Multiple Kinase Involvement in the Regulation of Vascular Growth

The initial discovery of protein phosphorylation as a regulatory mechanism for the control of glycogen metabolism has led to intense interest of protein phosphorylation in regulating protein function (Cohen et al., 2001). Kinases play a variety of roles in many physiological processes within cells and represent one of the largest families in the human genome with over 500 members comprising protein serine/threonine, tyrosine, and dual-specificity kinases (Manning et al., 2002). Phosphorylation of proteins is one of the most significant signal transduction mechanisms which regulate intracellular processes such as transport, growth, metabolism, apoptosis, cystoskeletal arrangement and hormone responses (Bononi et al., 2011; Heidenreich et al., 1991; Manning et al., 2002; Pawson et al., 2000). As such, abnormal phosphorylation of proteins can be either a cause or a consequence of disease. Kinases are regulated by activator and inhibitor proteins, ligand binding, and phosphorylation by other proteins or via autophosphorylation (Hanks et al., 1991; Hug et al., 1993; Scott, 1991; Taylor et al., 1990; Taylor et al., 1992). Since kinases play key functions in many cellular processes, they represent an attractive target for therapeutic interventions in many disease states such as cancer, inflammation, diabetes and arthritis (Cohen et al., 2010; Fry et al., 1994; Karin, 2005; Mayers et al., 2005). In particular, the serine/threonine family of kinases comprises approximately 125 of the 500 family of kinases and includes the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), the cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), and protein kinase C (PKC). These kinases are implicated in the regulation of cell growth and are the focus of this current study.We would like to acknowledge Jonathan C. Fox and Patti Shaver for assistance with isolation and culture of rat primary vascular smooth muscle cells. This project was supported by Award Number R01HL081720 from the National Institutes of Health National Heart, Lung, and Blood Institute (NHLBI), by ARRA Award Number R01HL081720-03S2, and by Post-doctoral Research Supplement Award Number R01HL081720-05S1 from the NHLBI

Phosphodiesterases Regulate BAY 41-2272-Induced VASP Phosphorylation in Vascular Smooth Muscle Cells

BAY 41-2272 (BAY), a stimulator of soluble guanylyl cyclase, increases cyclic nucleotides and inhibits proliferation of vascular smooth muscle cells (VSMCs). In this study, we elucidated mechanisms of action of BAY in its regulation of vasodilator-stimulated phosphoprotein (VASP) with an emphasis on VSMC phosphodiesterases (PDEs). BAY alone increased phosphorylation of VASPSer239 and VASPSer157, respective indicators of PKG and PKA signaling. IBMX, a non-selective inhibitor of PDEs, had no effect on BAY-induced phosphorylation at VASPSer239 but inhibited phosphorylation at VASPSer157. Selective inhibitors of PDE3 or PDE4 attenuated BAY-mediated increases at VASPSer239 and VASPSer157, whereas PDE5 inhibition potentiated BAY-mediated increases only at VASPSer157. In comparison, 8Br-cGMP increased phosphorylation at VASPSer239 and VASPSer157 which were not affected by selective PDE inhibitors. In the presence of 8Br-cAMP, inhibition of either PDE4 or PDE5 decreased VASPSer239 phosphorylation and inhibition of PDE3 increased phosphorylation at VASPSer239, while inhibition of PDE3 or PDE4 increased and PDE5 inhibition had no effect on VASPSer157 phosphorylation. These findings demonstrate that BAY operates via cAMP and cGMP along with regulation by PDEs to phosphorylate VASP in VSMCs and that the mechanism of action of BAY in VSMCs is different from that of direct cyclic nucleotide analogs with respect to VASP phosphorylation and the involvement of PDEs. Given a role for VASP as a critical cytoskeletal protein, these findings provide evidence for BAY as a regulator of VSMC growth and a potential therapeutic agent against vasculoproliferative disorders

Identification of cytosolic phosphodiesterases in the erythrocyte: A possible role for PDE5

Background Within erythrocytes (RBCs), cAMP levels are regulated by phosphodiesterases (PDEs). Increases in cAMP and ATP release associated with activation of β-adrenergic receptors (βARs) and prostacyclin receptors (IPRs) are regulated by PDEs 2, 4 and PDE 3, respectively. Here we establish the presence of cytosolic PDEs in RBCs and determine a role for PDE5 in regulating levels of cGMP. Material/Methods Purified cytosolic proteins were obtained from isolated human RBCs and western analysis was performed using antibodies against PDEs 3A, 4 and 5. Rabbit RBCs were incubated with dbcGMP, a cGMP analog, to determine the effect of cGMP on cAMP levels. To determine if cGMP affects receptor-mediated increases in cAMP, rabbit RBCs were incubated with dbcGMP prior to addition of isoproterenol (ISO), a βAR receptor agonist. To demonstrate that endogenous cGMP produces the same effect, rabbit and human RBCs were incubated with SpNONOate (SpNO), a nitric oxide donor, and YC1, a direct activator of soluble guanylyl cyclase (sGC), in the absence and presence of a selective PDE5 inhibitor, zaprinast (ZAP). Results Western analysis identified PDEs 3A, 4D and 5A. dbcGMP produced a concentration dependent increase in cAMP and ISO-induced increases in cAMP were potentiated by dbcGMP. In addition, incubation with YC1 and SpNO in the presence of ZAP potentiated βAR-induced increases in cAMP. Conclusions PDEs 2, 3A and 5 are present in the cytosol of human RBCs. PDE5 activity in RBCs regulates cGMP levels. Increases in intracellular cGMP augment cAMP levels. These studies suggest a novel role for PDE5 in erythrocytes

Activation of RhoA, but Not Rac1, Mediates Early Stages of S1P-Induced Endothelial Barrier Enhancement

<div><p>Compromised endothelial barrier function is a hallmark of inflammation. Rho family GTPases are critical in regulating endothelial barrier function, yet their precise roles, particularly in sphingosine-1-phosphate (S1P)-induced endothelial barrier enhancement, remain elusive. Confluent cultures of human umbilical vein endothelial cells (HUVEC) or human dermal microvascular endothelial cells (HDMEC) were used to model the endothelial barrier. Barrier function was assessed by determining the transendothelial electrical resistance (TER) using an electrical cell-substrate impedance sensor (ECIS). The roles of Rac1 and RhoA were tested in S1P-induced barrier enhancement. The results show that pharmacologic inhibition of Rac1 with Z62954982 failed to block S1P-induced barrier enhancement. Likewise, expression of a dominant negative form of Rac1, or knockdown of native Rac1 with siRNA, failed to block S1P-induced elevations in TER. In contrast, blockade of RhoA with the combination of the inhibitors Rhosin and Y16 significantly reduced S1P-induced increases in TER. Assessment of RhoA activation in real time using a fluorescence resonance energy transfer (FRET) biosensor showed that S1P increased RhoA activation primarily at the edges of cells, near junctions. This was complemented by myosin light chain-2 phosphorylation at cell edges, and increased F-actin and vinculin near intercellular junctions, which could all be blocked with pharmacologic inhibition of RhoA. The results suggest that S1P causes activation of RhoA at the cell periphery, stimulating local activation of the actin cytoskeleton and focal adhesions, and resulting in endothelial barrier enhancement. S1P-induced Rac1 activation, however, does not appear to have a significant role in this process.</p></div

Inhibition of RhoA attenuated S1P-induced barrier enhancement of endothelial monolayers.

<p><i>A</i>. The time course of changes in of TER of HUVEC monolayers pretreated with the 30 min with the combination of Rhosin and Y16 (5 μM of each) or vehicle control, followed by treatment with 2 μM S1P or vehicle (N = 8 for each group). <i>B</i>. Comparison of the mean maximal changes in TER of HUVEC monolayers (%) within the first 10 min after S1P or vehicle. The corresponding results for HDMEC monolayers are shown in panels <i>C & D</i> (N = 8 each group). *P<0.05, S1P vs. vehicle treated group. †P<0.05, inhibitor vs. vehicle pretreatments.</p

Histamine Activates p38 MAP Kinase and Alters Local Lamellipodia Dynamics, Reducing Endothelial Barrier Integrity and Eliciting Central Movement of Actin Fibers

The role of the actin cytoskeleton in endothelial barrier function has been debated for nearly four decades. Our previous investigation revealed spontaneous local lamellipodia in confluent endothelial monolayers that appear to increase overlap at intercellular junctions. We tested the hypothesis that the barrier-disrupting agent histamine would reduce local lamellipodia protrusions and investigated the potential involvement of p38 mitogen-activated protein (MAP) kinase activation and actin stress fiber formation. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein-actin were studied using time-lapse fluorescence microscopy. The protrusion and withdrawal characteristics of local lamellipodia were assessed before and after addition of histamine. Changes in barrier function were determined using electrical cell-substrate impedance sensing. Histamine initially decreased barrier function, lamellipodia protrusion frequency, and lamellipodia protrusion distance. A longer time for lamellipodia withdrawal and reduced withdrawal distance and velocity accompanied barrier recovery. After barrier recovery, a significant number of cortical fibers migrated centrally, eventually resembling actin stress fibers. The p38 MAP kinase inhibitor SB203580 attenuated the histamine-induced decreases in barrier function and lamellipodia protrusion frequency. SB203580 also inhibited the histamine-induced decreases in withdrawal distance and velocity, and the subsequent actin fiber migration. These data suggest that histamine can reduce local lamellipodia protrusion activity through activation of p38 MAP kinase. The findings also suggest that local lamellipodia have a role in maintaining endothelial barrier integrity. Furthermore, we provide evidence that actin stress fiber formation may be a reaction to, rather than a cause of, reduced endothelial barrier integrity. the endothelium plays a critical role in cardiovascular function, containing a relatively high-pressure closed-loop circulation while also permitting diffusive exchange between the blood and tissues through the capillaries and postcapillary venules. The mechanisms that control the permeability of microvessels, which permit significant leakage of plasma proteins during inflammation, have been debated for nearly a century (15). Evidence collected over the past few decades has highlighted the active role of endothelial cells in response to inflammatory stimuli, including remodeling of the actin cytoskeleton (26, 42, 45). One of the current prevailing theories from this line of investigation has been that endothelial cells adopt a contractile state during inflammation, which favors opening of junctions between cells, permitting increased paracellular flux of fluids and solutes (32, 33). The contractile theory is supported by evidence that certain agents that elevate permeability also cause the development of centripetal tension generated by the actin cytoskeleton, which can place stress on the junctions and limit their strength (26, 29). Several inflammatory mediators also promote development of actin stress fibers in endothelial cells (7, 32). Actin-mediated contraction in endothelial cells is promoted by phosphorylation of myosin regulatory light chains (MLC) on Thr-18/Ser-19, which is determined by the activities of MLC kinase (MLCK) and MLC phosphatase (MLCP). Inhibition of MLCK attenuates hyperpermeability caused by activated neutrophils (46), histamine (34), and ethanol (21), and shortens the time course of the thrombin-induced barrier dysfunction (29) in cultured endothelial cell monolayers. Inhibition of Rho kinase (ROCK), an upstream regulator of MLCP (7, 32), also attenuates hyperpermeability caused by activated neutrophils (11), thrombin (13, 38, 43), histamine (43), and vascular endothelial growth factor (35) in endothelial cell monolayer models. Inhibition of MLCK or ROCK also decreases actin stress fiber formation, typically observed in fixed cells by labeling F-actin with a fluorochrome-bound phalloidin (5, 6, 10, 11, 13, 19, 34, 35, 38, 43, 46). However, there is also evidence of histamine-induced increases in microvascular permeability that do not quite fit this paradigm. Histamine increases the permeability of postcapillary venules in the absence of actin stress fiber formation in endothelial cells (4). Interestingly, it was also noted that actin stress fibers did form after permeability had returned to normal (4). It was also apparent in early studies with cultured endothelial cells that histamine did not cause actin stress fiber formation within the time frame of elevated permeability in cultured endothelial cells. Rather, histamine decreased actin cables, which was postulated to be a cause of histamine-induced permeability of the endothelium (42). Likewise, histamine caused only mild phosphorylation of MLC compared with thrombin, and did not elicit an increase in the isometric tension of cultured endothelial cell monolayers (27–29). These findings indicate the importance of contraction-independent mechanisms in the control of the endothelial barrier (27, 43). Recent work has highlighted the importance of cortical actin for maintaining endothelial barrier integrity (7, 33). The small GTPase Rac1 promotes cortical actin structures, thus stabilizing intercellular junctions (1, 40, 41), and RhoA activation localized to the cell periphery promotes barrier integrity (36). To better understand the dynamics of the actin cytoskeleton, we recently used time-lapse imaging of endothelial cells expressing green fluorescent protein (GFP)-actin. We discovered that endothelial cells had frequent, brief protrusions of the plasma membrane localized at intercellular junctions, termed local lamellipodia, which were reduced during increases in endothelial permeability and restored during the restoration of barrier function (12, 17). The local lamellipodia were dependent upon myosin II activity, and decreases in their protrusion frequency correlated with reduced Rac1 activity (12). These findings are relevant because maintenance of an optimal distance of the junctional cleft between endothelial cells is important for the normal permeability of postcapillary venules (14). Our previous study provided evidence that local lamellipodia may contribute to the maintenance of endothelial junctions. However, that study was limited to an investigation of thrombin and sphingosine-1-phosphate (S1P), and it remains unclear whether additional agents that alter microvascular permeability impact this mechanism. To investigate the mechanism of action of histamine, we assessed the dynamic changes it induces in the actin cytoskeleton. We present evidence that histamine briefly reduced local lamellipodia formation. On the basis of our previous studies showing the importance of the p38 mitogen-activated protein (MAP) kinase in histamine-induced disruption of endothelial barrier integrity, we also determined the extent to which inhibition of p38 MAP kinase affects both histamine-induced changes in barrier function and lamellipodia protrusion/withdrawal. In addition, we developed a new understanding of the spatial mechanisms of stress fiber formation caused by histamine

Pharmacologic inhibition of Rac1 failed to block S1P-induced endothelial barrier enhancement in HUVEC and HDMEC monolayers.

<p><i>A</i>. The time course of changes in of TER of HUVEC monolayers pretreated with the 30 min with Rac1 inhibitor Z62954982 or vehicle control, followed by treatment with 2 μM S1P are shown (N = 8 for each group). <i>B</i>. Mean maximal change in TER (%) of HUVEC monolayers after S1P treatment within the first 10-min window. Panels <i>C & D</i> show corresponding results for HDMEC monolayers (N = 8 each group). *P<0.05 vs. S1P Vehicle pretreated groups.</p

RhoA inhibition abrogated S1P-induced F-actin formation and recruitment of vinculin near the cell periphery.

<p>The results showed that S1P increases F-actin and vinculin labeling in the peripheral areas of cells (10 min after the addition of S1P). This was inhibited after pretreatment with combined Rhosin and Y-16 (Inh; 5 μM each, 30 min). The inhibitors alone had no impact. All images are representative of 3 separate experiments.</p

Pharmacological inhibition or siRNA-mediated knockdown of Rac1 impaired baseline endothelial barrier integrity.

<p><i>A</i>. Treatment with the selective Rac1 inhibitor Z62954982 reduces TER in a concentration-dependent manner in HDMEC monolayers. <i>B</i>. Comparison of the mean maximum decreases in TER in the 30-min time window for each concentration of Z62954982 in HDMEC monolayers. Panels <i>C & D</i> show that Z62954982 produces a similar concentration-dependent TER in HUVEC monolayers. <i>E</i>. Western blot confirming knockdown (KD) with Rac1-specific siRNA, compared to sham and scrambled RNA (Scr) control groups. Bands for β-actin from re-probed blots confirmed equivalent loading of protein for each lane. <i>F</i>. Mean baseline TER values of HDMEC and HUVEC monolayers in Rac1 knockdown, scrambled control, and sham-transfected groups. *P<0.05 versus vehicle treated group. †P<0.05 versus other concentrations.</p