolves within plaques in consequence to neovascularization in particular. The outline evolution of individual atherosclerotic plaques would considerably modulate the dynamics of migration of smooth muscle cells to the intima and as a consequence of various agonists such as hypoxia, growth factors and coagulationanticoagulation-fibrinolysis systems. Also, insulin appears to exert toxicity on the vascular wall and possibly promote atherogenesis (Nandish et al., 2011).

#### **2. System pathways of injury**

System pathways constitute a representative sequence of events that depend on dysfunctional activation of endothelial cells. It is within scope of parameters of permeability and loss of endothelial cells that a full plethora of forms of injury converge as intimal cell proliferation and as deposition of protein matrix proteoglycans. Oxidative stress and chronic inflammation promote diabetes, hypertension and atherosclerosis (Sewon et al., 2011).

The individual roles played by various agonist actions in the definition of the atherosclerotic plaque would evolve within the specificity of focal injury to the intima in particular. The convergence of such injuries appears a constitutive attribute of the variable expression of sequence prototypes in lesion demarcation.

Developmental parameters of modelling include the delineation of individual pathogenic events in terms that integrally reconstitute the modified anatomy of the individual atherosclerotic plaque. In this regard, Ghrelin improves endothelial function, lowers blood pressure and regulates atherosclerosis (Zhang et al., 2011).

Significance in terms of complicated plaques as constitutive pathways in modelling of plaques includes the essential interactivity of endothelium with smooth muscle cell trophic effect. Macrophages in particular implicate a series of converging events that sequentially re-define in repetitive form the dynamics of atherogenesis. Dyslipidemmia increases lipid content in foam cells found in atherosclerotic plaques (Wong et al., 2011).

Parametric Determination of Hypoxic Ischemia in Evolution of Atherogenesis 73

The central core of the plaque is one dominated by influences exerted by transformations in terms of neovascularization and as compounded maturation leading to lipid core formation within the plaque. Insulin resistance and cardiovascular pathology may share a common

It is highly significant to view the trophic attributes of injury to the endothelium as source of the evolutionary traits of emerging atherosclerotic plaques; indeed, Insulin-like growth factor-1 stabilizes the atherosclerotic plaque by altering smooth muscle phenotype (von der Thusen et al., 2011). Stages in sequence pathway maturation are central to the outline demarcation of individual plaques in a manner that depends integrally on dynamics of

Hemodynamic shear stress contributes to a redistribution of actin microfilaments within endothelial cells in a manner that modulates dysfunctional issues of activated

A proliferative smooth muscle cellular response is further significant in the maturation of the atherosclerotic plaque within such sequence steps as redefined injury to the

Stages in preparation for subsequent events in the outline of the atherogenesis process would include the delivery of injurious agents to the intima. The sequence pathways are significant parametric factors in defining the dimensions of the endothelial participation in atherogenesis. Endoplasmic reticulum stress and the unfolded protein response characterize

It is further to the evolving forms of agonist action that atherogenesis modifies in repeatedly

Neovascularization proves to be a centrally operative agonist in the modified development of atheroma formation and deposition. The added parameters of consequence appear to implicate a primarily evolutionary role for hypoxia and ischemia as plaque re-definition,

The inflammatory nature of the intimal deposits elicits a responsive panorama that implicates derivative attributes of low density lipoproteins and cholesterol and as extended participation of the neovascularization of the plaque. MicroRNA-29a targets lipoprotein lipase in oxidized low density lipoprotein and modulates cytokines and scavenger receptors

Hypoxia is itself essential for the formation of new vessels within the plaque with the production of growth factors and would additionally contribute to the intimal thickening as

Directional re-orientation of active dysfunctional states of endothelial cells compounds hypoxia and ischemia within the vascular intima. By-products of matrix proteoglycans and of lipid metabolism indicate the essential staging events in plaque maturation and as derivative phenomenon to further atherogenesis. Chemokines produced by endothelial cells are associated with leukocyte recruitment and angiogenesis in atherosclerosis (Speyer &

staged sequence the pathways of dysfunctional activation of the endothelium.

both morphologically and in dysfunctional forms of endothelial activation.

further evidence for staged representation of injury to the intima.

endothelial susceptibility to atherogenesis (Civelek et al., 2011).

genetic background (Bacci et al., 2011).

neovascularization of the plaque core.

**5. Cellular proliferative kinetics**

**6. Low-density lipoproteins**

(Chen et al., 2011)

Ward, 2011).

endothelium.

endothelium.

Growth factors are instrumental in terms of the emerging morphologic features of the early atherosclerotic plaque and as derivative phenomena of endothelial injury and permeability. The constitutive parameters of re-distribution of trophic effect particularly interact with neovascularization within the plaque core.

Developmental sequences of multifactorial type in atherogenesis are particularly prone to a staged outline evolution that permeates the intima and sustains injury to the endothelium. Such significant interactivity contributes to outline emergence of new sequences in trophic effect and as proliferation and migration of smooth muscle cells.

The macrophage is central to such interactivity and operates primarily in chemotaxis and trophism, and also in terms that dominantly influence in significant fashion the attributes of lipid foam cells. Plant-derived alpha-linolenic acid, for example, restricts plaque T-cell proliferation, differentiation and inflammatory activity (Winnik et al., 2011).

#### **3. Focality of inflammation**

The inflammatory infiltrates include a representative response to injury as atherogenesis further compounds injury to the vascular intima. The role of endothelium is implicated as dysfunctional response with increased permeability to monocytes in particular. Proteoglycans retain lipoproteins subendothelially (Anggraeni et al., (2011).

Such recruitment of novel forms of injury includes the transforming ability of protein matrix proteoglycans as integral constitution of the injurious agents. It is within the dimensional redistribution of such injury that the endothelium plays a prominent role in sequence selectivity and in modulation of parameters of redefinition of activated dysfunctional states of inflammatory cells within the plaque.

The focality of injury is particularly significant within the neovascularized core of the individual plaque and as parameters in the growth and maturation of smooth muscle cells. Reparative processes allow for a sequential remodelling within such system pathways as coagulation and cellular migration within the vascular intima.

The variability of delineation of injurious agents indicates an activation of new parameters as the plaque evolves. The overall confines further extend parameters in plaque modelling that permits responsive elements in the creation of multiple sequence pathways that evolve in their own right; for example, deletion of microsomal prostaglandin E2 synthase-1 retards atherogenesis (Wang et al., 2011).

#### **4. The individual smooth muscle cell/macrophage**

The secretory dynamics of smooth muscle cells and of macrophages attribute a central pathogenic role to foam cells within the intima. Both smooth muscle cells and macrophages are recognized source for foam cells that, in turn, predominate in the mature atheromatous plaque. On the other hand, the matrix proteoglycans are also central players in re-defining attributes of the injured endothelium.

The reactivity of macrophages within the protein matrix that accumulate within the intima allows for the emergence of multiple converging agonists that characterize endothelial cell activation. The development of subsequent new forms of injury transforms such endothelial dysfunctionality as parameters of maturation of the plaque. Interleukin 18 is involved in plaque destabilization and regulates the innate immune response (Yamaoka-Tojo et al., 2011).

The central core of the plaque is one dominated by influences exerted by transformations in terms of neovascularization and as compounded maturation leading to lipid core formation within the plaque. Insulin resistance and cardiovascular pathology may share a common genetic background (Bacci et al., 2011).

It is highly significant to view the trophic attributes of injury to the endothelium as source of the evolutionary traits of emerging atherosclerotic plaques; indeed, Insulin-like growth factor-1 stabilizes the atherosclerotic plaque by altering smooth muscle phenotype (von der Thusen et al., 2011). Stages in sequence pathway maturation are central to the outline demarcation of individual plaques in a manner that depends integrally on dynamics of neovascularization of the plaque core.

Hemodynamic shear stress contributes to a redistribution of actin microfilaments within endothelial cells in a manner that modulates dysfunctional issues of activated endothelium.

### **5. Cellular proliferative kinetics**

72 Atherogenesis

Growth factors are instrumental in terms of the emerging morphologic features of the early atherosclerotic plaque and as derivative phenomena of endothelial injury and permeability. The constitutive parameters of re-distribution of trophic effect particularly interact with

Developmental sequences of multifactorial type in atherogenesis are particularly prone to a staged outline evolution that permeates the intima and sustains injury to the endothelium. Such significant interactivity contributes to outline emergence of new sequences in trophic

The macrophage is central to such interactivity and operates primarily in chemotaxis and trophism, and also in terms that dominantly influence in significant fashion the attributes of lipid foam cells. Plant-derived alpha-linolenic acid, for example, restricts plaque T-cell

The inflammatory infiltrates include a representative response to injury as atherogenesis further compounds injury to the vascular intima. The role of endothelium is implicated as dysfunctional response with increased permeability to monocytes in particular.

Such recruitment of novel forms of injury includes the transforming ability of protein matrix proteoglycans as integral constitution of the injurious agents. It is within the dimensional redistribution of such injury that the endothelium plays a prominent role in sequence selectivity and in modulation of parameters of redefinition of activated dysfunctional states

The focality of injury is particularly significant within the neovascularized core of the individual plaque and as parameters in the growth and maturation of smooth muscle cells. Reparative processes allow for a sequential remodelling within such system pathways as

The variability of delineation of injurious agents indicates an activation of new parameters as the plaque evolves. The overall confines further extend parameters in plaque modelling that permits responsive elements in the creation of multiple sequence pathways that evolve in their own right; for example, deletion of microsomal prostaglandin E2 synthase-1 retards

The secretory dynamics of smooth muscle cells and of macrophages attribute a central pathogenic role to foam cells within the intima. Both smooth muscle cells and macrophages are recognized source for foam cells that, in turn, predominate in the mature atheromatous plaque. On the other hand, the matrix proteoglycans are also central players in re-defining

The reactivity of macrophages within the protein matrix that accumulate within the intima allows for the emergence of multiple converging agonists that characterize endothelial cell activation. The development of subsequent new forms of injury transforms such endothelial dysfunctionality as parameters of maturation of the plaque. Interleukin 18 is involved in plaque destabilization and regulates the innate immune response (Yamaoka-Tojo et al.,

neovascularization within the plaque core.

**3. Focality of inflammation**

of inflammatory cells within the plaque.

atherogenesis (Wang et al., 2011).

attributes of the injured endothelium.

2011).

effect and as proliferation and migration of smooth muscle cells.

proliferation, differentiation and inflammatory activity (Winnik et al., 2011).

Proteoglycans retain lipoproteins subendothelially (Anggraeni et al., (2011).

coagulation and cellular migration within the vascular intima.

**4. The individual smooth muscle cell/macrophage**

A proliferative smooth muscle cellular response is further significant in the maturation of the atherosclerotic plaque within such sequence steps as redefined injury to the endothelium.

Stages in preparation for subsequent events in the outline of the atherogenesis process would include the delivery of injurious agents to the intima. The sequence pathways are significant parametric factors in defining the dimensions of the endothelial participation in atherogenesis. Endoplasmic reticulum stress and the unfolded protein response characterize endothelial susceptibility to atherogenesis (Civelek et al., 2011).

It is further to the evolving forms of agonist action that atherogenesis modifies in repeatedly staged sequence the pathways of dysfunctional activation of the endothelium.

#### **6. Low-density lipoproteins**

Neovascularization proves to be a centrally operative agonist in the modified development of atheroma formation and deposition. The added parameters of consequence appear to implicate a primarily evolutionary role for hypoxia and ischemia as plaque re-definition, both morphologically and in dysfunctional forms of endothelial activation.

The inflammatory nature of the intimal deposits elicits a responsive panorama that implicates derivative attributes of low density lipoproteins and cholesterol and as extended participation of the neovascularization of the plaque. MicroRNA-29a targets lipoprotein lipase in oxidized low density lipoprotein and modulates cytokines and scavenger receptors (Chen et al., 2011)

Hypoxia is itself essential for the formation of new vessels within the plaque with the production of growth factors and would additionally contribute to the intimal thickening as further evidence for staged representation of injury to the intima.

Directional re-orientation of active dysfunctional states of endothelial cells compounds hypoxia and ischemia within the vascular intima. By-products of matrix proteoglycans and of lipid metabolism indicate the essential staging events in plaque maturation and as derivative phenomenon to further atherogenesis. Chemokines produced by endothelial cells are associated with leukocyte recruitment and angiogenesis in atherosclerosis (Speyer & Ward, 2011).

Parametric Determination of Hypoxic Ischemia in Evolution of Atherogenesis 75

Procedural and technical specificity of individual plaques illustrate dynamic turnover within plaques in terms particularly of agonists and cellular parameters in redefinition of atherogenesis. In this regard, Interferon-alpha upregulates expression of scavenger-A in

The specificity of the inflammatory response is sequentially consequent to the interchangeability of agonist-induced parameters in creating a microenvironment of hypoxia and

The contributing roles of endothelium especially in cases of trauma to the vessel wall would indicate the prototypical attributes in lesion emergence and of subsequent maturation of the atherogenesis phenomenon. Atherogenesis is contributory phenomenon to an ongoing migratory involvement of the intima. This is well-testified by smooth muscle cells that synthesize and secrete matrix proteoglycans, and oxidative stress also induces production of superoxide by endothelial cells with nitric oxide synthase

Significant participation in atherogenesis involves mirror-imaged targeting of multiple component systems within the vessel wall that developmentally integrate as regions of hypoxia and ischemia, including the thioredoxin system that correlates with cellular apoptosis in endothelial cell lines in hypoxic stress (Park et al., 2011). Neovascularization proves a permissive phenomenon in development of gradients of ischemia that redefine the individual plaque as compounding parameters of progression to further injury to the vascular wall. Permissive dynamics are characteristic of oxidation of lipids and particularly of low-density lipoproteins and as evolution of deposition within the intima. Targeting of subsets of cells indicates a selectivity process of progression within sequential pathways of incremental further injury to the vessel wall. Disrupted endoplasmic reticulum equilibrium engages the

System reproduction indicates vulnerability selectivity in the evolution of atheromatous plaques in terms ranging from cell kinetics to proliferative migration of smooth muscle cells directed to the intima and a sensitivity of endothelial cells to responsive pathway generation and trophic factor production. The macrophage system is especially representative of novel pathway events that induce a sequential series of models in manipulative further

Within such scopes of pathogenic representation, there would emerge a parametric remodelling based on aberrant reconstitution of injury as further projected by responses to injury to the intima and endothelium. Within such context, the unfolded protein response is

The operative essentiality of the intimal involvement in atheroma formation calls into operation the developmental dimensions of both endothelium and also of medial smooth muscle cells. The sequence attributes of multiple different pathways contribute to the subsequent emergence of activation phenomena as well represented by the macrophage and

implicated in all stages of atherogenesis and plaque progression (Lhotak et al., 2011).

unfolded protein response in such cells as monocytes (Carroll et al., 2011).

monocytes/macrophages with foam cell formation (Li et al., 2011).

ischemia centered on the intima.

**10. Cellular endothelial injury**

uncoupling (Zweier et al., 2011).

**11. Vulnerability issues**

**12. Pathway activation**

compromise of viability of the endothelial cells.

#### **7. Intimal remodeling**

It is as remodelling of the injury to the intima that hypoxia and ischemia further modulate the migration of smooth muscle cells within the intima. The increased permeability of the endothelium is significant as a redefined series of further injuries to the underlying intima.

The orientational redistribution of the agonists in atherogenesis redefine a central plaque contribution to increasing profiles of further hypoxia/ischemia and as evidential remounting of parameters of sequence effect. The multi-factorial injurious events are converging agonists in hypoxic/ischemic core regions of the individual atherosclerotic plaque. In such manner, the multi-staged evolution of plaques correlates with interactive dynamics of further injury within the intima.

Dynamics of action of oxidized lipids correlate closely with emerging new roles for agonist action in developing plaques. The interaction of variably participating agonists contrast with the intimal emergence of incremental hypoxia/ischemia in terms of increasing matrix proteoglycan deposition and cellular proliferation of smooth muscle cells in particular. Atherosclerosis affecting different topographic sites correlate with the type of hyperlipidemia (Van Craeyveld et al., 2011).

The platelet/coagulation systems are incremental sequence events as trophic influence in staged convergence of multiple agonists in intimal injury.

Distributional parameters are particularly significant in the dimensional targeting of the intima in terms of the vasa vasorum supplying the arterial wall.

Component systems of sequential impact would contribute to the emergence of positive feedback effect in agonist action. The endothelial cells participate by the production of various agents such as growth factors, in particular Platelet-Derived Growth Factor. The semblance of such influence dominantly re-characterizes the atherosclerotic plaque that trophically redefines the form of hypoxic/ischemic injury to the intima. miRNA –mediated epigenetic regulation may be implicated in atherogenesis, involving oxidized low-density lipoproteins (Chen et al., 2011).

#### **8. Hemodynamics**

Hemodynamics within the neovascularized core of the plaque allows for a developmental evolution in terms of so-called complications such as hemorrhage and rupture of the plaque and as staged representation of the endothelial cell injury. Consequential pathways of significance would confirm the agonist nature of hypoxia/ischemia in terms of further emergence of intimal deposition and of cellular proliferation and migration.

A response to injury permits role redefinition as emerging parameters in pathogenesis of the deposition of proteoglycans within the vascular intima. Matrix metalloproteinases participate in plaque destabilization and rupture. Their overexpression is an independent factor in the pathogenesis of acute coronary syndromes (Kulach et al., 2010). A proliferative response in particular illustrates the nature of the vascular wall injury that incrementally progresses as gradients of hypoxia and ischemia within the vessel wall.

#### **9. Gradient parameters**

Consequential involvement of the lipid deposition phenomenon integrally permits the establishment of gradient parameters of hypoxia/ischemia within the operative fields of emerging neovascularization in the plaque core.

Procedural and technical specificity of individual plaques illustrate dynamic turnover within plaques in terms particularly of agonists and cellular parameters in redefinition of atherogenesis. In this regard, Interferon-alpha upregulates expression of scavenger-A in monocytes/macrophages with foam cell formation (Li et al., 2011).

The specificity of the inflammatory response is sequentially consequent to the interchangeability of agonist-induced parameters in creating a microenvironment of hypoxia and ischemia centered on the intima.

### **10. Cellular endothelial injury**

74 Atherogenesis

It is as remodelling of the injury to the intima that hypoxia and ischemia further modulate the migration of smooth muscle cells within the intima. The increased permeability of the endothelium is significant as a redefined series of further injuries to the underlying intima. The orientational redistribution of the agonists in atherogenesis redefine a central plaque contribution to increasing profiles of further hypoxia/ischemia and as evidential remounting of parameters of sequence effect. The multi-factorial injurious events are converging agonists in hypoxic/ischemic core regions of the individual atherosclerotic plaque. In such manner, the multi-staged evolution of plaques correlates with interactive

Dynamics of action of oxidized lipids correlate closely with emerging new roles for agonist action in developing plaques. The interaction of variably participating agonists contrast with the intimal emergence of incremental hypoxia/ischemia in terms of increasing matrix proteoglycan deposition and cellular proliferation of smooth muscle cells in particular. Atherosclerosis affecting different topographic sites correlate with the type of

The platelet/coagulation systems are incremental sequence events as trophic influence in

Distributional parameters are particularly significant in the dimensional targeting of the

Component systems of sequential impact would contribute to the emergence of positive feedback effect in agonist action. The endothelial cells participate by the production of various agents such as growth factors, in particular Platelet-Derived Growth Factor. The semblance of such influence dominantly re-characterizes the atherosclerotic plaque that trophically redefines the form of hypoxic/ischemic injury to the intima. miRNA –mediated epigenetic regulation may be implicated in atherogenesis, involving oxidized low-density

Hemodynamics within the neovascularized core of the plaque allows for a developmental evolution in terms of so-called complications such as hemorrhage and rupture of the plaque and as staged representation of the endothelial cell injury. Consequential pathways of significance would confirm the agonist nature of hypoxia/ischemia in terms of further

A response to injury permits role redefinition as emerging parameters in pathogenesis of the deposition of proteoglycans within the vascular intima. Matrix metalloproteinases participate in plaque destabilization and rupture. Their overexpression is an independent factor in the pathogenesis of acute coronary syndromes (Kulach et al., 2010). A proliferative response in particular illustrates the nature of the vascular wall injury that incrementally

Consequential involvement of the lipid deposition phenomenon integrally permits the establishment of gradient parameters of hypoxia/ischemia within the operative fields of

emergence of intimal deposition and of cellular proliferation and migration.

progresses as gradients of hypoxia and ischemia within the vessel wall.

**7. Intimal remodeling**

dynamics of further injury within the intima.

hyperlipidemia (Van Craeyveld et al., 2011).

lipoproteins (Chen et al., 2011).

**8. Hemodynamics**

**9. Gradient parameters**

emerging neovascularization in the plaque core.

staged convergence of multiple agonists in intimal injury.

intima in terms of the vasa vasorum supplying the arterial wall.

The contributing roles of endothelium especially in cases of trauma to the vessel wall would indicate the prototypical attributes in lesion emergence and of subsequent maturation of the atherogenesis phenomenon. Atherogenesis is contributory phenomenon to an ongoing migratory involvement of the intima. This is well-testified by smooth muscle cells that synthesize and secrete matrix proteoglycans, and oxidative stress also induces production of superoxide by endothelial cells with nitric oxide synthase uncoupling (Zweier et al., 2011).

Significant participation in atherogenesis involves mirror-imaged targeting of multiple component systems within the vessel wall that developmentally integrate as regions of hypoxia and ischemia, including the thioredoxin system that correlates with cellular apoptosis in endothelial cell lines in hypoxic stress (Park et al., 2011). Neovascularization proves a permissive phenomenon in development of gradients of ischemia that redefine the individual plaque as compounding parameters of progression to further injury to the vascular wall.

Permissive dynamics are characteristic of oxidation of lipids and particularly of low-density lipoproteins and as evolution of deposition within the intima. Targeting of subsets of cells indicates a selectivity process of progression within sequential pathways of incremental further injury to the vessel wall. Disrupted endoplasmic reticulum equilibrium engages the unfolded protein response in such cells as monocytes (Carroll et al., 2011).

#### **11. Vulnerability issues**

System reproduction indicates vulnerability selectivity in the evolution of atheromatous plaques in terms ranging from cell kinetics to proliferative migration of smooth muscle cells directed to the intima and a sensitivity of endothelial cells to responsive pathway generation and trophic factor production. The macrophage system is especially representative of novel pathway events that induce a sequential series of models in manipulative further compromise of viability of the endothelial cells.

Within such scopes of pathogenic representation, there would emerge a parametric remodelling based on aberrant reconstitution of injury as further projected by responses to injury to the intima and endothelium. Within such context, the unfolded protein response is implicated in all stages of atherogenesis and plaque progression (Lhotak et al., 2011).

#### **12. Pathway activation**

The operative essentiality of the intimal involvement in atheroma formation calls into operation the developmental dimensions of both endothelium and also of medial smooth muscle cells. The sequence attributes of multiple different pathways contribute to the subsequent emergence of activation phenomena as well represented by the macrophage and

Parametric Determination of Hypoxic Ischemia in Evolution of Atherogenesis 77

Macrophages induce transformational events within micro-environmental conditions of propagated susceptibility patterns that relate in particular to selective sites of vascular involvement such as near-arterial branch points of exit. It is such representation that illustrates the evolving vulnerability of focal sites of intima and endothelium in the generation of multiple atheromatous plaques; these subsequently promote self-involvement

Regional pathways of spread and further expansion contrast with the maturation of plaque morphology within such systems as macrophage and endothelial cell activation, with the creation of the synthetic/secretory phenotype of the individual smooth muscle cell within

Distributional dynamics in generation of the atheromatous plaque are developmental issues as indicated by activation of the proto-oncogenes c-fos and c-myc. The considerable heterogeneity of component cell subpopulations within any atheromatous plaque also permits the emergence of monoclonal groups of smooth muscle cells that trophically sustain growth of the plaque within dimensional confines of the involved intima and of injured

Hypoxia-ischemia is a powerful component series of systems in evolution of the susceptibility pattern determination of plaque localization and remodelling, as well-typified

Gradient generation is a key mechanistic system in generation of projected effects of hypoxia-ischemia that coordinate the convergence of injurious agonists in terms of trophic

Anggraemi VY, Emoto N, Yagi K, Mayasari DS, Nakayama K, Izumikawa T et al.

elongation in atherosclerosis" Biochem Biophys Res Commun 2011 Jan 29. Bacci S, Rizza S, Prudente S, Spoto B, Powers C, Facciorusso A et al. "The ENPPI Q121

Carroll TP, Greene CM, McEloaney NG "Measurement of the unfolded protein response

Chen KC, Wang YS, Hu CY, Chang WC, Liao YC, Dai CY et al. "OxLDL up-regulates

Chen T, Li Z, Tu J, Zhu W, Ge J, Zheng X et al. "MicroRNA-29a regulates pro-inflammatory

Chyu KY, Shah PK "Emerging therapies for atherosclerosis prevention and management"

Civelek M, Manduchi E, Grant GR, Stoeckert CJ Jr, Davies PF "Discovery approaches to UPR in athero-susceptible endothelium in vivo" Methods Enzymol 204;489:109-26. Kulach A, Dabek J, Glogowska-Ligus J, Garczorz W, Gasior Z "Effects of standard treatment

acute coronary syndromes" Pharmacol Rep 2010 Nov-Dec;62(6):1108-16.

interaction with obesity in diabetic patients" Diabetes 2011 Jan31.

novel mechanism for cardiovascular diseases" FASEB J 2011 Jan25.

(UPR) in monocytes" Methods Enzymol 2011;489:83-95.

stimulated dendritic cells" FEBS Lett 2011 Jan27.

Cardiol Clin 2011 Feb;29(1):123-35.

"Correlation of C4ST-1 and ChGn-2 expression with chondroitin sulphate chain

variant predicts major cardiovascular events in high-risk individuals: evidence for

microRNA-29b, leading to epigenetic modifications of MMP-2/MMP-9 genes: a

cytokine secretion and scavenger receptor expression by targeting LPL in oxLDL-

on the dynamics of matrix metalloproteinases gene expression in patients with

in dynamic transformation to the so-called complicated plaque.

by the marked eventual thickening of the involved intima.

and destructive elements within the intima of the arterial wall.

the intima.

overlying endothelium.

**15. References**

foam cell systems. Mast cells, macrophages and neutrophils release TNF-alpha, IFN-gamma and IL-6 with expression of adhesion molecules and leukocyte recruitment (Zhang et al., 2011). Synthetic and contractile phenotypes of the individual smooth muscle cell indicate specialized forms of series determination in reconstitution of the damaged or injured subintima and also modelled parametric fashioning of the overlying endothelium. Hemodynamics of blood flow localizes such injured endothelium as representative and constitutive foci of persistent pathway activation that delivers dysfunctional attributes to the multi-components of the early atheromatous plaque.

#### **13. Eventual sequence emergence**

Contrasting sequentiality is triggered by an aberrant selectivity for trophic effect reproduction in terms of ongoing creation of hypoxia/ischemic gradients across both the endothelium lining the vascular lumen and also within critical regions of operative effect in the involved vascular intima. Such representation calls into evidence gradient pathways of projected reproduction that specifically induce focality of involvement of the plaque within systems of cascade effect. A complex interaction of genetic and environmental factors operates (Chyu & Shah, 2011)

The platelet and coagulation systems conclusively demonstrate a participating series of roles culminating in organization of adherent thrombus within the plaque as incorporated dynamics of trophic potential.

Incremental attributes of further compromise of the viability of the intima are demonstrable as evidential pathways of increasing impact in terms of enhanced intimal thickness. In this regard, cells proliferate in atherosclerotic lesions and also in vascular tissue bordering the plaque (Zettler et al., 2010).

Scope of representative projection is conclusively constituted by the end-stage plaque with a central atheromatous core that consists of cholesterol lipid, lipoprotein and oxidized molecular entities of variable derivation. Increasing representation of inflammatory dynamics is largely dependent on initiating events within the foci of intima underlying dysfunctionally activated endothelium. The dynamics of spread and of replication of individual endothelial cells constitutes a further pathway model for gradient creation between flowing blood and vascular wall intima. Pancoronary arterial instability implicates multifocal disease in acute coronary syndromes (Puri et al., 2011).

#### **14. Concluding remarks**

Re-distribution and retargeting events are primary modelling systems in sequence pathways and as multi-staged involvement of the intima of arterial vessel walls. The intimal thickness and remodelling of pathways allow for incremental redistribution of agonists that target differential systems such as endothelium and smooth muscle cells. Macrophages are constitutive systemic parameters that focally re-orientate the targeting dynamics of hypoxia and ischemia in intimal lesion creation. Oxidation-specific epitopes present on apoptotic cells induce the selection of Pattern Recognition Receptors and damage-associated molecular patterns that may be targeted by innate immunity (Miller et al., 2011).

Only in terms of ensuing neovascularization of the individual atherosclerotic plaque can system specificity in atherogenesis permit the emergence of converging pathways of injury and attempted reconstitution of the vessel wall and endothelium.

Macrophages induce transformational events within micro-environmental conditions of propagated susceptibility patterns that relate in particular to selective sites of vascular involvement such as near-arterial branch points of exit. It is such representation that illustrates the evolving vulnerability of focal sites of intima and endothelium in the generation of multiple atheromatous plaques; these subsequently promote self-involvement in dynamic transformation to the so-called complicated plaque.

Regional pathways of spread and further expansion contrast with the maturation of plaque morphology within such systems as macrophage and endothelial cell activation, with the creation of the synthetic/secretory phenotype of the individual smooth muscle cell within the intima.

Distributional dynamics in generation of the atheromatous plaque are developmental issues as indicated by activation of the proto-oncogenes c-fos and c-myc. The considerable heterogeneity of component cell subpopulations within any atheromatous plaque also permits the emergence of monoclonal groups of smooth muscle cells that trophically sustain growth of the plaque within dimensional confines of the involved intima and of injured overlying endothelium.

Hypoxia-ischemia is a powerful component series of systems in evolution of the susceptibility pattern determination of plaque localization and remodelling, as well-typified by the marked eventual thickening of the involved intima.

Gradient generation is a key mechanistic system in generation of projected effects of hypoxia-ischemia that coordinate the convergence of injurious agonists in terms of trophic and destructive elements within the intima of the arterial wall.

#### **15. References**

76 Atherogenesis

foam cell systems. Mast cells, macrophages and neutrophils release TNF-alpha, IFN-gamma and IL-6 with expression of adhesion molecules and leukocyte recruitment (Zhang et al., 2011). Synthetic and contractile phenotypes of the individual smooth muscle cell indicate specialized forms of series determination in reconstitution of the damaged or injured subintima and also modelled parametric fashioning of the overlying endothelium. Hemodynamics of blood flow localizes such injured endothelium as representative and constitutive foci of persistent pathway activation that delivers dysfunctional attributes to the

Contrasting sequentiality is triggered by an aberrant selectivity for trophic effect reproduction in terms of ongoing creation of hypoxia/ischemic gradients across both the endothelium lining the vascular lumen and also within critical regions of operative effect in the involved vascular intima. Such representation calls into evidence gradient pathways of projected reproduction that specifically induce focality of involvement of the plaque within systems of cascade effect. A complex interaction of genetic and environmental factors

The platelet and coagulation systems conclusively demonstrate a participating series of roles culminating in organization of adherent thrombus within the plaque as incorporated

Incremental attributes of further compromise of the viability of the intima are demonstrable as evidential pathways of increasing impact in terms of enhanced intimal thickness. In this regard, cells proliferate in atherosclerotic lesions and also in vascular tissue bordering the

Scope of representative projection is conclusively constituted by the end-stage plaque with a central atheromatous core that consists of cholesterol lipid, lipoprotein and oxidized molecular entities of variable derivation. Increasing representation of inflammatory dynamics is largely dependent on initiating events within the foci of intima underlying dysfunctionally activated endothelium. The dynamics of spread and of replication of individual endothelial cells constitutes a further pathway model for gradient creation between flowing blood and vascular wall intima. Pancoronary arterial instability implicates

Re-distribution and retargeting events are primary modelling systems in sequence pathways and as multi-staged involvement of the intima of arterial vessel walls. The intimal thickness and remodelling of pathways allow for incremental redistribution of agonists that target differential systems such as endothelium and smooth muscle cells. Macrophages are constitutive systemic parameters that focally re-orientate the targeting dynamics of hypoxia and ischemia in intimal lesion creation. Oxidation-specific epitopes present on apoptotic cells induce the selection of Pattern Recognition Receptors and damage-associated

Only in terms of ensuing neovascularization of the individual atherosclerotic plaque can system specificity in atherogenesis permit the emergence of converging pathways of injury

molecular patterns that may be targeted by innate immunity (Miller et al., 2011).

multifocal disease in acute coronary syndromes (Puri et al., 2011).

and attempted reconstitution of the vessel wall and endothelium.

multi-components of the early atheromatous plaque.

**13. Eventual sequence emergence**

operates (Chyu & Shah, 2011)

dynamics of trophic potential.

plaque (Zettler et al., 2010).

**14. Concluding remarks**

**5**

*USA*

**Emerging Epigenetic Therapy for Vascular**

*2University of Texas Health Science Center at Houston, Texas, Department of Neurology*

Atherosclerosis and restenosis, complex pathologies of blood vessels, are multifactorial diseases triggered by the inflammatory response to injury of endothelium. Remodeling of the injured vessel, proliferation and migration of vascular smooth muscle cells (VSMC) and elaboration and accumulation of extracellular matrix proteins are main traits of these diseases (Dzau et al., 2002; Libby, 2002; Pons et al., 2009; Ranganna et al., 2006; Ross, 1995;). Despite the substantial progress in understanding the etiology and the clinical management of atherosclerosis and restenosis, they are still life threatening diseases. Precise reasons are not still fully transparent. Different cell types; distinct cellular pathways and processes; and multiple genes within each participating cell types that are vulnerable to both genetic and environmental risk factors participate in the pathogenesis of atherosclerosis and restenosis. Recently, it is recognized that besides the genetic control epigenetic mechanisms regulate development and maintenance of organisms or their interaction with surrounding environment through the coordination of a set of reversible modifications that turn parts of the genome 'off' and 'on' at strategic times and at specific sites causing changes in gene expression with no changes in DNA sequences (Ekstrom, 2009; Pons et al., 2009; Ranganna et al., 2006; Turunen, 2009). The two well-known epigenetic mechanisms, DNA methylation and histone modifications change the chromatin structure and dynamics that alter gene functions by influencing gene expressions. Dysregulation of epigenetic processes has been linked to human diseases, which influences many aspects of cell biology including cell growth, cell cycle control, proliferation, differentiation, and cell death. Reversing the dysregulation of epigenetic mechanisms may offer effective treatment strategy for many diseases including cardiovascular disease due to atherosclerosis and restenosis. This review presents the current advancement in the epigenetics of VSMC proliferation and potential use of histone epigenetic modifiers in the intervention of atherosclerosis and restenosis.

Atherosclerosis, a disease of medium to large arterial vessels, accounts for over 55% of all deaths in western countries. It is typically asymptomatic for decades but ultimately result in life-threatening pathological outcomes like myocardial infarction and stroke, both with tissue infarction because of intra-arterial thrombosis provoked by atherosclerosis.

**2. Overview of pathogenesis of atherosclerosis**

**1. Introduction**

Kasturi Ranganna1, Frank M. Yatsu2 and Omana P. Mathew1 *1Texas Southern University, Department of Pharmaceutical Sciences,*

**Proliferative Diseases**

### **Emerging Epigenetic Therapy for Vascular Proliferative Diseases**

Kasturi Ranganna1, Frank M. Yatsu2 and Omana P. Mathew1 *1Texas Southern University, Department of Pharmaceutical Sciences, 2University of Texas Health Science Center at Houston, Texas, Department of Neurology USA*

#### **1. Introduction**

78 Atherogenesis

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and macrophage-derived foam cell formation: A novel link between interferon-alpha

epitopes are danger-associated molecular patterns recognized by pattern recognition receptors of innate immunity" Circ Res 2011 Jan21; 108(2):235-48. Nandish S, Barlon O, Wyatt J, Smith J, Stevens A, Lujan M et al. "Vasculotoxic effects of insulin and its role in atherosclerosis: what is the evidence?" Curr Atheroscler Rep 2011 Feb2. Park KJ, Kim YJ, Choi EJ, Park NK, Kim GH, Kim SM et al. "Expression pattern of the

thioredoxin system in human endothelial progenitor cells and endothelial cells

different topographic sites is highly dependent on the type of hyperlipidemia"

al. "IGF-1 has plaque stabilizing effects in atherosclerosis by altering vascular

prostaglandin E2 synthase-1 modulates the response to vascular injury" Circulation

linolenic acid diminishes experimental atherogenesis and restricts T-cell-driven

fatty acid content and composition by exposure to dyslipidemia serum in vitro"

18: A specific biomarker for atherosclerosis-prone patients with metabolic

adhesion molecule expression by mast cells, macrophages, and neutrophils » PLoS

Atherosclerosis and restenosis, complex pathologies of blood vessels, are multifactorial diseases triggered by the inflammatory response to injury of endothelium. Remodeling of the injured vessel, proliferation and migration of vascular smooth muscle cells (VSMC) and elaboration and accumulation of extracellular matrix proteins are main traits of these diseases (Dzau et al., 2002; Libby, 2002; Pons et al., 2009; Ranganna et al., 2006; Ross, 1995;). Despite the substantial progress in understanding the etiology and the clinical management of atherosclerosis and restenosis, they are still life threatening diseases. Precise reasons are not still fully transparent. Different cell types; distinct cellular pathways and processes; and multiple genes within each participating cell types that are vulnerable to both genetic and environmental risk factors participate in the pathogenesis of atherosclerosis and restenosis. Recently, it is recognized that besides the genetic control epigenetic mechanisms regulate development and maintenance of organisms or their interaction with surrounding environment through the coordination of a set of reversible modifications that turn parts of the genome 'off' and 'on' at strategic times and at specific sites causing changes in gene expression with no changes in DNA sequences (Ekstrom, 2009; Pons et al., 2009; Ranganna et al., 2006; Turunen, 2009). The two well-known epigenetic mechanisms, DNA methylation and histone modifications change the chromatin structure and dynamics that alter gene functions by influencing gene expressions. Dysregulation of epigenetic processes has been linked to human diseases, which influences many aspects of cell biology including cell growth, cell cycle control, proliferation, differentiation, and cell death. Reversing the dysregulation of epigenetic mechanisms may offer effective treatment strategy for many diseases including cardiovascular disease due to atherosclerosis and restenosis. This review presents the current advancement in the epigenetics of VSMC proliferation and potential use of histone epigenetic modifiers in the intervention of atherosclerosis and restenosis.

#### **2. Overview of pathogenesis of atherosclerosis**

Atherosclerosis, a disease of medium to large arterial vessels, accounts for over 55% of all deaths in western countries. It is typically asymptomatic for decades but ultimately result in life-threatening pathological outcomes like myocardial infarction and stroke, both with tissue infarction because of intra-arterial thrombosis provoked by atherosclerosis.

Emerging Epigenetic Therapy for Vascular Proliferative Diseases 81

Atherosclerosis and restenosis both are multifactorial vascular occlusive processes but exhibit certain similarities and differences in the origin and progression of their development (Dzau et al., 2002; Pons et al., 2009). Inflammatory response of activated endothelial cells to injury or insults elicits both these processes. Activation of endothelial cells leads to a cascade of events, which promotes vascular remodeling by changing the size, structure and composition of vessel wall. Moreover, both processes involve proliferation, migration and activation of VSMC and modulation of extracellular matrix by elaborating and accumulating extracellular matrix proteins. Although they share some of the risk factors such as hypertension and diabetes, there is a consensus that atherosclerosis develops in response to elevated low-density lipoproteins (LDL) and cigarette smoke. On the other hand, VSMC proliferation is the primary pathophysiological mechanism in restenosis, which is largely due to transcending wound healing response to clinical procedures such as balloon angioplasty, stent placement and vein graft surgery [Dzau et al., 2002; Pons et al., 2009]. While restenosis appears to be insensitive to circulating lipids, accumulation of oxidized LDL is the characteristic feature of atherosclerosis. Additionally, while development and progression of atherosclerosis is a gradual process, the restenotic process is a relatively a rapid process caused by surgical revascularization procedures such as angioplasty and stent placement. Despite substantial progress in understanding the etiology and the clinical management of atherosclerosis and restenosis, they are still life-threatening diseases. Possible reasons are multiple factors, different cellular pathways and processes,

and multiple genes are contributory to these complex vascular disease processes.

cell number and composition of vessel wall (Pons et al., 2009; Ross, 1993).

VSMC are highly specialized cells, which play vital roles in the regulation of blood pressure, blood flow and in many pathological states. In mature individuals, the typical function of arterial VSMC is contraction and maintenance of vascular tone. As such, VSMC in adult artery exhibit contractile or differentiated phenotype displaying quiescent proliferation state, decreased synthetic activity and expression of proteins unique to contractile phenotype like contractile proteins, ion channels and signaling proteins. However, VSMC retain their remarkable plasticity to undergo reversible phenotypic change in response to alterations in the local environment like during development, physiological conditions like pregnancy or in response to vascular injury. This remarkable flexible persona of VSMC makes them vulnerable to phenotypic modification from contractile to proliferative, synthetic, or de-differentiated phenotype in conjunction with vessel remodeling by altering

VSMC proliferation is also the primary pathophysiological mechanism in different clinical pathologies such as postangioplasty restenosis, in-stent restenosis, vein bypass graft failure and transplant vasculopathy (Dzau et al., 2002; Holmes, 2003). The clinical procedures performed to clear the occluded vessel fortuitously become precursors for restenosis in 30- 40% of the patients, mainly due to proliferation and activation of VSMC. While entry into cell cycle followed by proliferation of VSMC contributes to the formation of neointima, activation of VSMC induces expression of proinflammatory cytokines, adhesion molecules, chemoattractants, proteolytic enzymes and other molecules not usually present in normal, quiescent, contractile VSMC of the medial layer (Kleemann et al., 2008; Li et al., 1993; O'Brien et al., 1996; Zeffer et al., 2004). Expression of these molecules amplifies the

**2.2 VSMC proliferation**

**2.1 Vascular remodeling in atherosclerosis and restenosis**

Atherosclerosis is a complex progressive disease in which intimal thickening of the arterial wall promotes luminal stenosis by vascular remodeling, accumulation of cellular and extracellular substances and VSMC proliferation and migration. Integrity of arterial wall is crucial for the regulation of vascular tone, control inflammation, thrombosis, and angiogenesis, enhance regional blood flow, and inhibit cancer metastasis. Arterial wall is composed of three tunics that surround a central lumen through which blood flows. The innermost layer is the tunica intima composed of endothelial cells that form a smooth lining that minimizes interaction with circulating cellular and non-cellular components as blood moves through the vessel. The middle layer, tunic media, is composed of vascular smooth muscle cells (VSMC) and layers of flexible proteins, which enables the lumen to contract and dilate to regulate blood flow in the body. The outer layer, tunica adventitia is a protective layer of connective tissue that anchors the blood vessel to surrounding structures.

Under normal conditions, a delicate balance between proliferation and apoptosis of local vascular cell types maintains the thickness of arterial vessel wall. A number of regulatory factors produced by the endothelial cells are responsible for the homeostatic balance by controlling vessel tone, coagulation state, leukocyte trafficking, and cellular proliferative response. Any damage to the vessel wall by mechanical, biochemical, or immunological insults triggers endothelial dysfunction or denudation of endothelial layer overwhelming the normal homeostatic balance, thus, upsets the normal vascular tone setting the stage for the activation of proinflammatory and immune response. Escalating evidence indicates that inflammatory or atherogenic stimuli promote ROS generation in endothelial milieu causing oxidative stress (Freeman & Crapo, 1982; Kehrer, 1993; Kunsch & Medford, 1999; Madamanchi et al., 2005). Inflammatory response fueled by the oxidative stress is also linked to oxidation of lipoproteins. LDL molecules that enter the subendothelial space are oxidized to form oxidized LDL (OxLDL) by different mechanisms including enzymatic and nonenzymatic pathways, which are taken up by macrophages via scavenger receptors to become foam cells. Besides stimulating proinflammatory and proatherogenic effects, OxLDL also appears to elicit highly immunogenic response resulting in the generation of autoantibodies that appears to be of pathogenic significance (Hansson, 2009; Klingenberg, R., & Hansson, G.K. 2009; Steinberg & Witztum, 2010; Witztum, 1997). Moreover, elevated levels of ROS appear to function as second-messenger molecules transmitting the extracellular signals to nucleus via redox-sensitive signaling pathways to turn on the expression of atherogenic gene products such as adhesion molecules and inflammatory cytokines. Expression of these gene products elicit changes in the vessel wall promoting inflammation, infiltration of monocytes and T cells, proliferation, migration and activation of VSMC and matrix alteration (Freeman & Crapo, 1982; Hansson, 2007; Kehrer, 1993; Klingenberg & Hansson, 2009; Kunsch & Medford, 1999; Madamanchi et al., 2005; Steinberg & Witztum, 2010; Witztum, 1997). These processes involve synthesis and release of a host of regulatory molecules, both by cellular components in the blood and vascular cells of the arterial wall triggering autocrine, paracrine, and endocrine type of interactions between cells and the molecules they produce. Outcome of these complex interactions leads to migration of VSMCs from their normal residence in the arterial media to the intima where they change their phenotype from a contractile to a proliferative type (Libby, 2002; March et al., 1999; Ross, 1995). This phenotypic change, in conjunction with excessive production and accumulation of extracellular matrix proteins, is the main contributor to vascular remodeling.

#### **2.1 Vascular remodeling in atherosclerosis and restenosis**

Atherosclerosis and restenosis both are multifactorial vascular occlusive processes but exhibit certain similarities and differences in the origin and progression of their development (Dzau et al., 2002; Pons et al., 2009). Inflammatory response of activated endothelial cells to injury or insults elicits both these processes. Activation of endothelial cells leads to a cascade of events, which promotes vascular remodeling by changing the size, structure and composition of vessel wall. Moreover, both processes involve proliferation, migration and activation of VSMC and modulation of extracellular matrix by elaborating and accumulating extracellular matrix proteins. Although they share some of the risk factors such as hypertension and diabetes, there is a consensus that atherosclerosis develops in response to elevated low-density lipoproteins (LDL) and cigarette smoke. On the other hand, VSMC proliferation is the primary pathophysiological mechanism in restenosis, which is largely due to transcending wound healing response to clinical procedures such as balloon angioplasty, stent placement and vein graft surgery [Dzau et al., 2002; Pons et al., 2009]. While restenosis appears to be insensitive to circulating lipids, accumulation of oxidized LDL is the characteristic feature of atherosclerosis. Additionally, while development and progression of atherosclerosis is a gradual process, the restenotic process is a relatively a rapid process caused by surgical revascularization procedures such as angioplasty and stent placement. Despite substantial progress in understanding the etiology and the clinical management of atherosclerosis and restenosis, they are still life-threatening diseases. Possible reasons are multiple factors, different cellular pathways and processes, and multiple genes are contributory to these complex vascular disease processes.

#### **2.2 VSMC proliferation**

80 Atherogenesis

Atherosclerosis is a complex progressive disease in which intimal thickening of the arterial wall promotes luminal stenosis by vascular remodeling, accumulation of cellular and extracellular substances and VSMC proliferation and migration. Integrity of arterial wall is crucial for the regulation of vascular tone, control inflammation, thrombosis, and angiogenesis, enhance regional blood flow, and inhibit cancer metastasis. Arterial wall is composed of three tunics that surround a central lumen through which blood flows. The innermost layer is the tunica intima composed of endothelial cells that form a smooth lining that minimizes interaction with circulating cellular and non-cellular components as blood moves through the vessel. The middle layer, tunic media, is composed of vascular smooth muscle cells (VSMC) and layers of flexible proteins, which enables the lumen to contract and dilate to regulate blood flow in the body. The outer layer, tunica adventitia is a protective

layer of connective tissue that anchors the blood vessel to surrounding structures.

remodeling.

Under normal conditions, a delicate balance between proliferation and apoptosis of local vascular cell types maintains the thickness of arterial vessel wall. A number of regulatory factors produced by the endothelial cells are responsible for the homeostatic balance by controlling vessel tone, coagulation state, leukocyte trafficking, and cellular proliferative response. Any damage to the vessel wall by mechanical, biochemical, or immunological insults triggers endothelial dysfunction or denudation of endothelial layer overwhelming the normal homeostatic balance, thus, upsets the normal vascular tone setting the stage for the activation of proinflammatory and immune response. Escalating evidence indicates that inflammatory or atherogenic stimuli promote ROS generation in endothelial milieu causing oxidative stress (Freeman & Crapo, 1982; Kehrer, 1993; Kunsch & Medford, 1999; Madamanchi et al., 2005). Inflammatory response fueled by the oxidative stress is also linked to oxidation of lipoproteins. LDL molecules that enter the subendothelial space are oxidized to form oxidized LDL (OxLDL) by different mechanisms including enzymatic and nonenzymatic pathways, which are taken up by macrophages via scavenger receptors to become foam cells. Besides stimulating proinflammatory and proatherogenic effects, OxLDL also appears to elicit highly immunogenic response resulting in the generation of autoantibodies that appears to be of pathogenic significance (Hansson, 2009; Klingenberg, R., & Hansson, G.K. 2009; Steinberg & Witztum, 2010; Witztum, 1997). Moreover, elevated levels of ROS appear to function as second-messenger molecules transmitting the extracellular signals to nucleus via redox-sensitive signaling pathways to turn on the expression of atherogenic gene products such as adhesion molecules and inflammatory cytokines. Expression of these gene products elicit changes in the vessel wall promoting inflammation, infiltration of monocytes and T cells, proliferation, migration and activation of VSMC and matrix alteration (Freeman & Crapo, 1982; Hansson, 2007; Kehrer, 1993; Klingenberg & Hansson, 2009; Kunsch & Medford, 1999; Madamanchi et al., 2005; Steinberg & Witztum, 2010; Witztum, 1997). These processes involve synthesis and release of a host of regulatory molecules, both by cellular components in the blood and vascular cells of the arterial wall triggering autocrine, paracrine, and endocrine type of interactions between cells and the molecules they produce. Outcome of these complex interactions leads to migration of VSMCs from their normal residence in the arterial media to the intima where they change their phenotype from a contractile to a proliferative type (Libby, 2002; March et al., 1999; Ross, 1995). This phenotypic change, in conjunction with excessive production and accumulation of extracellular matrix proteins, is the main contributor to vascular

VSMC are highly specialized cells, which play vital roles in the regulation of blood pressure, blood flow and in many pathological states. In mature individuals, the typical function of arterial VSMC is contraction and maintenance of vascular tone. As such, VSMC in adult artery exhibit contractile or differentiated phenotype displaying quiescent proliferation state, decreased synthetic activity and expression of proteins unique to contractile phenotype like contractile proteins, ion channels and signaling proteins. However, VSMC retain their remarkable plasticity to undergo reversible phenotypic change in response to alterations in the local environment like during development, physiological conditions like pregnancy or in response to vascular injury. This remarkable flexible persona of VSMC makes them vulnerable to phenotypic modification from contractile to proliferative, synthetic, or de-differentiated phenotype in conjunction with vessel remodeling by altering cell number and composition of vessel wall (Pons et al., 2009; Ross, 1993).

VSMC proliferation is also the primary pathophysiological mechanism in different clinical pathologies such as postangioplasty restenosis, in-stent restenosis, vein bypass graft failure and transplant vasculopathy (Dzau et al., 2002; Holmes, 2003). The clinical procedures performed to clear the occluded vessel fortuitously become precursors for restenosis in 30- 40% of the patients, mainly due to proliferation and activation of VSMC. While entry into cell cycle followed by proliferation of VSMC contributes to the formation of neointima, activation of VSMC induces expression of proinflammatory cytokines, adhesion molecules, chemoattractants, proteolytic enzymes and other molecules not usually present in normal, quiescent, contractile VSMC of the medial layer (Kleemann et al., 2008; Li et al., 1993; O'Brien et al., 1996; Zeffer et al., 2004). Expression of these molecules amplifies the

Emerging Epigenetic Therapy for Vascular Proliferative Diseases 83

radiation sources are used to diminish restenosis in patients with post-angioplasty restenosis or with in-stent restenosis. The rationale for using radiation for treating restenosis is that uncontrolled proliferation of VSMC is similar to neoplastic cells that can be targeted for radiation therapy just as transformed cells in cancer tissue. Brachytherapy-induced DNA damage of VSMC can result in arrest of VSMC at the G1 checkpoint or induction of apoptosis through p53 induced p21Cip1 upregulation. A key feature of brachytherapy is that the irradiation only affects a precise localized area around the radiation sources. Exposure to radiation of healthy tissues further away from the sources is therefore, reduced. In addition, brachytherapy is associated with a low risk of serious adverse side effects. More than a dozen randomized trials established its safety and efficacy. However, it exhibits two radiotherapy-related problems, arterial narrowing adjacent to the edge of the target site and

Gene therapy techniques provide a unique opportunity to genetically engineer vessels and grafts to become impervious to atherosclerosis and neointimal formation that contributes to arterial restenosis, in-stent restenosis and vein graft failure (Dzau et al., 2002; Khanna, 2008; Kishore & Losordo, 2007; Gaffney et al., 2007; Melo et al., 2005; Von der Leyen & Dzau, 2001). Gene therapy approach has potential not only against monogenic diseases, but also against complex diseases where multiple genes are involved in the disease pathogenesis like in cardiovascular diseases and cancer. One of the key challenges of the gene therapy is appropriate vector for the delivery of functional gene or a concoction of genes in multigenic diseases as in cardiovascular diseases. Besides the choice of vector, other parameters such as, appropriate gene targets and efficient methods of vector delivery for a specific target have to be optimized. Vectors can be either viral or non-viral. The ideal vector is the one, which is nonpathogenic, less immunogenic, more efficient, and enhanced tissue specificity. Delivery of therapeutic genes to the cardiovascular tissues is challenging. To facilitate local gene delivery to lesions in the vasculature catheter-based vector delivery has been tried using a variety of balloon catheters in animal models and human trials (Khanna, 2008; Kishore & Losordo, 2007; Gaffney et al., 2007; Melo et al., 2005). Stents are ideal gear for localized gene delivery to the vascular wall because of their widespread use, safety and permanent scaffold structure. Stents can be coated with genetically engineered cells or plasmid or adenoviral vectors carrying therapeutic genes (Khanna, 2008; Kishore & Losordo, 2007; Gaffney et al., 2007; Melo et al., 2005). Experimental studies have demonstrated usefulness of gene therapy in treating atherosclerosis and restenosis in various animal models and in some clinical trials. It can be used to transfer exogenous genes to express functional gene products to overcome defective or downregulated endogenous gene expressions through vector-based delivery system. It also can be used to knockdown or suppress the expression of gene products that contribute to pathogenesis of disease by one of the several methods of gene silencing. These include antisense oligonucleotides (ODNs), short segments of RNA with enzymatic activity (ribozymes) and small interfering RNAs

unexpected late coronary thrombo-occlusive events (Raizner, 2000).

[siRNA] (Dzau et al., 2002; J.M. Li et al., 2010; Banno, et al., 2006).

A number of studies have shown that gene therapies can be targeted for reducing cholesterol levels, inflammation and thrombosis (Feldman & Isner, 1995); for upregulating apo-A1 and downregulating chemoattractant protein-1 (MCP1)receptor expression (Tangirala, 1999); transferring pleiotropic atheroprotective nitric oxide synthase [NOS]

**2.4.2 Gene therapy**

inflammatory response, and in turn, increases further proliferation of VSMC and elaboration of vessel remodeling.

#### **2.3 Antiproliferative therapeutics to target VSMC proliferation**

The current therapies used for atherosclerosis aim to minimize the risk factors that promote atherosclerosis, such as reducing elevated levels of cholesterol or enhancing the blood flow by surgical intervention of an already occluded vessel. Ironically, the surgical procedures performed to clear the occluded vessel become a precursor for restenosis, mainly due to VSMC proliferation, in significant number of patients (Dzau et al., 2002; Ferns et al., 1991). Because proliferation of VSMC is the hallmark of atherosclerosis and clinical conditions such as arterial stenosis, transplant vasculopathy, and bypass graft failure, the suitable therapeutic approach is to develop strategies that inhibit or block VSMC proliferation. Based on the current understanding of the molecular basis of vascular proliferative diseases, there is an abundance of potential therapeutic possibilities. Accordingly, a number of agents are tested for antiproliferative activity including heparins, cytostatic agents, inhibitors of angiotensin converting enzyme, and antagonists to growth factors (Dzau et al., 2002; Ferns et al., 1991; Gershlick, 2002; Stephen et al., 2005; Toshiro et al., 2005). Although some of these agents have shown promise in animal models, they failed to elicit any protection in human clinical studies (Gershlick, 2002). Species differences, potential toxicity, and lack of potency are possible culprits. Furthermore, the probability of successfully treating a multifactorial disease by targeting a single factor is unlikely. Additionally, all vascular cell types secrete growth factors and cytokines that activate signaling pathways that are redundant and thus prevent the success of targeting one or two factors.

#### **2.4 Cell cycle as the therapeutic target**

Based on the current knowledge of cell cycle mechanisms, it appears that targeting specific parts of the cell cycle is a better strategy to inhibit or block the development of vascular proliferative diseases such as arterial restenosis; in-stent-stenosis and vein bypass graft failure. Moreover, cell cycle is the final common pathway where all the growth regulatory signals converge, and thus, makes a rational target of antiproliferative therapeutics to inhibit vascular proliferative diseases. Some of the experimental studies indeed reveal that inhibition of cell cycle progression emerges as an important therapeutic target for prevention of vascular proliferative diseases (Dzau et al., 2002; Ranganna et al., 2006; Von der Leyen & Dzau, 2001). Different approaches such as pharmacological agents, irradiation, and gene therapy have been used for arresting VSMC proliferation. These approaches inhibit proliferation by cytostatic or cytotoxic mechanism. However, cytostatic mechanism of cell cycle arrest is desired over cytotoxic mechanism to avoid unintended damage to the vessel wall due to cytotoxic treatment. Three different approaches have been tried for arresting VSMC proliferation by targeting cell cycle, which include: 1) brachytherapy, 2) gene therapy, and 3) pharmacotherapy.

#### **2.4.1. Brachytherapy**

Endovascular radiotherapy is a promising method for effective antiproliferative treatment of restenosis (Teirstein & King, 2003; Waksman, 2000). Radiotherapy directed at restenosis has two objectives, one to treat restenosis by killing the cells that re-occluded and to prevent further restenosis by inhibiting tissue growth. Brachytherapy with either beta or gamma radiation sources are used to diminish restenosis in patients with post-angioplasty restenosis or with in-stent restenosis. The rationale for using radiation for treating restenosis is that uncontrolled proliferation of VSMC is similar to neoplastic cells that can be targeted for radiation therapy just as transformed cells in cancer tissue. Brachytherapy-induced DNA damage of VSMC can result in arrest of VSMC at the G1 checkpoint or induction of apoptosis through p53 induced p21Cip1 upregulation. A key feature of brachytherapy is that the irradiation only affects a precise localized area around the radiation sources. Exposure to radiation of healthy tissues further away from the sources is therefore, reduced. In addition, brachytherapy is associated with a low risk of serious adverse side effects. More than a dozen randomized trials established its safety and efficacy. However, it exhibits two radiotherapy-related problems, arterial narrowing adjacent to the edge of the target site and unexpected late coronary thrombo-occlusive events (Raizner, 2000).

#### **2.4.2 Gene therapy**

82 Atherogenesis

inflammatory response, and in turn, increases further proliferation of VSMC and elaboration

The current therapies used for atherosclerosis aim to minimize the risk factors that promote atherosclerosis, such as reducing elevated levels of cholesterol or enhancing the blood flow by surgical intervention of an already occluded vessel. Ironically, the surgical procedures performed to clear the occluded vessel become a precursor for restenosis, mainly due to VSMC proliferation, in significant number of patients (Dzau et al., 2002; Ferns et al., 1991). Because proliferation of VSMC is the hallmark of atherosclerosis and clinical conditions such as arterial stenosis, transplant vasculopathy, and bypass graft failure, the suitable therapeutic approach is to develop strategies that inhibit or block VSMC proliferation. Based on the current understanding of the molecular basis of vascular proliferative diseases, there is an abundance of potential therapeutic possibilities. Accordingly, a number of agents are tested for antiproliferative activity including heparins, cytostatic agents, inhibitors of angiotensin converting enzyme, and antagonists to growth factors (Dzau et al., 2002; Ferns et al., 1991; Gershlick, 2002; Stephen et al., 2005; Toshiro et al., 2005). Although some of these agents have shown promise in animal models, they failed to elicit any protection in human clinical studies (Gershlick, 2002). Species differences, potential toxicity, and lack of potency are possible culprits. Furthermore, the probability of successfully treating a multifactorial disease by targeting a single factor is unlikely. Additionally, all vascular cell types secrete growth factors and cytokines that activate signaling pathways that are redundant and thus

Based on the current knowledge of cell cycle mechanisms, it appears that targeting specific parts of the cell cycle is a better strategy to inhibit or block the development of vascular proliferative diseases such as arterial restenosis; in-stent-stenosis and vein bypass graft failure. Moreover, cell cycle is the final common pathway where all the growth regulatory signals converge, and thus, makes a rational target of antiproliferative therapeutics to inhibit vascular proliferative diseases. Some of the experimental studies indeed reveal that inhibition of cell cycle progression emerges as an important therapeutic target for prevention of vascular proliferative diseases (Dzau et al., 2002; Ranganna et al., 2006; Von der Leyen & Dzau, 2001). Different approaches such as pharmacological agents, irradiation, and gene therapy have been used for arresting VSMC proliferation. These approaches inhibit proliferation by cytostatic or cytotoxic mechanism. However, cytostatic mechanism of cell cycle arrest is desired over cytotoxic mechanism to avoid unintended damage to the vessel wall due to cytotoxic treatment. Three different approaches have been tried for arresting VSMC proliferation by targeting cell cycle, which include: 1) brachytherapy, 2)

Endovascular radiotherapy is a promising method for effective antiproliferative treatment of restenosis (Teirstein & King, 2003; Waksman, 2000). Radiotherapy directed at restenosis has two objectives, one to treat restenosis by killing the cells that re-occluded and to prevent further restenosis by inhibiting tissue growth. Brachytherapy with either beta or gamma

**2.3 Antiproliferative therapeutics to target VSMC proliferation**

prevent the success of targeting one or two factors.

**2.4 Cell cycle as the therapeutic target**

gene therapy, and 3) pharmacotherapy.

**2.4.1. Brachytherapy**

of vessel remodeling.

Gene therapy techniques provide a unique opportunity to genetically engineer vessels and grafts to become impervious to atherosclerosis and neointimal formation that contributes to arterial restenosis, in-stent restenosis and vein graft failure (Dzau et al., 2002; Khanna, 2008; Kishore & Losordo, 2007; Gaffney et al., 2007; Melo et al., 2005; Von der Leyen & Dzau, 2001). Gene therapy approach has potential not only against monogenic diseases, but also against complex diseases where multiple genes are involved in the disease pathogenesis like in cardiovascular diseases and cancer. One of the key challenges of the gene therapy is appropriate vector for the delivery of functional gene or a concoction of genes in multigenic diseases as in cardiovascular diseases. Besides the choice of vector, other parameters such as, appropriate gene targets and efficient methods of vector delivery for a specific target have to be optimized. Vectors can be either viral or non-viral. The ideal vector is the one, which is nonpathogenic, less immunogenic, more efficient, and enhanced tissue specificity.

Delivery of therapeutic genes to the cardiovascular tissues is challenging. To facilitate local gene delivery to lesions in the vasculature catheter-based vector delivery has been tried using a variety of balloon catheters in animal models and human trials (Khanna, 2008; Kishore & Losordo, 2007; Gaffney et al., 2007; Melo et al., 2005). Stents are ideal gear for localized gene delivery to the vascular wall because of their widespread use, safety and permanent scaffold structure. Stents can be coated with genetically engineered cells or plasmid or adenoviral vectors carrying therapeutic genes (Khanna, 2008; Kishore & Losordo, 2007; Gaffney et al., 2007; Melo et al., 2005). Experimental studies have demonstrated usefulness of gene therapy in treating atherosclerosis and restenosis in various animal models and in some clinical trials. It can be used to transfer exogenous genes to express functional gene products to overcome defective or downregulated endogenous gene expressions through vector-based delivery system. It also can be used to knockdown or suppress the expression of gene products that contribute to pathogenesis of disease by one of the several methods of gene silencing. These include antisense oligonucleotides (ODNs), short segments of RNA with enzymatic activity (ribozymes) and small interfering RNAs [siRNA] (Dzau et al., 2002; J.M. Li et al., 2010; Banno, et al., 2006).

A number of studies have shown that gene therapies can be targeted for reducing cholesterol levels, inflammation and thrombosis (Feldman & Isner, 1995); for upregulating apo-A1 and downregulating chemoattractant protein-1 (MCP1)receptor expression (Tangirala, 1999); transferring pleiotropic atheroprotective nitric oxide synthase [NOS]

Emerging Epigenetic Therapy for Vascular Proliferative Diseases 85

PREVENT trial for human vascular bypass grafts, which resulted in about 75% reduction in VSMC proliferation and fewer graft occlusions. Similar gene manipulation approach was used for coronary bypass grafts in PREVENT II trial. Although the phase 1 trial (PREVENT Trial) showed promising results, later studies were less positive including the phase III, multicentre, randomized double-blinded, placebo-controlled trial of 3014 patients undergoing primary coronary artery bypass graft surgery with at least two planned saphenous grafts (Alexander et al., 2005; Conte et al., 2005, 2006). Although appears to be promising, the use of gene therapy in the treatment of vascular proliferative diseases is still in infancy. Various feasibility and efficacy issues as well as design and delivery of the genes have to be addressed taking into account the complexity of the pathological processes

A number of pharmacological agents have been used to target injury-induced VSMC proliferation that contributes to neointimal growth in balloon-injured arteries. Among these rapamycin or sirolimus, a cytostatic agent, arrests VSMC proliferation and migration *in vitro* and reduces neointimal growth in animal models of balloon-injury (Dzau et al., 2002; Guerin et al., 2005). Its action appears to be mediated through the inhibition of mammalian target of rapamycin (mTOR). One of the downstream events induced by the inhibition of mTOR is induction of p27Kip1, an inhibitor of cyclin-dependent kinases (cdk), causes cells to arrest in G1 phase of the cell cycle. In doing so, it inhibits cell proliferation. Paclitaxel, a derivative of Taxol, is another promising agent for proliferation arrest, which by collapsing the mitotic spindle formation causes mitotic arrest (Jordan et al., 1993). It prevents neointima formation in animal models, and its clinical effect in the blockade of restenosis is investigated in several human trials (the ELUTES, TAXUS and ASPECT trials) via local delivery through

Over the past several years, accumulating data have identified involvement of several antigens in the initiation of immune response during atherosclerosis. These include exogenous infectious microbial pathogens like, cytomegalovirus and chlamydia pneumonia and endogenous proteins such as oxLDL, heat shock proteins (HSPs) and β2-glycoprotein-1b (Habets et al., 2010). Among these, the epitopes recognized on oxLDL are important because of the role of oxLDL in the pathogenesis of atherosclerosis ((Hansson, 1997; 2007; Steinberg & Witztum, 2010; Witztum, 1997). In addition to its proinflammatory and proatherogenic effects, and participation in the formation of foam cells, oxLDL is also immunogenic due to the presence of several neoepitopes. A number of neoepitopes generated during the oxidation of LDL are highly immunogenic and cause the generation of autoantibodies, which are detected in atherosclerotic lesions. Since the different epitopes of oxLDL induce atherogenic immune response, it may be possible to inhibit proatherogenic effects of oxLDL by modulating the immune response towards oxLDL through the immunization against oxLDL. Several antigens have been identified and investigated for immunization against atherosclerosis in animal models. Immunization against oxLDL show reduction in atherosclerosis in several animal models (Habets et al., 2010). This discovery of atheroprotective immunity has resulted in the emergence of immunotherapy approach against atherosclerosis. Indeed, several animal studies indicate that immunization against

stents coated with paclitaxel (Finn et al., 2007; Wilson et al., 2007).

leading to atherosclerosis and restenosis.

**2.4.3 Pharmacotherapy**

**2.4.4 Immunotherapy**

(Qian, 1999); targeting vascular redox biology through heme oxygenase-1, superoxide dismutase, catalase and glutathione peroxidase antioxidant gene therapy to attenuate oxidative stress (Van Assche, 2011); and lipid-lowering gene therapy to reduce plasma LDL levels (Grossman et al., 1995). Furthermore, neointimal hyperplasia that contributes to pathogenesis of arterial stenosis, in-stent stenosis and vein graft failure is also a good target for gene therapy. A number of potential therapeutic genes, which are key to the development of neointimal hyperplasia, have been identified. The ones that are promising for gene therapy include tissue inhibitors of matrix metalloproteinases (Akowuah et al., 2005; Gaffney et al., 2007; Khanna, 2008); NOS (Cooney, 2006; Dzau et al., 2002; Khanna, 2008; Kishore & Losordo, 2007; Gaffney et al., 2007; Melo et al., 2005; Von der Leyen & Dzau, 2001) and p53 (Gaffney et al., 2007). Importantly, delivery of antiproliferative genes such as those coding for p21Cip1, p27Kip1, and iNOS are used to inhibit stenosis and neointimal hyperplasia (Dzau et al., 2002; Von der Leyen & Dzau, 2001]. Conversely, silencing the genes that contribute to proliferation via antisense ODNs (Dzau et al., 2002; Khanna, 2008; Kishore & Losordo, 2007; Gaffney et al., 2007; Melo et al., 2005) or siRNA approach is also effective in preventing in-stent and graft neointimal hyperplasia (Banno, et al., 2006; F. Li et al., 2005; J.M. Li et al., 2010; Matsumae et al., 2008). Antisense ODNs–based inhibition of cell proliferation-related genes such as PCNA, c-myc, c-myb or different cyclin-dependent kinases (cdks) have been successfully carried out in experimental models of vascular lesion formation (Braun-Dullaeus et al., 1998; Dzau et al., 2002; Morishita et al., 1993; Simons et al., 1994).

RNA interference (RNAi) technology is becoming popular approach to alter gene expressions to interrogate their role in pathogenesis of disease, which has utility in the inhibition of VSMC proliferation and neointimal hyperplasia (Banno, et al., 2006; F. Li et al., 2005; J.M. Li et al., 2010; Matsumae et al., 2008). To determine whether Angiotensin II (ANG II)-induced neointimal thickening is mediated via cytoplasmic phospholipase A2 (cPLA2) and phospholipase D2 (PLD2)-activated Akt, injured carotid arteries were exposed to a retrovirus containing cPLA2 siRNA or PLD2 siRNA to test whether their knockdown will result in the reduction of ANG II-induced neointimal thickening (F. Li et al., 2005). SiRNAmediated downregulation of cPLA2 and PLD2 resulted in the reduction of ANG II-induced neointimal thickening. The involvement of CCN1, an extracellular matrix-associated protein, in the development of neointimal hyperplasia is confirmed by siRNA-mediated knockdown approach (Matsumae et al., 2008). The atheroprotective role of midkine (MK), a heparin-binding growth factor, is corroborated by the use of MK-siRNA (Banno, et al., 2006). NADPH oxidase has a critical role in the development of neointimal hyperplasia and restenosis due to its contribution to oxidative stress, which is blocked by siRNA specific to NOX2 gene *Cybb,* an important component of NADPH oxidase (J.M. Li, et al., 2010).

Several experimental gene transfer and gene silencing strategies are evaluated as potential treatments for cardiovascular disease, which resulted in Phase I, and Phase I to Phase III clinical studies for inducible iNOS (Tzeng, 1996; Von der Leyen & Dzau, 2001) and transcription factor E2F, respectively (Dzau et al., 2002; McCarthy, 2001; Mann et al., 1999). E2F, a transcription factor that leads to upregulation of up to 12 cell-cycle genes is an ideal target for cell-cycle blockade. A double-stranded E2F decoy ODN that bears the consensus E2F-binding site (*cis*-elements) was designed as an agent for prevention of vein graft disease. In rabbits, treatment of vein grafts with E2F decoy ODN resulted in inhibition of neointimal hyperplasia and graft atherosclerosis for up to 6 months. This led to phase I PREVENT trial for human vascular bypass grafts, which resulted in about 75% reduction in VSMC proliferation and fewer graft occlusions. Similar gene manipulation approach was used for coronary bypass grafts in PREVENT II trial. Although the phase 1 trial (PREVENT Trial) showed promising results, later studies were less positive including the phase III, multicentre, randomized double-blinded, placebo-controlled trial of 3014 patients undergoing primary coronary artery bypass graft surgery with at least two planned saphenous grafts (Alexander et al., 2005; Conte et al., 2005, 2006). Although appears to be promising, the use of gene therapy in the treatment of vascular proliferative diseases is still in infancy. Various feasibility and efficacy issues as well as design and delivery of the genes have to be addressed taking into account the complexity of the pathological processes leading to atherosclerosis and restenosis.

#### **2.4.3 Pharmacotherapy**

84 Atherogenesis

(Qian, 1999); targeting vascular redox biology through heme oxygenase-1, superoxide dismutase, catalase and glutathione peroxidase antioxidant gene therapy to attenuate oxidative stress (Van Assche, 2011); and lipid-lowering gene therapy to reduce plasma LDL levels (Grossman et al., 1995). Furthermore, neointimal hyperplasia that contributes to pathogenesis of arterial stenosis, in-stent stenosis and vein graft failure is also a good target for gene therapy. A number of potential therapeutic genes, which are key to the development of neointimal hyperplasia, have been identified. The ones that are promising for gene therapy include tissue inhibitors of matrix metalloproteinases (Akowuah et al., 2005; Gaffney et al., 2007; Khanna, 2008); NOS (Cooney, 2006; Dzau et al., 2002; Khanna, 2008; Kishore & Losordo, 2007; Gaffney et al., 2007; Melo et al., 2005; Von der Leyen & Dzau, 2001) and p53 (Gaffney et al., 2007). Importantly, delivery of antiproliferative genes such as those coding for p21Cip1, p27Kip1, and iNOS are used to inhibit stenosis and neointimal hyperplasia (Dzau et al., 2002; Von der Leyen & Dzau, 2001]. Conversely, silencing the genes that contribute to proliferation via antisense ODNs (Dzau et al., 2002; Khanna, 2008; Kishore & Losordo, 2007; Gaffney et al., 2007; Melo et al., 2005) or siRNA approach is also effective in preventing in-stent and graft neointimal hyperplasia (Banno, et al., 2006; F. Li et al., 2005; J.M. Li et al., 2010; Matsumae et al., 2008). Antisense ODNs–based inhibition of cell proliferation-related genes such as PCNA, c-myc, c-myb or different cyclin-dependent kinases (cdks) have been successfully carried out in experimental models of vascular lesion formation (Braun-Dullaeus et al., 1998; Dzau et al., 2002; Morishita et al., 1993; Simons et al.,

RNA interference (RNAi) technology is becoming popular approach to alter gene expressions to interrogate their role in pathogenesis of disease, which has utility in the inhibition of VSMC proliferation and neointimal hyperplasia (Banno, et al., 2006; F. Li et al., 2005; J.M. Li et al., 2010; Matsumae et al., 2008). To determine whether Angiotensin II (ANG II)-induced neointimal thickening is mediated via cytoplasmic phospholipase A2 (cPLA2) and phospholipase D2 (PLD2)-activated Akt, injured carotid arteries were exposed to a retrovirus containing cPLA2 siRNA or PLD2 siRNA to test whether their knockdown will result in the reduction of ANG II-induced neointimal thickening (F. Li et al., 2005). SiRNAmediated downregulation of cPLA2 and PLD2 resulted in the reduction of ANG II-induced neointimal thickening. The involvement of CCN1, an extracellular matrix-associated protein, in the development of neointimal hyperplasia is confirmed by siRNA-mediated knockdown approach (Matsumae et al., 2008). The atheroprotective role of midkine (MK), a heparin-binding growth factor, is corroborated by the use of MK-siRNA (Banno, et al., 2006). NADPH oxidase has a critical role in the development of neointimal hyperplasia and restenosis due to its contribution to oxidative stress, which is blocked by siRNA specific to

NOX2 gene *Cybb,* an important component of NADPH oxidase (J.M. Li, et al., 2010).

Several experimental gene transfer and gene silencing strategies are evaluated as potential treatments for cardiovascular disease, which resulted in Phase I, and Phase I to Phase III clinical studies for inducible iNOS (Tzeng, 1996; Von der Leyen & Dzau, 2001) and transcription factor E2F, respectively (Dzau et al., 2002; McCarthy, 2001; Mann et al., 1999). E2F, a transcription factor that leads to upregulation of up to 12 cell-cycle genes is an ideal target for cell-cycle blockade. A double-stranded E2F decoy ODN that bears the consensus E2F-binding site (*cis*-elements) was designed as an agent for prevention of vein graft disease. In rabbits, treatment of vein grafts with E2F decoy ODN resulted in inhibition of neointimal hyperplasia and graft atherosclerosis for up to 6 months. This led to phase I

1994).

A number of pharmacological agents have been used to target injury-induced VSMC proliferation that contributes to neointimal growth in balloon-injured arteries. Among these rapamycin or sirolimus, a cytostatic agent, arrests VSMC proliferation and migration *in vitro* and reduces neointimal growth in animal models of balloon-injury (Dzau et al., 2002; Guerin et al., 2005). Its action appears to be mediated through the inhibition of mammalian target of rapamycin (mTOR). One of the downstream events induced by the inhibition of mTOR is induction of p27Kip1, an inhibitor of cyclin-dependent kinases (cdk), causes cells to arrest in G1 phase of the cell cycle. In doing so, it inhibits cell proliferation. Paclitaxel, a derivative of Taxol, is another promising agent for proliferation arrest, which by collapsing the mitotic spindle formation causes mitotic arrest (Jordan et al., 1993). It prevents neointima formation in animal models, and its clinical effect in the blockade of restenosis is investigated in several human trials (the ELUTES, TAXUS and ASPECT trials) via local delivery through stents coated with paclitaxel (Finn et al., 2007; Wilson et al., 2007).

#### **2.4.4 Immunotherapy**

Over the past several years, accumulating data have identified involvement of several antigens in the initiation of immune response during atherosclerosis. These include exogenous infectious microbial pathogens like, cytomegalovirus and chlamydia pneumonia and endogenous proteins such as oxLDL, heat shock proteins (HSPs) and β2-glycoprotein-1b (Habets et al., 2010). Among these, the epitopes recognized on oxLDL are important because of the role of oxLDL in the pathogenesis of atherosclerosis ((Hansson, 1997; 2007; Steinberg & Witztum, 2010; Witztum, 1997). In addition to its proinflammatory and proatherogenic effects, and participation in the formation of foam cells, oxLDL is also immunogenic due to the presence of several neoepitopes. A number of neoepitopes generated during the oxidation of LDL are highly immunogenic and cause the generation of autoantibodies, which are detected in atherosclerotic lesions. Since the different epitopes of oxLDL induce atherogenic immune response, it may be possible to inhibit proatherogenic effects of oxLDL by modulating the immune response towards oxLDL through the immunization against oxLDL. Several antigens have been identified and investigated for immunization against atherosclerosis in animal models. Immunization against oxLDL show reduction in atherosclerosis in several animal models (Habets et al., 2010). This discovery of atheroprotective immunity has resulted in the emergence of immunotherapy approach against atherosclerosis. Indeed, several animal studies indicate that immunization against

Emerging Epigenetic Therapy for Vascular Proliferative Diseases 87

epigenetics of VSMC proliferation and in particular their susceptibility to perturbation by the epigenetic modifiers may offer novel insights into disease pathogenesis and epigenetic therapeutic approaches. Therefore, it is appropriate to review the current knowledge of

Curiosity in epigenetics has surged during past decade even though the principle question it aims to address has been there for decades. That is, how a multicellular organism maintain drastically different gene expression profile in different cell types of the organism, while all the different cell types of the organism have exactly the same DNA. This is where epigenetics come into picture. Epigenetics refers to the inheritance of gene function/activity/expression that may be stable over long periods, last through several cell divisions or inherit through several generations, all without any change in their primary DNA (Ng & Gurdon, 2008; Probst, 2009). The three interrelated epigenetic mechanisms, which involve: methylation of DNA at CpG dinucleotides at specific position in the DNA molecule suppress expression of nearby genes (Esteller, 2008); posttranslational modifications of histones alters chromatin structure and changes promoter accessibility (Kouzarides, 2007); and small RNA molecules generated from noncoding RNAs (ncRNAs) inhibit gene expression (Mattick et al., 2009). All these mechanisms involved in epigenetic regulation contribute to epigenome. This review focuses on the role of posttranslational modifications of histones in the regulation of VSMC proliferation, and on the epigenetic regulators of histone modifications as potential candidates for drug targeting in the treatment and management of vascular proliferative