title: Pyk2 inhibition promotes contractile differentiation in arterial smooth muscle.
abstract: Modulation from contractile to synthetic phenotype of vascular smooth muscle cells is a central process in disorders involving compromised integrity of the vascular wall. Phenotype modulation has been shown to include transition from voltage-dependent toward voltage-independent regulation of the intracellular calcium level, and inhibition of non-voltage dependent calcium influx contributes to maintenance of the contractile phenotype. One possible mediator of calcium-dependent signaling is the FAK-family non-receptor protein kinase Pyk2, which is activated by a number of stimuli in a calcium-dependent manner. We used the Pyk2 inhibitor PF-4594755 and Pyk2 siRNA to investigate the role of Pyk2 in phenotype modulation in rat carotid artery smooth muscle cells and in cultured intact arteries. Pyk2 inhibition promoted the expression of smooth muscle markers at the mRNA and protein levels under stimulation by FBS or PDGF-BB and counteracted phenotype shift in cultured intact carotid arteries and balloon injury ex vivo. During long-term (24-96 hr) treatment with PF-4594755, smooth muscle markers increased before cell proliferation was inhibited, correlating with decreased KLF4 expression and differing from effects of MEK inhibition. The Pyk2 inhibitor reduced Orai1 and preserved SERCA2a expression in carotid artery segments in organ culture, and eliminated the inhibitory effect of PDGF stimulation on L-type calcium channel and large-conductance calcium-activated potassium channel expression in carotid cells. Basal intracellular calcium level, calcium wave activity, and store-operated calcium influx were reduced after Pyk2 inhibition of growth-stimulated cells. Pyk2 inhibition may provide an interesting approach for preserving vascular smooth muscle differentiation under pathophysiological conditions.
authors.affiliation: Department of Experimental Medical Science, Lund University, Lund, Sweden.
authors.forename: Mario
authors.initials: M
authors.lastname: Grossi
authors.affiliation: Department of Experimental Medical Science, Lund University, Lund, Sweden.
authors.forename: Anirban
authors.initials: A
authors.lastname: Bhattachariya
authors.affiliation: Department of Experimental Medical Science, Lund University, Lund, Sweden.
authors.forename: Ina
authors.initials: I
authors.lastname: Nordström
authors.affiliation: Department of Experimental Medical Science, Lund University, Lund, Sweden.
authors.forename: Karolina M
authors.initials: KM
authors.lastname: Turczyńska
authors.affiliation: Department of Experimental Medical Science, Lund University, Lund, Sweden.
authors.forename: Daniel
authors.initials: D
authors.lastname: Svensson
authors.affiliation: Department of Experimental Medical Science, Lund University, Lund, Sweden.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Albinsson
authors.affiliation: Department of Experimental Medical Science, Lund University, Lund, Sweden.
authors.forename: Bengt-Olof
authors.initials: BO
authors.lastname: Nilsson
authors.affiliation: Department of Experimental Medical Science, Lund University, Lund, Sweden.
authors.forename: Per
authors.identifier: http://orcid.org/0000-0003-2081-2666
authors.initials: P
authors.lastname: Hellstrand
mesh_terms: D000818:Animals; D000077214:Becaplermin; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D020212:Carotid Artery Injuries / Q000201:enzymology* / Q000235:genetics / Q000503:physiopathology; D017536:Carotid Artery, Common / Q000187:drug effects / Q000201:enzymology / Q000503:physiopathology; D002454:Cell Differentiation / Q000187:drug effects*; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D051418:Focal Adhesion Kinase 2 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits / Q000235:genetics / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000201:enzymology / Q000503:physiopathology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000201:enzymology; D000071740:ORAI1 Protein / Q000235:genetics / Q000378:metabolism; D009924:Organ Culture Techniques; D010641:Phenotype; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D020574:Proto-Oncogene Proteins c-sis / Q000494:pharmacology; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014661:Vasoconstriction / Q000187:drug effects*
chemical_list: C506948:Atp2a2 protein, rat; C504728:Cacna1c protein, rat; D020746:Calcium Channels, L-Type; C545899:KCNMB1 protein, rat; C000715469:Klf4 protein, rat; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits; D000071740:ORAI1 Protein; C532493:Orai1 protein, rat; D047428:Protein Kinase Inhibitors; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger; D000077214:Becaplermin; D051418:Focal Adhesion Kinase 2; C496054:Ptk2b protein, rat; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases
keywords: PF-459475; SERCA; calcium; phenotype; vascular remodeling