Protease-Activated Receptor 2 Controls Vascular Smooth Muscle Cell Proliferation in Cyclic AMP-Dependent Protein Kinase/Mitogen-Activated Protein Kinase Kinase 1/2-Dependent Manner.

INTRODUCTION: Cardiovascular disorders are characterized by vascular smooth muscle (VSM) transition from a contractile to proliferative state. Protease-activated receptor 2 (PAR2) involvement in this phenotypic conversion remains unclear. We hypothesized that PAR2 controls VSM cell proliferation in phenotype-dependent manner and through specific protein kinases. METHODS: Rat clonal low (PLo; P3-P6) and high passage (PHi; P10-P15) VSM cells were established as respective models of quiescent and proliferative cells, based on reduced PKG-1 and VASP. Western blotting determined expression of cytoskeletal/contractile proteins, PAR2, and select protein kinases. DNA synthesis and cell proliferation were measured 24-72 h following PAR2 agonism (SLIGRL; 100 nM-10 µm) with/without PKA (PKI; 10 µm), MEK1/2 (PD98059; 10 µm), and PI3K (LY294002; 1 µm) blockade. RESULTS: PKG-1, VASP, SM22α, calponin, cofilin, and PAR2 were reduced in PHi versus PLo cells. Following PAR2 agonism, DNA synthesis and cell proliferation increased in PLo cells but decreased in PHi cells. Western analyses showed reduced PKA, MEK1/2, and PI3K in PHi versus PLo cells, and kinase blockade revealed PAR2 controls VSM cell proliferation through PKA/MEK1/2. DISCUSSION: Findings highlight PAR2 and PAR2-driven PKA/MEK1/2 in control of VSM cell growth and provide evidence for continued investigation of PAR2 in VSM pathology.

Making the cut: Innovative methods for optimizing perfusion-based migration assays.

Application of fluid shear stress to adherent cells dramatically influences their cytoskeletal makeup and differentially regulates their migratory phenotype. Because cytoskeletal rearrangements are necessary for cell motility and migration, preserving these adaptations under in vitro conditions and in the presence of fluid flow are physiologically essential. With this in mind, parallel plate flow chambers and microchannels are often used to conduct in vitro perfusion experiments. However, both of these systems currently lack capacity to accurately study cell migration in the same location where cells were perfused. The most common perfusion/migration assays involve cell perfusion followed by trypsinization which can compromise adaptive cytoskeletal geometry and lead to misleading phenotypic conclusions. The purpose of this study was to quantitatively highlight some limitations commonly found with currently used cell migration approaches and to introduce two new advances which use additive manufacturing (3D printing) or laser capture microdissection (LCM) technology. The residue-free 3D printed insert allows accurate cell seeding within defined areas, increases cell yield for downstream analyses, and more closely resembles the reported levels of fluid shear stress calculated with computational fluid dynamics as compared to other residue-free cell seeding techniques. The LCM approach uses an ultraviolet laser for "touchless technology" to rapidly and accurately introduce a custom-sized wound area in otherwise inaccessible perfusion microchannels. The wound area introduced by LCM elicits comparable migration characteristics compared to traditional pipette tip-induced injuries. When used in perfusion experiments, both of these newly characterized tools were effective in yielding similar results yet without the limitations of the traditional modalities. These innovative methods provide valuable tools for exploring mechanisms of clinically important aspects of cell migration fundamental to the pathogenesis of many flow-mediated disorders and are applicable to other perfusion-based models where migration is of central importance. © 2016 International Society for Advancement of Cytometry.

AMP-activated protein kinase inhibits transforming growth factor-ß-mediated vascular smooth muscle cell growth: implications for a Smad-3-dependent mechanism.

Dysfunctional vascular growth is a major contributor to cardiovascular disease, the leading cause of morbidity and mortality worldwide. Growth factor-induced activation of vascular smooth muscle cells (VSMCs) results in a phenotypic switch from a quiescent, contractile state to a proliferative state foundational to vessel pathology. Transforming growth factor-ß (TGF-ß) is a multifunctional signaling protein capable of growth stimulation via Smad signaling. Although Smad signaling is well characterized in many tissues, its role in VSM growth disorders remains controversial. Recent data from our lab and others implicate the metabolic regulator AMP-activated protein kinase (AMPK) in VSM growth inhibition. We hypothesized that AMPK inhibits VSMC proliferation by reducing TGF-ß-mediated growth in a Smad-dependent fashion. Treatment of rat VSMCs with the AMPK agonist AICAR significantly decreased TGF-ß-mediated activation of synthetic Smad2 and Smad3 and increased inhibitory Smad7. Flow cytometry and automated cell counting revealed that AICAR reversed TGF-ß-mediated cell cycle progression at 24 h and elevated cell numbers at 48 h. TGF-ß/Smad signaling increased the G0/G1 inducers cyclin D1/cyclin-dependent kinase (CDK) 4 and cyclin E/CDK2; however, AICAR reversed these events while increasing cytostatic p21. The specific role of Smad3 in AMPK-mediated reversal of TGF-ß-induced growth was then explored using adenovirus-mediated Smad3 overexpression (Ad-Smad3). Ad-Smad3 cells increased cell cycle progression and cell numbers compared with Ad-GFP control cells, and these were restored to basal levels with concomitant AICAR treatment. These findings support a novel AMPK target in TGF-ß/Smad3 for VSMC growth control and support continued investigation of AMPK as a possible therapeutic target for reducing vascular growth disorders.

AMP-activated protein kinase inhibits vascular smooth muscle cell proliferation and migration and vascular remodeling following injury.

Vascular smooth muscle cell (VSMC) activation promotes a synthetic phenotype that underlies many vessel growth disorders. In this regard it has been suggested that the metabolic sensor adenosine 5'-monophosphate-activated protein kinase (AMPK) has significant antigrowth and antimetastatic properties and may serve as a viable therapeutic target. In the current study we hypothesized that AMPK reduces neointima formation following balloon injury and that this occurs through reduction in VSMC proliferation and migration. Data reveal that local or systemic dosing with the AMPK agonist 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) significantly increased AMPK activity in vivo and inhibited neointima formation in rat carotid arteries 2 wk after injury. In primary VSMCs, AICAR inhibited migration and induced cytostatic growth arrest through increased protein phosphatase 2A-mediated inhibition of mitosis-promoting cyclin B. AICAR also significantly enhanced AMPK-specific T278 phosphorylation of the actin anticapping vasodilator-activated serum phosphoprotein, increased G- to F-actin ratios and stress fiber formation, and abrogated PDGF-stimulated S397 autophosphorylation of focal adhesion kinase, promigratory cytoplasmic accumulation of paxillin, and extracellular matrix proteolysis by matrix metalloproteinase-9. Together, these results provide compelling evidence that AMPK serves to inhibit vascular smooth muscle migration and proliferation through regulation of cytoskeletal/focal adhesion/ECM stability, increasing our knowledge of this important metabolic regulator and providing support for its continued investigation in the treatment of vascular growth disorders.

FoxO3 normalizes Smad3-induced arterial smooth muscle cell growth.

Transition of arterial smooth muscle (ASM) from a quiescent, contractile state to a growth-promoting state is a hallmark of cardiovascular disease (CVD), a leading cause of death and disability in the United States and worldwide. While many individual signals have been identified as important mechanisms in this phenotypic conversion, the combined impact of the transcription factors Smad3 and FoxO3 in ASM growth is not known. The purpose of this study was to determine that a coordinated, phosphorylation-specific relationship exists between Smad3 and FoxO3 in the control of ASM cell growth. Using a rat in vivo arterial injury model and rat primary ASM cell lysates and fractions, validated low and high serum in vitro models of respective quiescent and growth states, and adenoviral (Ad-) gene delivery for overexpression (OE) of individual and combined Smad3 and/or FoxO3, we hypothesized that FoxO3 can moderate Smad3-induced ASM cell growth. Key findings revealed unique cellular distribution of Smad3 and FoxO3 under growth conditions, with induction of both nuclear and cytosolic Smad3 yet primarily cytosolic FoxO3; Ad-Smad3 OE leading to cytosolic and nuclear expression of phosphorylated and total Smad3, with almost complete reversal of each with Ad-FoxO3 co-infection in quiescent and growth conditions; Ad-FoxO3 OE leading to enhanced cytosolic expression of phosphorylated and total FoxO3, both reduced with Ad-Smad3 co-infection in quiescent and growth conditions; Ad-FoxO3 inducing expression and activity of the ubiquitin ligase MuRF-1, which was reversed with concomitant Ad-Smad3 OE; and combined Smad3/FoxO3 OE reversing both the pro-growth impact of singular Smad3 and the cytostatic impact of singular FoxO3. A primary takeaway from these observations is the capacity of FoxO3 to reverse growth-promoting effects of Smad3 in ASM cells. Additional findings lend support for reciprocal antagonism of Smad3 on FoxO3-induced cytostasis, and these effects are dependent upon discrete phosphorylation states and cellular localization and involve MuRF-1 in the control of ASM cell growth. Lastly, results showing capacity of FoxO3 to normalize Smad3-induced ASM cell growth largely support our hypothesis, and overall findings provide evidence for utility of Smad3 and/or FoxO3 as potential therapeutic targets against abnormal ASM growth in the context of CVD.

Maternal Aerobic Exercise, but Not Blood Docosahexaenoic Acid and Eicosapentaenoic Acid Concentrations, during Pregnancy Influence Infant Body Composition.

Although discrete maternal exercise and polyunsaturated fatty acid (PUFA) supplementation individually are beneficial for infant body composition, the effects of exercise and PUFA during pregnancy on infant body composition have not been studied. This study evaluated the body composition of infants born to women participating in a randomized control exercise intervention study. Participants were randomized to aerobic exercise (n = 25) or control (stretching and breathing) groups (n = 10). From 16 weeks of gestation until delivery, the groups met 3×/week. At 16 and 36 weeks of gestation, maternal blood was collected and analyzed for Docosahexaenoic Acid (DHA) and Eicosapentaenoic Acid (EPA). At 1 month postnatal, infant body composition was assessed via skinfolds (SFs) and circumferences. Data from 35 pregnant women and infants were analyzed via t-tests, correlations, and regression. In a per protocol analysis, infants born to aerobic exercisers exhibited lower SF thicknesses of triceps (p = 0.008), subscapular (p = 0.04), SF sum (p = 0.01), and body fat (BF) percentage (%) (p = 0.006) compared with controls. After controlling for 36-week DHA and EPA levels, exercise dose was determined to be a negative predictor for infant skinfolds of triceps (p = 0.001, r2 = 0.27), subscapular (p = 0.008, r2 = 0.19), SF sum (p = 0.001, r2 = 0.28), mid-upper arm circumference (p = 0.049, r2 = 0.11), and BF% (p = 0.001, r2 = 0.32). There were no significant findings for PUFAs and infant measures: during pregnancy, exercise dose, but not blood DHA or EPA levels, reduces infant adiposity.

The Influence of Maternal Aerobic Exercise, Blood DHA and EPA Concentrations on Maternal Lipid Profiles.

Exercise and polyunsaturated fatty acid (PUFA) supplementation independently improve lipid profiles. The influence of both exercise and PUFAs on lipids during pregnancy remains unknown. This study evaluated exercise, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) concentrations on lipids during pregnancy. Participants were randomized to aerobic exercise or control groups. From 16 weeks gestation until delivery, groups met 3x/week; exercisers performed moderate-intensity aerobic activity, controls performed low-intensity stretching and breathing. At 16 and 36 weeks' gestation, maternal blood was analyzed for lipids (total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TG)), DHA and EPA. In intent-to-treat analysis, the aerobic group (n = 20), relative to controls (n = 10), exhibited a higher HDL change across gestation (p = 0.03). In a per protocol analysis, the aerobic group, relative to controls, exhibited 21.2% lower TG at 36 weeks (p = 0.04). After controlling for 36-week DHA and EPA, exercise dose predicts 36 weeks' TG (F (1,36) = 6.977, p = 0.012, r2 = 0.16). Aerobic exercise normalizes late pregnancy TG. During pregnancy, exercise dose controls the rise in TG, therefore maintaining normal levels. DHA and EPA do not have measurable effects on lipids. Regardless of PUFA levels, exercise at recommended levels maintains appropriate TG levels in pregnant women. Normal TG levels are critical for pregnancy outcomes, and further studies are warranted to investigate this association in broader populations.

The soluble guanylate cyclase stimulator BAY 41-2272 inhibits vascular smooth muscle growth through the cAMP-dependent protein kinase and cGMP-dependent protein kinase pathways.

Vascular smooth muscle (VSM) proliferation and migration are key components in vessel remodeling. Cyclic nucleotide signaling is protective and has long-served as a therapeutic target against undesired VSM growth. The present work analyzed the effects of the soluble guanylate cyclase (sGC) stimulator 3-(4-amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine [BAY 41-2272 (BAY)] on VSM growth, and we hypothesize that BAY has the capacity to reduce proliferation and migration via cyclic nucleotide-driven kinase signaling. Perivascular BAY postballoon injury reduced neointimal growth by ∼ 40% compared with vehicle controls after 2 weeks. In VSM cells, BAY (10 µM) reduced proliferation by ∼ 40% after 72 h and migration by ∼ 40% after 6 h and ∼ 60% after 18 h without deleterious effects on cell viability. cGMP content peaked (248 ×) 20 min after BAY treatment and remained elevated (140 ×) through 60 min; however, BAY did not affect cAMP levels compared with controls. Conventional and In-Cell Western analyses showed increases in vasodilator-stimulated phosphoprotein (VASP) phosphorylation (pVASP) at serines 239 (3 ×) and 157 (2 ×), respective markers of cGMP- and cAMP-directed protein kinases (PKG and PKA, respectively). The PKG inhibitor YGRKKRRQRRRPPLRKKKKKH peptide (DT-2) completely reversed BAY-mediated increases in pVASPSer(239) and BAY-mediated inhibition of migration. In comparison, the PKA inhibitor peptide PKI further potentiated BAY-stimulated pVASPSer(157) and pVASPSer(239) and partially reversed the antiproliferative effects of BAY. This is the first report demonstrating the effectiveness of BAY in reducing neointimal growth with direct evidence for PKG-specific antimigratory and PKA-specific antiproliferative mechanisms. Conclusively, the sGC stimulator BAY reduces VSM growth through cGMP-dependent PKG and PKA processes, providing support for continued evaluation of its clinical utility.

Arginase promotes neointima formation in rat injured carotid arteries.

OBJECTIVE: Arginase stimulates the proliferation of cultured vascular smooth muscle cells (VSMCs); however, the influence of arginase on VSMC growth in vivo is not known. This study investigated the impact of arginase on cell cycle progression and neointima formation after experimental arterial injury. METHODS AND RESULTS: Balloon injury of rat carotid arteries resulted in a sustained increase in arginase activity in the vessel wall and the induction of arginase I protein in both the media and neointima of injured vessels. Furthermore, local perivascular application of the potent and selective arginase inhibitors S-(2-boronoethyl)-L-cysteine (BEC) or N(G)-hydroxy-nor-L-arginine (L-OHNA) immediately after injury markedly attenuated medial and neointimal DNA synthesis and neointima formation. Substantial arginase I protein and arginase activity was also detected in rat cultured aortic VSMCs. Moreover, treatment of VSMCs with BEC or L-OHNA, or knockdown of arginase I protein, arrested cells in the G(0)/G(1) phase of the cell cycle and induced the expression of the cyclin-dependent protein kinase inhibitor, p21. CONCLUSIONS: This study demonstrates that arginase is essential for VSMCs to enter the cell cycle and that arginase I contributes to the remodeling response after arterial injury. Arginase I represents a potentially new therapeutic target for the treatment of vasculoproliferative disorders.

Increased AMP deaminase activity decreases ATP content and slows protein degradation in cultured skeletal muscle.

BACKGROUND: Protein degradation is an energy-dependent process, requiring ATP at multiple steps. However, reports conflict as to the relationship between intracellular energetics and the rate of proteasome-mediated protein degradation. METHODS: To determine whether the concentration of the adenine nucleotide pool (ATP + ADP + AMP) affects protein degradation in muscle cells, we overexpressed an AMP degrading enzyme, AMP deaminase 3 (AMPD3), via adenovirus in C2C12 myotubes. RESULTS: Overexpression of AMPD3 resulted in a dose- and time-dependent reduction of total adenine nucleotides (ATP, ADP and AMP) without increasing the ADP/ATP or AMP/ATP ratios. In agreement, the reduction of total adenine nucleotide concentration did not result in increased Thr172 phosphorylation of AMP-activated protein kinase (AMPK), a common indicator of intracellular energetic state. Furthermore, LC3 protein accumulation and ULK1 (Ser 555) phosphorylation were not induced. However, overall protein degradation and ubiquitin-dependent proteolysis were slowed by overexpression of AMPD3, despite unchanged content of several proteasome subunit proteins and proteasome activity in vitro under standard conditions. CONCLUSIONS: Altogether, these findings indicate that a physiologically relevant decrease in ATP content, without a concomitant increase in ADP or AMP, is sufficient to decrease the rate of protein degradation and activity of the ubiquitin-proteasome system in muscle cells. This suggests that adenine nucleotide degrading enzymes, such as AMPD3, may be a viable target to control muscle protein degradation and perhaps muscle mass.

YC-1 stimulates the expression of gaseous monoxide-generating enzymes in vascular smooth muscle cells.

The benzylindazole derivative 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) is an allosteric stimulator of soluble guanylate cyclase (sGC) that sensitizes the enzyme to the gaseous ligands carbon monoxide (CO) and nitric oxide (NO). In this study, we examined whether YC-1 also promotes the production of these gaseous monoxides by stimulating the expression of the inducible isoforms of heme oxygenase (HO-1) and NO synthase (iNOS) in vascular smooth muscle cells (SMCs). YC-1 increased HO-1 mRNA, protein, and promoter activity and potentiated cytokine-mediated expression of iNOS protein and NO synthesis by SMCs. The induction of HO-1 by YC-1 was unchanged by the sGC inhibitor, 1H-(1,2,4)oxadiazolo[4,3-alpha]quinozalin-1-one (ODQ) or by the protein kinase G inhibitors (8R,9S,11S)-(-)-2-methyl-9-methoxyl-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cyclocta9(cde)trinen-1-one (KT 5823) and YGRKKRRQRRRPPLRKKKKKH-amide (DT-2) and was not duplicated by 8-bromo-cGMP or the NO-independent sGC stimulator 5-cyclopropyl-2[1-(2-fluorobenzyl)-1H-pyrazolo [3,4-b] pyridine-3-yl] pyrimidin-4-ylamine (BAY 41-2272). However, the YC-1-mediated induction of HO-1 was inhibited by the phosphatidylinositol-3-kinase (PI3K) inhibitors wortmannin and 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002). In contrast, the enhancement of cytokine-stimulated iNOS expression and NO production by YC-1 was prevented by ODQ and the protein kinase A inhibitor (9S,10S, 12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9, 12-epoxy-1H-diindolo(1,2,3-fg:3',2',1'-kl)pyrrolo(3,4-i)(1,6)-benzodiazocine-10-carboxylic acid hexyl ester (KT 5720) and was mimicked by 8-bromo-cGMP and BAY 41-2272. In conclusion, these studies demonstrate that YC-1 stimulates the expression of HO-1 and iNOS in vascular SMCs via the PI3K and sGC-cGMP-protein kinase A pathway, respectively. The ability of YC-1 to sensitize sGC to gaseous monoxides and simultaneously stimulate their production through the induction of HO-1 and iNOS provides a potent mechanism by which the cGMP-dependent and -independent biological actions of this agent are amplified.

Antigrowth properties of BAY 41-2272 in vascular smooth muscle cells.

Vascular smooth muscle (VSM) growth is integral in the pathophysiology of blood vessel diseases, and identifying approaches that have capacity to regulate VSM growth is critically essential. Cyclic nucleotide signaling has been generally considered protective in cardiac and vascular tissues and has been the target of numerous basic science and clinical studies. In this project, the influence of BAY 41-2272 (BAY), a recently described soluble guanylate cyclase stimulator and inducer of cyclic guanosine monophosphate (cGMP) synthesis, on VSM cell growth was analyzed. In rat A7R5 VSM cells, BAY significantly reduced proliferation in a dose- and time-dependent fashion. BAY activated cGMP and cyclic adenosine monophosphate (cAMP) signaling evidenced through elevated cGMP and cAMP content, increased expression of cyclic nucleotide-dependent protein kinases, and differential vasodilator-stimulated phosphoprotein phosphorylation. BAY significantly elevated cyclin E expression, decreased expression of the regulatory cyclin-dependent kinases -2 and -6, increased expression of cell cycle inhibitory p21 WAF1/Cip1 and p27 Kip1, and reduced expression of phosphorylated focal adhesion kinase. These comprehensive findings provide first evidence for the antigrowth cell cycle-regulatory properties of the neoteric agent, BAY 41-2272, in VSM and lend support for its continued study in the clinical and basic cardiovascular sciences.

The cyclic GMP modulators YC-1 and zaprinast reduce vessel remodeling through antiproliferative and proapoptotic effects.

Guanosine-specific cyclic nucleotide signaling is suggested to serve protective actions in the vasculature; however, the influence of selective pharmacologic modulation of cyclic guanosine monophosphate- synthesizing soluble guanylate cyclase or cyclic guanosine monophosphate-degrading phosphodiesterase on vessel remodeling has not been thoroughly examined. In this study, rat carotid artery balloon injury was performed and the growth-modulating effects of the soluble guanylate cyclase stimulator YC-1 or the cyclic guanosine monophosphate-dependent phosphodiesterase-V inhibitor zaprinast were examined. YC-1 or zaprinast elevated vessel cyclic guanosine monophosphate content, reduced medial wall and neointimal cell proliferation, stimulated medial and neointimal cellular apoptosis, and markedly attenuated neointimal remodeling in comparable fashion. Interestingly, soluble guanylate cyclase inhibition by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one failed to noticeably alter neointimal growth, and concomitant zaprinast with YC-1 did not modify any parameter compared to individual treatments. These results provide novel in vivo evidence that YC-1 and zaprinast inhibit injury-induced vascular remodeling through antimitogenic and proapoptotic actions and may offer promising therapeutic approaches against vasoproliferative disorders.

Novel therapies for cyclic GMP control of vascular smooth muscle growth.

Cyclic GMP, guanosine 3',5'-cyclic monophosphate, is a critical and multifunctional second-messenger molecule that mediates diverse physiological and pathophysiological functions in cardiac and vascular tissues. Synthesized through nitric oxide, carbon monoxide, and/or natriuretic peptide-mediated guanylate cyclase stimulation and guanosine triphosphate dephosphorylation, cyclic GMP is capable of stimulating a cascade of serine/threonine kinase events, including signaling through cyclic GMP- and/or cyclic AMP-dependent protein kinases, eliciting protein kinase-independent actions such as modulation of ion channels or transporters, or undergoing hydrolytic degradation through actions of cyclic GMP-regulated phosphodiesterases. Substrates, enzymes, cofactors, and associated variables in this multifaceted system have historically been targets of vital pharmacotherapies with perhaps most common the use of vascular smooth muscle-targeting organonitrates in cardiac patients and phosphodiesterase inhibitors in individuals with erectile dysfunction. Accumulating basic science and clinical evidence, however, suggests that cyclic GMP signaling is compromised under conditions of disease or elevated physiological stresses. Moreover, nitric oxide can stimulate an array of cytotoxic effects and nitric oxide-based therapies can be limited by diminished bioactivity and the development of tachyphylaxis or tolerance after prolonged use. Consequently, an emerging area for clinical drug development and therapeutic drug evaluation for conditions of cardiovascular adversity has focused on identification of cyclic GMP signaling pathways that act under oxidized or nitric oxide-unresponsive conditions and/or that operate irrespective of nitric oxide-induced complications. The aim of this therapeutic review is to describe novel, nitric oxide-alternate avenues for cyclic GMP signaling in vascular smooth muscle growth with particular emphasis on pharmacotherapeutics of recently characterized cyclic GMP-specific approaches.

Cyclic Nucleotide-Directed Protein Kinases in Cardiovascular Inflammation and Growth.

Cardiovascular disease (CVD), including myocardial infarction (MI) and peripheral or coronary artery disease (PAD, CAD), remains the number one killer of individuals in the United States and worldwide, accounting for nearly 18 million (>30%) global deaths annually. Despite considerable basic science and clinical investigation aimed at identifying key etiologic components of and potential therapeutic targets for CVD, the number of individuals afflicted with these dreaded diseases continues to rise. Of the many biochemical, molecular, and cellular elements and processes characterized to date that have potential to control foundational facets of CVD, the multifaceted cyclic nucleotide pathways continue to be of primary basic science and clinical interest. Cyclic adenosine monophosphate (cyclic AMP) and cyclic guanosine monophosphate (cyclic GMP) and their plethora of downstream protein kinase effectors serve ubiquitous roles not only in cardiovascular homeostasis but also in the pathogenesis of CVD. Already a major target for clinical pharmacotherapy for CVD as well as other pathologies, novel and potentially clinically appealing actions of cyclic nucleotides and their downstream targets are still being discovered. With this in mind, this review article focuses on our current state of knowledge of the cyclic nucleotide-driven serine (Ser)/threonine (Thr) protein kinases in CVD with particular emphasis on cyclic AMP-dependent protein kinase (PKA) and cyclic GMP-dependent protein kinase (PKG). Attention is given to the regulatory interactions of these kinases with inflammatory components including interleukin 6 signals, with G protein-coupled receptor and growth factor signals, and with growth and synthetic transcriptional platforms underlying CVD pathogenesis. This article concludes with a brief discussion of potential future directions and highlights the importance for continued basic science and clinical study of cyclic nucleotide-directed protein kinases as emerging and crucial controllers of cardiac and vascular disease pathologies.

Pharmacologic modulators of soluble guanylate cyclase/cyclic guanosine monophosphate in the vascular system - from bench top to bedside.

Guanosine-dependent cyclic nucleotide second messenger signaling has been implicated as a pivotal mediator of vascular function under both homeostatic eutrophic conditions as well as in the inimical environs of injury and/or disease. This biological system is highly regulated through reciprocal, complimentary, and often redundant upstream and downstream molecular and cellular elements and feedback controls. Key endogenous factors of the guanosine-dependent cyclic nucleotide cascade include upstream gaseous activating ligands (nitric oxide, carbon monoxide), downstream substrates (cGMP-gated ion channels, cGMP-dependent protein kinases), and cGMP hydrolyzing phosphodiesterases. This intricate system also has capacity to "cross-talk" with parallel adenosine-dependent cyclic nucleotide machinery. Numerous complexes of ligands, enzymes, cofactors, and substrates present significant targets for pharmacologic modulation at the cellular, genetic, and/or molecular level eventuating therapeutically as constructive functional responses observed in vascular physiology and/or pathophysiology. Interestingly, emerging evidence based largely on transgenic mouse models challenges the historically accepted concept that this signaling system functions principally as a therapeutic modality in cardiac and vascular tissues. The general purpose of this update is to provide current information on recently described neoteric agents that impact multifaceted and critical cGMP-dependent signaling in the vascular system. Emphasis will be placed on novel agents that exert significant and often multiple actions on upstream and downstream sites and are capable of eliciting robust effects on guanosine-dependent cellular actions. Individual sections will be devoted to agents that rely on an intact and functional cyclase heme and those that operate independently of the sGC heme. Attention will be placed on the physiologic and pathophysiologic clinical manifestations of these pharmacologic regimens. This review will conclude with some thoughts for future directions for study and continued discovery of novel sGC/cGMP controllers in the vascular system at the basic science and clinical levels.

Curcumin inhibits platelet-derived growth factor-stimulated vascular smooth muscle cell function and injury-induced neointima formation.

OBJECTIVE: Vascular smooth muscle cell (VSMC) migration, proliferation, and collagen synthesis are key events involved in the pathogenesis of cardiovascular disease. Growth factors, such as platelet-derived growth factor (PDGF) and fibroblast growth factor, released during vascular injury plays a pivotal role in regulating these events. Curcumin (diferuloyl methane), a major component of the spice turmeric (Curcuma longa), has been shown recently to have beneficial effects in chronic conditions, such as inflammation, cancer, cystic fibrosis, and Alzheimer's disease. The objective of this study was to investigate the ability of curcumin to inhibit PDGF-stimulated migration, proliferation, and collagen synthesis in cultured VSMCs and neointima formation after carotid artery injury in rats. METHODS AND RESULTS: Curcumin (1 to 25 microM) produced a concentration-dependent inhibition of PDGF-elicited VSMC migration, proliferation, and collagen synthesis assessed by chemotaxis, [3H]thymidine incorporation, and [3H]-L-proline incorporation, respectively. Curcumin blocked PDGF-induced VSMC actin-cytoskeleton reorganization, attenuated PDGF signal transduction, and inhibited the binding of PDGF to its receptors. Carotid artery neointima formation was significantly attenuated by perivascular curcumin compared with vehicle controls 14 days after injury, characterized by reduced DNA synthesis, collagen synthesis, and PDGF receptor phosphorylation. CONCLUSIONS: These data suggest that curcumin is a potent inhibitor of key PDGF-stimulated VSMC functions and may play a critical role in regulating these events after vascular injury.

Histological and morphometric analyses for rat carotid balloon injury model.

Experiments aimed at analyzing the response of blood vessels to mechanical injury and ensuing remodeling responses often employ the highly characterized carotid artery balloon injury model in laboratory rats. This approach utilizes luminal insertion of a balloon embolectomy catheter into the common carotid artery with inflation and withdrawal resulting in an injury characterized by vascular endothelial cell (EC) denudation and medial wall distension. The adaptive response to this injury is typified by robust vascular smooth muscle cell (SMC) replication and migration, SMC apoptosis and necrosis, enhanced synthesis and deposition of extracellular matrix (ECM) components, partial vascular EC regeneration from the border zones, luminal narrowing, and establishment of a neointima in time-dependent fashion. Evaluation of these adaptive responses to blood vessel injury can include acute and longer term qualitative and quantitative measures including expression analyses, activity assays, immunostaining for a plethora of factors and signals, and morphometry of neointima formation and gross mural remodeling. This chapter presents a logical continuation of Chapter 1 that offers details for performing the rat carotid artery balloon injury model in a standard laboratory setting by providing commonly used protocols for performing histological and morphometric analyses in such studies. Moreover, procedures, caveats, and considerations included in this chapter are highly relevant for alternative animal vascular physiology/pathophysiology studies and in particular those related to mechanisms of vascular injury and repair. Included in this chapter are specifics for in situ perfusion-fixation, tissue harvesting and processing for both snap-frozen and paraffin-embedded protocols, specimen embedding and sectioning, slide preparation, several standard histological staining steps, and routine morphological assessment.

Rat carotid artery balloon injury model.

Numerous and diverse experimental animal models have been used over the years to examine reactions to various forms of blood vessel disease and/or injury across species and in multiple vascular beds in a cumulative effort to relate these findings to the human condition. In this context, the rat carotid artery balloon injury model is highly characterized and commonly used for investigating gross morphological, cellular, biochemical, and molecular components of the response to experimentally induced arterial injury. The mechanical damage caused by the balloon catheter completely removes the intimal endothelial lining and creates a distending mural injury in the operated vessel. This elicits a reproducible remodeling response characterized by vascular smooth muscle cell (SMC) mitogenesis and migration (through phenotypic switching), SMC apoptosis, partial vascular endothelial cell regeneration, enhanced matrix synthesis, and establishment of an invasive neointima in time-dependent fashion. This multi-factorial process allows for investigation of these many important pathophysiological processes and can serve as a valuable "proof-of-concept" tool to verify and substantiate in vitro results; however, inherent anatomical and adaptive constraints of this in vivo model ration comparison to the diseased human system (see Note 1). In this chapter, brief overview of the materials needed and the methodologies commonly employed for successful routine performance of this important experimental animal model is provided. Individual sub-sections will cover animal care and handling, pre-operative and post-operative procedures, and the surgery proper. Protocols for histopathology and morphometry and procedures for data management and interpretation pertinent to the rat carotid artery balloon injury model are discussed in Chapter 2.