**3.1.1 Profilin & vascular smooth muscle cells migration & proliferation**

Migration of smooth muscle cell takes place throughout vascular development, as a result of vascular injury, and throughout atherogenesis. Throughout vascular development, plateletderived growth factor promotes migration of pericyte or other precursors of smooth muscle that is required for the formation of correct vessel wall (Hellstrom et al., 1999). Clinically, vascular injury takes place after angioplasty, vascular stent implantation, or organ transplantation. In vascular injury in animals, thickening of intima and media has been attributed to VSM proliferation and migration from media to intima (Clowes et al., 1989; Majesky & Schwartz, 1990; Reidy, 1992). Throughout atherogenesis, VSMCs migrate to

Fig. 6. The involvement of profilin (red) in different signaling routes. This schematic drawing depicts only the main connections established so far. Molecules and second messengers of the polyphosphoinositide signaling pathway are indicated in yellow, protein members of signaling routes are marked green, proline-cluster proteins identified as profilin ligands are marked purple, the actin cycle is seen in blue, Ca2+ in intracellular stores and Ca2+ regulated microfilament proteins are marked grey. For simplicity, the solid arrows indicate either direct interactions between components, as shown by biochemical assays, or point to pathways. Broken arrows indicate suspected or indirect interactions. Abbreviations:

PI-3,4,5-P3: phosphatidylinositol 3,4,5-trisphosphate; PIP2: phosphatidylinositol 4,5 bisphosphate; RTK: receptor tyrosine kinase; DAG: diacylglycerol; PLCγ 1: phospholipase Cγ 1; cAMP/cGMP- PK: cAMP/cGMP dependent protein kinase; IP3: inositol 1,4,5-

trisphosphate, adapted from Schlüter et al., (1997) with permission.

**3.1 Role of profilin in vascular smooth muscle & endothelial cells** 

**3.1.1 Profilin & vascular smooth muscle cells migration & proliferation** 

Migration of smooth muscle cell takes place throughout vascular development, as a result of vascular injury, and throughout atherogenesis. Throughout vascular development, plateletderived growth factor promotes migration of pericyte or other precursors of smooth muscle that is required for the formation of correct vessel wall (Hellstrom et al., 1999). Clinically, vascular injury takes place after angioplasty, vascular stent implantation, or organ transplantation. In vascular injury in animals, thickening of intima and media has been attributed to VSM proliferation and migration from media to intima (Clowes et al., 1989; Majesky & Schwartz, 1990; Reidy, 1992). Throughout atherogenesis, VSMCs migrate to

**3. Profilin & vascular diseases** 

occupy the intima, either from the media (Murry et al., 1997) or from the circulation via CD34+ hematopoietic progenitor cells migration, resulting in smooth muscle progenitor cells (Yeh et al., 2003). Figure 7 shows the inner lining of a normal artery.

#### Fig. 7. Inner lining of normal artery

VSMC migration requires the extension of lamellipodia toward the stimulus via actin polymerization, trailing edge detachment via focal contacts degradation, and force generation via myosin II in the cellular body to drive the cell forward (Gerthoffer, 2007). Initiation of new filaments nucleation is achieved by actin-related proteins such as ARP2/3 complex interacting with the minus end and elimination at the plus end of capping proteins that are PIP2-sensitive. Extension of new actin filaments is improved by formin-related proteins such as mDia1 and mDia2 that operate along with profilin on the plus end. Activation of The formins mDia1 and mDia2 is achieved by RhoA and Cdc42, respectively. Profilin released from the binding sites of membrane phospholipid enhances nucleotide exchange on G-actin monomers and promotes actin polymerization. Stimulation of filament branching is accomplished via activating WAVE complex and WASP by Rac and Cdc42, respectively. WAVE and WASP increase nucleation and branching through activating actin-related proteins such as ARP2/3 complex. Severing of Actin-filament by gelsolin is stimulated by Ca2+, and nucleation is favored via liberating gelsolin from plus ends of F-actin by PtdIns 4,5-P2. Stimulation of actin depolymerization is executed by cofilin at the minus end. Cofilin acts to limit the filaments length and to induce the existing filaments turnover. These operations have been reported to be sufficient for force generation to expand the leading edge of the cell toward the stimulus (Mogilner & Oster, 2003; Prass et al., 2006). Consistent with these findings our recent data confirmed the significant role of profilin-I in VSMC migration. Migration assays performed on VSMC isolated from the aorta of transgenic mice that overexpress the cDNA of profilin-I or profilin-I-dominant negative mutant (88R/L) and nontransgenic controls showed that the rate of cell migration of profilin-I VSMCs is significantly higher than that of the control and 88R/L. Conversely, 88R/L mice exhibited a significantly lower rates compared to nontransgenic controls (Figure 8) (Hassanain HH, unpublished).

On the other hand, it has been shown that profilin plays a vital role in the proliferation and differentiation of normal cell. Disruption in the profilin results in embryonic lethality due to gross impairment in growth, motility, and cytokinesis in single cells (Haugwitz et al., 1994; Witke, 2004; Witke et al., 2001). Also, profilin-1 was demonstrated to exert cellular responses such as DNA synthesis and increasing the binding activity of AP-1 DNA in mesangial cells via activating putative cell surface receptors (Tamura et al., 2000).

Fig. 8. The MetaMorph image analysis of the mean speed of the individual cells (μm/min) of profilin-I, 88 R/L and control VSMCs. The differences in mean were determined by ANOVA. \*P < 0.05, compared with corresponding control, is considered to be significant (Hassanain HH, unpublished).

In line with the established role of profilin in cellular migration and proliferation, it has been shown that recombinant profilin-I stimulates DNA-synthesis and migration of both rat and human VSMCs in a concentration-dependent manner (Caglayan et al., 2010). The same study indicated that profilin-induced VSMCs migration is dependent on PI3K activity (Caglayan et al., 2010). Moreover, Cheng et al., (2011) found that profilin-I plays a key role in Angiotensin (Ang) II-induced VSMCs proliferation. They also suggested that Ang-II increases profilin-I expression and promotes VSMCs proliferation via activating AT1 receptor/JAK2/STAT3 pathway (Cheng et al., 2011). On the contrary, other studies described the involvement of phospho-extracellular signal-regulated kinase1/2 (P-ERK1/2) and phospho-c-Jun NH2-terminal kinase (P-JNK) in Ang-II-induced profilin-I expression (Zhong et al., 2011), and that PI3-kinase, Src, and, to a lesser extent, P-ERK1/2 are required for profilin-I-dependent VSMCs proliferation (Caglayan et al., 2010). Consequently, Cheng et al., (2011) proposed that the interaction of these signaling pathways mediating the role of profilin-I in VSMCs proliferation requires further investigation. Consistent with these data, we observed that the treatment of mouse aortic VSMCs with Ang-II (100 nM/10 min) resulted in increased profilin-I expression (Hassanain HH, unpublished)

#### **3.1.2 Profilin & vascular smooth muscle contraction**

Regulation of smooth muscle contraction has been thought to be only dependent on the 20 kDa regulatory light chain of myosin (MLC20) that in turn modulates cross-bridge cycling of actomyosin. Numerous studies showed that contractile stimulation promotes actin polymerization in vascular and airway smooth muscle tissues (Cipolla & Osol, 1998; Jones et

Fig. 8. The MetaMorph image analysis of the mean speed of the individual cells (μm/min) of

In line with the established role of profilin in cellular migration and proliferation, it has been shown that recombinant profilin-I stimulates DNA-synthesis and migration of both rat and human VSMCs in a concentration-dependent manner (Caglayan et al., 2010). The same study indicated that profilin-induced VSMCs migration is dependent on PI3K activity (Caglayan et al., 2010). Moreover, Cheng et al., (2011) found that profilin-I plays a key role in Angiotensin (Ang) II-induced VSMCs proliferation. They also suggested that Ang-II increases profilin-I expression and promotes VSMCs proliferation via activating AT1 receptor/JAK2/STAT3 pathway (Cheng et al., 2011). On the contrary, other studies described the involvement of phospho-extracellular signal-regulated kinase1/2 (P-ERK1/2) and phospho-c-Jun NH2-terminal kinase (P-JNK) in Ang-II-induced profilin-I expression (Zhong et al., 2011), and that PI3-kinase, Src, and, to a lesser extent, P-ERK1/2 are required for profilin-I-dependent VSMCs proliferation (Caglayan et al., 2010). Consequently, Cheng et al., (2011) proposed that the interaction of these signaling pathways mediating the role of profilin-I in VSMCs proliferation requires further investigation. Consistent with these data, we observed that the treatment of mouse aortic VSMCs with Ang-II (100 nM/10 min) resulted in increased profilin-I expression

Regulation of smooth muscle contraction has been thought to be only dependent on the 20 kDa regulatory light chain of myosin (MLC20) that in turn modulates cross-bridge cycling of actomyosin. Numerous studies showed that contractile stimulation promotes actin polymerization in vascular and airway smooth muscle tissues (Cipolla & Osol, 1998; Jones et

profilin-I, 88 R/L and control VSMCs. The differences in mean were determined by ANOVA. \*P < 0.05, compared with corresponding control, is considered to be significant

(Hassanain HH, unpublished).

(Hassanain HH, unpublished)

**3.1.2 Profilin & vascular smooth muscle contraction** 

al., 1999; D. Mehta & Gunst, 1999) and in cultured smooth muscle cells (An et al., 2002; Barany, et al., 2001; Hirshman & Emala, 1999). In addition, inhibition of actin polymerization by specific inhibitors such as latrunculin decreases the contractile stimuli- activated force development in smooth muscle (Cipolla & Osol, 1998; D. Mehta & Gunst, 1999; Youn et al., 1998). However, this does not affect contractile stimulation-induced MLC20 phosphorylation (34), suggesting that actin polymerization plays a central role during smooth muscle contraction. Tang & Tan, (2003) investigated the effect of profilin, the main actin-regulatory protein on the regulation of smooth muscle contraction. They demonstrated that profilin downregulation with antisense repressed force generation, without affecting MLC20 phosphorylation, signifying that profilin is crucial for smooth muscle contraction and that it does not regulate the activation of contractile protein. Yet, profilin downregulation repressed increases in the F-actin/G-actin ratio in return to agonist stimulation, showing that profilin is essential for actin dynamics during contractile stimulation of smooth muscle (Tang & Tan, 2003). In harmony with these finding our results showed higher expression of stress fibers and membrane ruffling in vascular smooth muscle cells from profilin-I transgenic mice compared with nontransgenic control and 88R/L. The 88R/L cells, however, showed lower expression of stress fiber formation and ruffling than the nontransgenic controls (Figure 9*A*) (Moustafa-Bayoumi et al., 2007). In addition, we confirmed these findings by assessing the ratio of F-actin/G-actin in the aortic smooth muscle cells from profilin-I. Our results showed a significant increase in F/G actin ratio in the aortic smooth muscle cells from profilin-I mice compared with the nontransgenic controls (Figure 9B) (Moustafa-Bayoumi et al., 2007). Furthermore, we showed that profilin-I plays a significant role in increased contractility and force development in the mesenteric arteries of profilin-I mice via activating Rho/ROCK pathway and MLC20 (Hassona et al., 2010). Activated Rho elevates MLC20 phosphorylation by *1*) directly phosphorylating MLC20 and *2*) phosphorylation and inhibition of the MBS of MLC20 phosphatase (Higgs & Pollard, 2001; Pollard & Borisy, 2003). This increases myosin contractility and tension contributing to stress fibers. In conclusion, our results indicate that overexpression of profilin-I in smooth muscle cells leads to increased contractility and force development via increasing actin polymerization (Moustafa-Bayoumi et al., 2007) and MLC20 activation(Hassona et al., 2010), which in turn induce mechanical stress that is considered as the main initiator for arterial stiffness and hypertension observed in these mice.

Fig. 9. Overexpression of profilin-I induced actin polymerization in vascular smooth muscle cells. Rhodamine-phalloidin staining of smooth muscle cell confluent monolayers shows increased stress fibers in vascular smooth muscle cells from profilin-I transgenic mouse as compared with nontransgenic control (*a*). The 88R/L cells, however, show lower expression of stress fiber formation than the control (*a*). Analysis of the F-actin/G-actin ratio shows significant increase in F-actin/G-actin (*F*/*G*) ratio in the aortic smooth muscle cells from profilin-I mice compared with the nontransgenic controls (*b*) (Moustafa-Bayoumi et al., 2007).

#### **3.1.3 Profilin & vascular endothelial cells adhesion**

Endothelial cells survival neatly depends on their ability to anchor to extracellular matrix proteins. Suppression of endothelial cell adhesion has been shown to induce apoptosis in these cells (Meredith et al., 1993; Re et al., 1994; Zang et al., 1995). It has been found that transient overexpression of profilin in cultured human aortic endothelial cells using replication-incompetent adenovirus enhances endothelial cells adhesion to the extracellular matrix via promoting the binding of extracellular fibronectin to its receptor on the surface of these cells. Additionally, it was revealed that profilin-mediated enhancement of endothelial cell adhesion has a protective role in situations of focal contacts disruption due to shear, stretch or other focal injuries (Moldovan et al., 1997).

Moreover, the authors, Moldovan et al., (1997) proposed that the profilin-mediated effect seems to be stimulated via recruiting integrins α5β1 to the endothelial cell surface. Numerous mechanisms may explain this later effect. One possibility is that profilin might cause improvement in the access of receptor molecules to the cell surface. Instead, profilin might cause impairment in the internalization of membrane receptors. These effects may be achieved in 1) actin-dependent manner, where profilin might decrease receptor internalization via disrupting actin stress fibers or it might offer a stronger anchor for fibronectin receptor molecules in focal contacts via stabilizing actin filaments that are not stress fibers (Finkel et al., 1994), or 2) actin-independent manner, where profilin interacts

(b)

Endothelial cells survival neatly depends on their ability to anchor to extracellular matrix proteins. Suppression of endothelial cell adhesion has been shown to induce apoptosis in these cells (Meredith et al., 1993; Re et al., 1994; Zang et al., 1995). It has been found that transient overexpression of profilin in cultured human aortic endothelial cells using replication-incompetent adenovirus enhances endothelial cells adhesion to the extracellular matrix via promoting the binding of extracellular fibronectin to its receptor on the surface of these cells. Additionally, it was revealed that profilin-mediated enhancement of endothelial cell adhesion has a protective role in situations of focal contacts disruption due to shear,

Moreover, the authors, Moldovan et al., (1997) proposed that the profilin-mediated effect seems to be stimulated via recruiting integrins α5β1 to the endothelial cell surface. Numerous mechanisms may explain this later effect. One possibility is that profilin might cause improvement in the access of receptor molecules to the cell surface. Instead, profilin might cause impairment in the internalization of membrane receptors. These effects may be achieved in 1) actin-dependent manner, where profilin might decrease receptor internalization via disrupting actin stress fibers or it might offer a stronger anchor for fibronectin receptor molecules in focal contacts via stabilizing actin filaments that are not stress fibers (Finkel et al., 1994), or 2) actin-independent manner, where profilin interacts

Fig. 9. Overexpression of profilin-I induced actin polymerization in vascular smooth muscle cells. Rhodamine-phalloidin staining of smooth muscle cell confluent monolayers shows increased stress fibers in vascular smooth muscle cells from profilin-I transgenic mouse as compared with nontransgenic control (*a*). The 88R/L cells, however, show lower expression of stress fiber formation than the control (*a*). Analysis of the F-actin/G-actin ratio shows significant increase in F-actin/G-actin (*F*/*G*) ratio in the aortic smooth muscle

cells from profilin-I mice compared with the nontransgenic controls (*b*) (Moustafa-

**3.1.3 Profilin & vascular endothelial cells adhesion** 

stretch or other focal injuries (Moldovan et al., 1997).

Bayoumi et al., 2007).

with PtdIns 4,5-P2 and inhibits its hydrolysis by phospholipase C (Goldschmidt-Clermont et al., 1990, 1991; Lassing & Lindberg, 1985). Increased concentrations of PtdIns 4,5-P2 could stimulate the stabilization of newly formed focal contacts including the fibronectin receptor via an unknown mechanism or profilin overexpression could overcome other actin-binding proteins for interacting with PtdIns 4,5-P2 and thus enhance their binding to actin filaments (Hartwig et al., 1995).

#### **3.1.4 Role of profilin in vascular endothelial cells migration, proliferation & capillary morphogenesis**

Vascular endothelial cell (VEC) migration is vital for capillary outgrowth from preexisting blood vessels during angiogenesis (Bauer, et al., 2005). During cell migration, actin cytoskeleton reorganization is a dynamic process that includes both actin polymerization and depolymerization in an accurate spatiotemporal manner. Regulation of this actin remodeling process is achieved by a large number of actin binding proteins such as those involved in monomer sequestering, nucleating, elongating, severing, depolymerizing, and capping of actin filaments (Pollard & Borisy, 2003). Expression profiles in VEC experiencing capillary morphogenesis identified some of the key actinbinding proteins that have been previously involved in angiogenesis such as thymosin β4, profilin, gelsolin and VASP. Among these proteins, as a minimum thymosin β4 has been established as a proangiogenic molecule *in vivo* (Philp et al., 2004; Salazar et al., 1999). In addition, it has been reported that silencing profilin-I expression in human umbilical vein endothelial cells significantly decreases their capability of forming planar cord-like structures on matrigel (a commonly adopted *in vitro* representation for angiogenesis). These findings proposed for the first time that profilin-I might play a key role in VEC capillary morphogenesis (Ding et al., 2006).

In a more recent report for the same group they adopted a knockdown–knockin experimental system to stably express either fully functional form or mutants of profilin-I that are deficient in binding to actin and proteins containing polyproline domains, in a human dermal microvascular cell line. They showed that silencing endogenous profilin-I expression in this cell line results in slow rate of random migration, decreased membrane protrusion velocity and a significant reduction in matrigel-induced cord formation. These defects were rescued only via re-expression of fully functional but not any of the two ligand-binding deficient mutants of profilin-I. They also showed that loss of profilin-I expression in VEC inhibits three dimensional capillary morphogenesis, MMP2 secretion and ECM invasion. Disruption of actin and polyproline interactions of profilin-I inhibited VEC invasion through ECM, as well. They concluded that profilin-I regulates VEC migration, invasion and capillary morphogenesis through its binding to both actin and proline-rich ligands (Ding et al., 2009). Furthermore, they indicated that these *in vitro* findings pave the way for future *in vivo* studies to investigate the role of profilin-I in angiogenesis.

Interestingly, cutaneous wound healing experiments in our profilin-I and 88R/L transgenic mice showed a significant increase in blood vessel density in profilin-I transgenic mice compared to 88R/L transgenic mice and nontransgenic control at post wound day 7 (Figure 10) (Hassanain HH, unpublished). These data could indicate the importance of profilin-I in angiogenic reponse in VEC.

Fig. 10. Stimulation of angiogenesis in the wound area of profilin-I mice. Distribution of capillaries along the margin of the excision wound in Profilin-I, 88R/L and nontransgenic control mice at post wound days 0 and 7. High magnification of capillaries in the skin was obtained with a 2X objective lens light microscope (Hassanain HH, unpublished).

#### **3.2 Role of profilin in vascular remodeling & hypertension**

Hypertension represents a major risk factor for cardiovascular events such as stroke and myocardial infarction. It is well established that hypertension leads to remodeling of large and small arteries (Folkow, 1982; Simon, 2004). Remodeling of the vasculature is an active process of structural changes that involves alterations in cellular processes, including growth and changes in the extracellular matrix integrin-cytoskeleton axis, resulting in an increase in the media-to-lumen ratio (Gimbrone et al., 1997; Intengan & Schiffrin, 2001). Physiological remodeling is an adaptive process occurring in response to hemodynamic changes and aging. However, when this process becomes maladaptive, it plays a role in hypertension's complications (Ming et al, 2002; Touyz, 2007). Increased mechanical strain/hypertension in the vessel wall triggers the hypertrophic signaling pathway resulting in structural remodeling of vasculature. Increased actin polymerization and stress fiber formation generate mechanical force that represents an important modulator of cellular morphology and function in a variety of tissues and is an important contributor to hypertrophy in the cardiovascular system (Ruwhof &van der Laarse, 2000). Also, it has been shown that actin polymerization within VSMCs in response to increased intravascular pressure is a novel mechanism underlying arterial myogenic behavior. The cytosolic concentration of G-actin is significantly reduced by an elevation in intravascular pressure, demonstrating the dynamic nature of actin within VSMCs and implying a shift in the F:G equilibrium in favor of F-actin. Profilin-I which is a key actin-regulatory protein that plays an essential role in regulating de novo actin polymerization, particularly actin treadmilling (Carlier & Pantaloni, 2007; Suetsugu et al., 1999) could be vital in regulating all of these vascular events. Indeed, our report in the Journal of Biological Chemistry (Moustafa-Bayoumi et al., 2007) established the feasibility of our proposal. We showed that elevated expression of profilin-I gene in VSMCs of profilin-I mice favoring F-actin induces stress fiber formation (Figure 11) and plays an important role in vascular hypertrophy by inducing

Fig. 10. Stimulation of angiogenesis in the wound area of profilin-I mice. Distribution of capillaries along the margin of the excision wound in Profilin-I, 88R/L and nontransgenic control mice at post wound days 0 and 7. High magnification of capillaries in the skin was

Hypertension represents a major risk factor for cardiovascular events such as stroke and myocardial infarction. It is well established that hypertension leads to remodeling of large and small arteries (Folkow, 1982; Simon, 2004). Remodeling of the vasculature is an active process of structural changes that involves alterations in cellular processes, including growth and changes in the extracellular matrix integrin-cytoskeleton axis, resulting in an increase in the media-to-lumen ratio (Gimbrone et al., 1997; Intengan & Schiffrin, 2001). Physiological remodeling is an adaptive process occurring in response to hemodynamic changes and aging. However, when this process becomes maladaptive, it plays a role in hypertension's complications (Ming et al, 2002; Touyz, 2007). Increased mechanical strain/hypertension in the vessel wall triggers the hypertrophic signaling pathway resulting in structural remodeling of vasculature. Increased actin polymerization and stress fiber formation generate mechanical force that represents an important modulator of cellular morphology and function in a variety of tissues and is an important contributor to hypertrophy in the cardiovascular system (Ruwhof &van der Laarse, 2000). Also, it has been shown that actin polymerization within VSMCs in response to increased intravascular pressure is a novel mechanism underlying arterial myogenic behavior. The cytosolic concentration of G-actin is significantly reduced by an elevation in intravascular pressure, demonstrating the dynamic nature of actin within VSMCs and implying a shift in the F:G equilibrium in favor of F-actin. Profilin-I which is a key actin-regulatory protein that plays an essential role in regulating de novo actin polymerization, particularly actin treadmilling (Carlier & Pantaloni, 2007; Suetsugu et al., 1999) could be vital in regulating all of these vascular events. Indeed, our report in the Journal of Biological Chemistry (Moustafa-Bayoumi et al., 2007) established the feasibility of our proposal. We showed that elevated expression of profilin-I gene in VSMCs of profilin-I mice favoring F-actin induces stress fiber formation (Figure 11) and plays an important role in vascular hypertrophy by inducing

obtained with a 2X objective lens light microscope (Hassanain HH, unpublished).

**3.2 Role of profilin in vascular remodeling & hypertension** 

internal mechanical stress and triggering the hypertrophic signaling pathways, integrinsα1β1/Rho-ROCK/MAPKs e.g. P-ERK and P-JNK, leading to vascular remodeling in both large (e.g. aorta) and small (e.g. mesenteric) arteries (Figure 12A, B) of profilin transgenic mice (Hassona et al., 2010; Moustafa-Bayoumi et al., 2007).

Fig. 11. Hypertension or increased profilin-I expression in VSMCs leads to a shift in the F:G equilibrium in favor of F-actin and an elevation in intravascular pressure. This pathway can be reversed by F-actin inhibitor, cytochalasin D or profilin-I mutant, 88R/L.

Consistent with our finding, very recent studies showed increased profilin-I expression in hypertrophic thoracic aorta and mesenteric arteries of spontaneously hypertensive rats with subsequent elevation in both P-ERK and P-JNK, suggesting that profilin-I may contribute to the vascular remodeling in these rats (Cheng et al., 2011; Zhong et al., 2011). In this context, previous studies suggested that mechanical stretch is closely related to JNK and ERK1/2 activation (Hu et al., 1997; Pyles et al., 1997). These cascades play an important role in remodeling of blood vessels, as well. In addition, this pathway is activated by Ang-II and has been implicated in the pathogenesis of cardiovascular diseases (P.K. Mehta & Griendling, 2007). Interestingly, it has been recently reported that profilin-I is a key component in the Ang-II-induced vascular remodeling (Cheng et al., 2011; Zhong et al., 2011).

As it was mentioned above that hypertension is a major cause of vascular remodeling. The primary aim of anti-hypertensive drugs, particularly Ang-converting enzyme inhibitors and Ang receptor subtype 1 antagonists, is to lower the blood pressure with the hope of reversing this remodeling (Schiffrin, 2001). Importantly, In our profilin-I model we demonstrate that the reverse can be true as well, i.e. alteration in cytoskeleton dynamics favoring increased actin polymerization can contribute to vascular adaptations with aging resulting in increased systolic blood pressure by the time the profilin-I mice were six months old (Figure 12C) (Moustafa-Bayoumi et al., 2007). The blood pressure in the profilin-I mice was elevated 25–30 mm Hg higher than nontransgenic controls. In agreement with our findings, it has been demonstrated that profilin speeds up the actin remodeling and accordingly improves the growth and invasion force of VSMCs resulting in increased vascular resistance and accelerated formation of pulmonary hypertension (Dai et al., 2006).

Fig. 12. Profilin overexpression induced vascular hypertrophy and hypertension. Hematoxylin and eosin staining shows clear signs of remodeling and vascular hypertrophy in the aorta of profilin-I transgenic mice (yellow arrows; **A**) and mesenteric arteries (white arrows; **B**). There are no differences, however, between 88R/L and nontransgenic control aortic sections (**A**). Tail cuff measurements of blood pressure show significant increase in the systolic blood pressure (BP) in profilin-I transgenic mice at 6 months and older compared with nontransgenic control mice (C) (Hassona et al., 2010; Moustafa-Bayoumi et al., 2007).

On the other hand, the blood pressure in 88R/L mice was below the control littermates; however, it did not reach statistical significance. The absence of a hypotensive phenotype in the 88R/L mice could be due to the lack of significant vascular remodeling as a result of decreased actin polymerization. Our results showed a decrease in stress fibers formation in 88R/L mice (Figure 9); however, these changes did not translate into significant alterations in the vasculature. This might be due to an activation of a compensatory mechanism to maintain the integrity of vessel structure and thus keep the blood pressure at a survival level. Additionally, our preliminary data showed that inhibition of profilin-I-induced stress fibers by cytochalasin D lowered blood pressure in profilin-I mice. As a pilot study the profilin-I mice were injected with a single dose of cytochalasin D (0.5 µg/gram body weight) which led to lowered blood pressure within 10 minutes in these mice from 140 mmHg to 70 mmHg and the effect was sustained for more than 1.5 hours. Then the mice were recovered without any sign of sickness. To make sure that cytochalasin D had no damaging effect on the endothelium, we assessed the functionality of the endothelium using Ach and wiremyography. Our results showed no damage in the endothelium after cytochalasin D treatment (Hassanain HH, unpublished). We should note that cytochalasin D was used before by other investigators in different studies with much higher doses and no toxicity was observed (Speirs & Kaufman, 1989).

Furthermore, stress fiber formation could affect the relaxation/contraction process of the smooth muscles, making it more constrictive and/or less responsive to vasodilators such as nitric oxide. That could be an important factor contributing to hypertension besides the vascular hypertrophy in the profilin-I transgenic mice. Our recent report in the American Journal of Physiology confirmed this proposal. We showed that vascular hypertrophy-

Fig. 12. Profilin overexpression induced vascular hypertrophy and hypertension.

was observed (Speirs & Kaufman, 1989).

Hematoxylin and eosin staining shows clear signs of remodeling and vascular hypertrophy in the aorta of profilin-I transgenic mice (yellow arrows; **A**) and mesenteric arteries (white arrows; **B**). There are no differences, however, between 88R/L and nontransgenic control aortic sections (**A**). Tail cuff measurements of blood pressure show significant increase in the systolic blood pressure (BP) in profilin-I transgenic mice at 6 months and older compared with nontransgenic control mice (C) (Hassona et al., 2010; Moustafa-Bayoumi et al., 2007).

On the other hand, the blood pressure in 88R/L mice was below the control littermates; however, it did not reach statistical significance. The absence of a hypotensive phenotype in the 88R/L mice could be due to the lack of significant vascular remodeling as a result of decreased actin polymerization. Our results showed a decrease in stress fibers formation in 88R/L mice (Figure 9); however, these changes did not translate into significant alterations in the vasculature. This might be due to an activation of a compensatory mechanism to maintain the integrity of vessel structure and thus keep the blood pressure at a survival level. Additionally, our preliminary data showed that inhibition of profilin-I-induced stress fibers by cytochalasin D lowered blood pressure in profilin-I mice. As a pilot study the profilin-I mice were injected with a single dose of cytochalasin D (0.5 µg/gram body weight) which led to lowered blood pressure within 10 minutes in these mice from 140 mmHg to 70 mmHg and the effect was sustained for more than 1.5 hours. Then the mice were recovered without any sign of sickness. To make sure that cytochalasin D had no damaging effect on the endothelium, we assessed the functionality of the endothelium using Ach and wiremyography. Our results showed no damage in the endothelium after cytochalasin D treatment (Hassanain HH, unpublished). We should note that cytochalasin D was used before by other investigators in different studies with much higher doses and no toxicity

Furthermore, stress fiber formation could affect the relaxation/contraction process of the smooth muscles, making it more constrictive and/or less responsive to vasodilators such as nitric oxide. That could be an important factor contributing to hypertension besides the vascular hypertrophy in the profilin-I transgenic mice. Our recent report in the American Journal of Physiology confirmed this proposal. We showed that vascular hypertrophyassociated hypertension of profilin-I transgenic mice led to functional remodeling of peripheral arteries. Our results showed a significant increase in the contraction response of profilin-I mesenteric arteries toward phenylephrine and significant decreases in the relaxation response toward ACh and sodium nitrite compared with nontransgenic controls (Hassona et al, 2010). Additionally, inhibiting stress fibers formation with cytochalasin D significantly relaxes the phenylephrine-contracted mesenteric arteries, suggesting that the increased constriction of mesenteric arteries to phenylephrine could be because of the increased F- to G actin ratio; however, cytochalasin D treatment reduced this ratio (Hassona et al., 2010).

Moreover, it has been reported that in addition to the role of hypertension in vascular remodeling, there are pressure-independent genes that play a key role in vascular remodeling. This concept is supported by the observation that despite blood pressure control in hypertensive patients, the rate of restenosis (attributable to remodeling) remains high (Gurlek et al., 1995). In harmony with this concept we recently showed that normalization of blood pressure by selected anti-hypertensive agents is not enough to correct the structural and functional remodeling of profilin-I transgenic mice (Hassona et al., 2011). Our results demonstrated that there is only correction in the functional remodeling and signaling cascades of the mesenteric arteries of losartan- and amlodipine-treated, but not those of atenolol-treated profilin-I transgenic mice, where losartan and amlodipine decrease the F-actin and stress fibers formation, proposing that the stress fibers seem to play a major role in the development and progression of the vascular remodeling-associated hypertension. We finally concluded that profilin-I gene, which is the key player controlling stress fiber formation may be a good target to treat not only hypertension but also the vascular remodeling in hypertensive patients (Hassona et al., 2011).

#### **3.3 Role of profilin in atherosclerosis & vascular complication in diabetes**

Vascular endothelium dysfunction goes before, and may participate in atheroma formation in return to various cardiovascular risk factors such as diabetes (Johnstone et al., 1993; Tesfamariam et al., 1990;), hyperlipidemia (Chikani et al., 2004; Steinberg et al., 1997), and both local and systemic inflammatory mediators (Libby, 2002). Interestingly, Romeo et al., (2004) revealed that profilin-I levels are improved in the endothelium of diabetic aorta of both human and experimental animals. They also demonstrated that profilin overexpression in primary aorta EC was capable of triggering indicators of endothelial dysfunction such as apoptosis, ICAM-1 up-regulation, and decreased VASP phosphorylation. In addition, profilin was found to be required for LDL-mediated ICAM-1 up-regulation and it can be regulated by LDL/cholesterol signaling, but not high glucose (Romeo et al., 2004). Although, Clarkson et al., (2002) reported that exposure to high glucose was able to increase profilin-I mRNA in mesangial cells and in the diabetic rat kidney. Romeo et al., (2004) suggested that the inability of high glucose to enhance profilin-I protein levels in EC is in line with a multifactorial etiology of endothelial dysfunction coupled with the metabolic syndrome and may reveal the inadequate effect of glucose-lowering monotherapy to prevent macrovascular complications in type 2 diabetic patients (U.K. Prospective Diabetes Study (UKPDS) Group, 1998). On the other hand, our preliminary data showed that mouse aortic VSMCs treated with glucose (25 mM/24 hours) increased profilin-I expression (Hassanain HH, unpublished).

Furthermore, Romeo et al., (2004) showed that profilin was clearly increased in EC and macrophages within atherosclerotic lesions of apoE null mice. In a more recent report, the same group specified the significance of profilin-I for atherogenesis *in vivo* as profilin-I heterozygosity resulted in protection from atherosclerosis in LDL receptor-null mice (Romeo et al., 2007). In this report, a variety of atheroprotective indicators were recognized in mice with heterozygous deficiency of profilin-I, as compared to profilin-I wild-type mice. Aortas from these heterozygous mice exhibited preserved activation of endothelial nitric oxide synthase (eNOS) and nitric oxide-dependent signaling, decreased expression of vascular cell adhesion molecule (VCAM)-1 and decreased accumulation of macrophage at the sites of injury. Correspondingly, profilin-I knockdown in cultured aortic ECs was able to protect against endothelial dysfunction induced by oxidized lowdensity lipoproteins (oxLDL). Additionally, macrophages from bone marrow of profilin-Ideficient heterozygous mice exhibited diminished internalization of oxLDL and oxLDLinduced inflammation. These studies concluded that profilin-I plays a vital role in early atheroma formation and that decreasing profilin-I levels is atheroprotective. Finally, profilin-I atheroprotective effect is mediated via combined mechanisms that depend on both endothelium and macrophages (Romeo et al., 2007).

Moreover, the same group addressed the pathways responsible for profilin-I gene expression in 7-ketocholesterol (oxysterol)-stimulated endothelial cells and in the diabetic aorta. They showed that oxysterol-binding protein-1 (OSBP1) is required for oxysterol-dependent nucleation and activation of the JAK2/STAT3 pathway, which in turn regulates profilin-I gene expression in endothelial cells. Similarly, diabetes increases the activation of STAT3 and its recruitment to the profilin-I promoter in large vessels *in vivo* (Romeo et al., 2008)

Very recently, it has been reported that profilin-I expression is markedly increased in human atherosclerotic plaques compared to the normal vessel wall (Caglayan et al., 2010). A correlation was found between profilin-I serum levels and the degree of atherosclerosis, as well. The atherogenic effects of profilin-I on VSMCs imply an auto-/paracrine role within the plaque. In addition, it was found that profilin-I acts as an extracellular ligand and triggers atherogenic effects in VSMCs including DNA synthesis and migration. Besides, profilin-1 stimulates typical signaling pathways such as the PI3K/AKT and RAS-RAf-MEK-ERK pathways. These findings revealed that profilin-I might play a critical role in atherogenesis and may represent a novel therapeutic target in human patients (Caglayan et al., 2010).

#### **3.4 Role of profilin in age-associated vascular problems**

Aging is a major risk factor for the development of vascular diseases, such as hypertension and arteriosclerosis, which lead to stroke and heart failure (Spagnoli et al., 1991). Aging is also linked with decreased stress tolerance. Susceptibility to a variety of physiological stresses such as infection, inflammation, and oxidative damage enhances with age and is causally coupled with clinical problems in the elderly (Starr et al., 2011). So far, the mechanism of age-related changes in vasculature has not been completely understood. On the top of that, the role of profilin in these age related changes remains largely unstudied.

Recently, it has been reported that protein nitration levels increased in aged mice compared to young mice. Also, particularly strong nitration was found in the pulmonary vascular endothelium during systemic inflammatory response syndrome (SIRS). Age- and SIRSdependent increased protein nitration was evident in proteins related to the actin cytoskeleton that are responsible for maintaining pulmonary vascular permeability such as transgelin-2, LASP 1, tropomyosin, myosin and profilin-I. Recognizing the nitrated proteins

same group specified the significance of profilin-I for atherogenesis *in vivo* as profilin-I heterozygosity resulted in protection from atherosclerosis in LDL receptor-null mice (Romeo et al., 2007). In this report, a variety of atheroprotective indicators were recognized in mice with heterozygous deficiency of profilin-I, as compared to profilin-I wild-type mice. Aortas from these heterozygous mice exhibited preserved activation of endothelial nitric oxide synthase (eNOS) and nitric oxide-dependent signaling, decreased expression of vascular cell adhesion molecule (VCAM)-1 and decreased accumulation of macrophage at the sites of injury. Correspondingly, profilin-I knockdown in cultured aortic ECs was able to protect against endothelial dysfunction induced by oxidized lowdensity lipoproteins (oxLDL). Additionally, macrophages from bone marrow of profilin-Ideficient heterozygous mice exhibited diminished internalization of oxLDL and oxLDLinduced inflammation. These studies concluded that profilin-I plays a vital role in early atheroma formation and that decreasing profilin-I levels is atheroprotective. Finally, profilin-I atheroprotective effect is mediated via combined mechanisms that depend on

Moreover, the same group addressed the pathways responsible for profilin-I gene expression in 7-ketocholesterol (oxysterol)-stimulated endothelial cells and in the diabetic aorta. They showed that oxysterol-binding protein-1 (OSBP1) is required for oxysterol-dependent nucleation and activation of the JAK2/STAT3 pathway, which in turn regulates profilin-I gene expression in endothelial cells. Similarly, diabetes increases the activation of STAT3 and its

Very recently, it has been reported that profilin-I expression is markedly increased in human atherosclerotic plaques compared to the normal vessel wall (Caglayan et al., 2010). A correlation was found between profilin-I serum levels and the degree of atherosclerosis, as well. The atherogenic effects of profilin-I on VSMCs imply an auto-/paracrine role within the plaque. In addition, it was found that profilin-I acts as an extracellular ligand and triggers atherogenic effects in VSMCs including DNA synthesis and migration. Besides, profilin-1 stimulates typical signaling pathways such as the PI3K/AKT and RAS-RAf-MEK-ERK pathways. These findings revealed that profilin-I might play a critical role in atherogenesis and

Aging is a major risk factor for the development of vascular diseases, such as hypertension and arteriosclerosis, which lead to stroke and heart failure (Spagnoli et al., 1991). Aging is also linked with decreased stress tolerance. Susceptibility to a variety of physiological stresses such as infection, inflammation, and oxidative damage enhances with age and is causally coupled with clinical problems in the elderly (Starr et al., 2011). So far, the mechanism of age-related changes in vasculature has not been completely understood. On the top of that, the role of profilin in these age related changes remains largely unstudied. Recently, it has been reported that protein nitration levels increased in aged mice compared to young mice. Also, particularly strong nitration was found in the pulmonary vascular endothelium during systemic inflammatory response syndrome (SIRS). Age- and SIRSdependent increased protein nitration was evident in proteins related to the actin cytoskeleton that are responsible for maintaining pulmonary vascular permeability such as transgelin-2, LASP 1, tropomyosin, myosin and profilin-I. Recognizing the nitrated proteins

recruitment to the profilin-I promoter in large vessels *in vivo* (Romeo et al., 2008)

may represent a novel therapeutic target in human patients (Caglayan et al., 2010).

both endothelium and macrophages (Romeo et al., 2007).

**3.4 Role of profilin in age-associated vascular problems** 

indicated important modifications to the vascular endothelial cytoskeleton, which potentially participates in the barrier dysfunction, enhanced vascular permeability, and pulmonary edema (Starr et al., 2011).

It has been established that deficiency in plasma fibronectin increases lung vascular permeability (Wheatley et al., 1993); consequently, as adhesion of endothelial cell to fibronectin depends on profilin expression (Moldovan et al., 1997), lack of functional profilin may be to some extent responsible for vascular permeability as a result of inefficient barrier integrity. These data can fairly elucidate the age-associated enhancement in susceptibility to systemic inflammation, acute lung injury, and respiratory failure (Starr et al., 2011).

Fig. 13. JAK2/STAT3 pathway activation increases profilin-I (Romeo et al., 2008; Cheng et al., 2011) in the vessel media induced stress fiber formation and increased internal mechanical stress in the vessel walls (Moustafa-Bayoumi et al., 2007) which modulates changes in ECM and integrins (Abouelnaga et al., 2009; Hassona et al., 2010). These changes led to activation of FAK (Abouelnaga et al., 2009) that in turn activate Rho/ROCKII (Hassona et al, 2010; Moustafa-Bayoumi et al., 2007), PI3 kinase and AKT (Caglayan et al., 2010). Activation of Rac1/NADPH pathway (Abouelnaga et al., 2009) results in increased superoxide production and increases oxidative stress (Hassanain HH, unpublished) in vessel walls which could contribute to hypertension. The activation of Rho/ROCKII and AKT result in activation of MLC20 (Hassona et al., 2010), and increases in protein synthesis (Gingras et al., 1998; Kitamura et al., 1998; Ushio-Fukai et al., 1999) and calcification (Byon et al., 2008), respectively. These changes in the media of the vessel walls result in arterial stiffening and hypertension (Moustafa-Bayoumi et al., 2007). Profilin-I inhibitor can block the stress fiber formation in this pathway (Moustafa-Bayoumi et al., 2007 ) and dehydroepiandrosterone (DHEA) can inhibit AKT kinase pathway (Bonnet et al., 2009).

Conversely, other indirect evidence showed that profilin-I increased with age; a recent study using proteomic and genomic analyses of hippocampus from young and old rats showed a significant increase in profilin-I expression in aged rat hippocampus (Weinreb et al., 2007). Another study investigating differential protein expression profiles in chronically stimulated T cell clones found that profilin-I was widely and highly expressed in cytoplasm (Mazzatti et al., 2007). The study concluded that differential expression of profilin-I in aging may contribute directly to immunosenescence via disrupting the intracellular signaling and intercellular communication (Egerton et al., 1992; Witke et al., 1998). Consistent with these findings our preliminary data showed an increase in profilin-I expression in the aortic medial layers of older wild-type mice compared with young mice (Hassanain HH, unpublished).

Taken together, this review shed some light on the important role of profilin-I in vascular diseases. However, more studies need to be done in order to fully understand the profilin-I signaling pathway and its mechanism(s) of regulation. Figure 13 summarize some of the proposed signaling molecules involved in profilin-induced vascular complications.
