Multiple Kinase Involvement in the Regulation of Vascular Growth

Adderley, Shaquria; Joshi, Chintamani N; Martin, Danielle N; Mooney, Shayna; Tulis, David A.

doi:10.5772/37206

Cited by 3 publications

(17 citation statements)

References 60 publications

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“…Thoracic aortae from male CD ® (Sprague-Dawley IGS) rats (100–125 g; 4–5 weeks old; strain 001; Charles River) were digested with collagenase/elastase and derived VSM cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; #10-013-CM; Corning) with 10% fetal bovine serum (FBS; #900-108-500; GeminiBio), 2 m m l -Glutamine, sodium pyruvate, 5 m m HEPES, and Primocin (100 mg/L; #ant-pm-2; InvivoGen) at 37°C in 95% air/5% CO 2 [ 4 , 8 ]. Cells were serially split (at 80–100% confluence; ∼3 cell doublings per passage [P]) from the same parental clones into two cohorts: P Lo cells between P3 and P6, and P Hi cells between P10 and P15, recognized passages for distinguishing contractile versus synthetic VSM cells [ 15 , 16 ].…”

Section: Methodsmentioning

confidence: 99%

“…The Pierce Coomassie (Bradford) Protein Assay Kit (#23200; Thermo Fisher) was used to determine protein quantification of cell homogenates per manufacturer’s instructions. Lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using stain-free gels (#5678123; Bio-Rad) [ 4 , 8 ]. Separated protein was transferred to polyvinylidene fluoride membranes using the TransBlot Turbo Transfer System (Bio-Rad) at a mixed MW setting (7 min, 2.5 A up to 25 V).…”

Section: Methodsmentioning

confidence: 99%

“…P Lo and P Hi VSM cells were seeded in 96-well plates (10,000 cells/well) and incubated with SLIGRL (100nM–10 µ m ), with/without 30-min pretreatment with the following inhibitors: PKI for PKA (10 μ m ; #BML-P203-0500; Enzo, Farmingdale, NY, USA); LY294002 (LY) for PI3K (1 μ m ; #440202; Sigma Aldrich, St. Louis, MO); PD98059 (PD) for MEK1/2 (10 μ m ; #1213; Tocris); or vehicle (appropriate for each inhibitor) as detailed [ 4 ] for 24 h, after which media was removed and a colorimetric BrdU cell proliferation ELISA (#2752; Millipore Sigma) was performed per manufacturer’s guidelines.…”

Section: Methodsmentioning

confidence: 99%

“…Following PAR-specific interventions, cells were trypsinized and cell numbers were determined through automated quantification with trypan blue exclusion (ViCell; Beckman Coulter) to provide objective measures of viability and total and viable cell numbers as detailed [ 4 , 8 ].…”

Section: Methodsmentioning

confidence: 99%

“…GPCRs represent the largest group of cell surface receptors in the human genome and constitute ∼34% of all FDA-approved drug targets [ 7 ]. Considering their wide-ranging intracellular targets, GPCRs can impact numerous effectors, including members of the equally broad family of protein kinases [ 3 , 4 , 8 ]. Protease-activated receptors (PARs), a GPCR subfamily, have been theorized as important in cardiovascular (patho)physiology [ 9 , 10 ], and of emerging interest in VSM is PAR2.…”

Section: Introductionmentioning

confidence: 99%

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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

Williams,

Bullock,

Johnson

et al. 2023

J Vasc Res

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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 μ<sc>m</sc>) with/without PKA (PKI; 10 μ<sc>m</sc>), MEK1/2 (PD98059; 10 μ<sc>m</sc>), and PI3K (LY294002; 1 μ<sc>m</sc>) 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.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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

Williams,

Bullock,

Johnson

et al. 2023

J Vasc Res

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FoxO3 normalizes Smad3-induced arterial smooth muscle cell growth

Francisco,

Holt,

Bullock

et al. 2023

Front. Physiol.

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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.

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Vascular Smooth Muscle as a Therapeutic Target in Disease Pathology

Holt¹,

Tulis²

2015

Muscle Cell and Tissue

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Our circulatory system is composed of numerous elements that are responsible for transport of blood and delivery of essential nutrients and gases to vital downstream tissues. "mong these components that make up our circulation is vascular smooth muscle VSM , the primary muscular and contractile element of blood vessels and regulator of many blood vessel functions. This is of particular importance as cardiovascular disease CVD , the number one killer of individuals in "merica and worldwide, is primarily vascular in origin. Logically, identifying and characterizing feasible targets that could control CVD are highly appealing and much desired. With this in mind and given its centrality in control of vascular physiology, VSM has gained wide attention as a plausible target to combat elements of CVD. This book chapter focuses on VSM as a potential therapeutic target against CVD and will provide overview of vascular anatomy and physiology and brief discussions about the pivotal roles of VSM in CVD pathology, the influence of abnormal blood flow mechanics and hemodynamics in CVD, neural control of VSM and the vasculature, and possible novel cellular and molecular signaling targets that could be used to control and/or minimize CVD. This chapter hopes to serve as a valuable resource for basic and applied scientists as well as clinicians interested in understanding the crucial roles that VSM plays in vessel physiology and pathology.

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Multiple Kinase Involvement in the Regulation of Vascular Growth

Cited by 3 publications

References 60 publications

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

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

FoxO3 normalizes Smad3-induced arterial smooth muscle cell growth

Vascular Smooth Muscle as a Therapeutic Target in Disease Pathology

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