Vascular smooth muscle cells (VSM) dedifferentiate from the contractile to synthetic phenotype in response to acute vascular diseases such as restenosis and chronic vascular diseases such as atherosclerosis and contribute to growth of the neointima. We recently demonstrated that balloon catheter injury of rat carotid arteries resulted in increased expression of CaMKIIδ2 in the medial wall and the expanding neointima. These findings led us to hypothesize that increased expression of CaMKIIδ2 is a positive mediator of synthetic VSM. HDAC4 and HDAC5 function as transcriptional corepressers and are regulated in a CaMKII-dependent manner. In this study, we report that endogenous HDAC4 and HDAC5 in VSM are activated in a Ca2+- and CaMKIIδ2-dependent manner. We further show that AngII-and PDGF-dependent phosphorylation of HDAC4 and HDAC5 is reduced when CaMKIIδ2 expression is suppressed or CaMKIIδ2 activity is attenuated. The transcriptional activator MEF2 is an important determinant of VSM phenotype and is regulated in an HDAC-dependent manner. Herein, we report that stimulation of VSM cells with ionomycin or Ang II potentiates MEF2's ability to bind DNA and increases the expression of established MEF2 target genes Nur77 and MCP1. Suppression of CaMKIIδ2 attenuates increased MEF2 DNA binding activity and upregulation of Nur77 and MCP1. Finally, we show that HDAC5 is regulated by HDAC4 in VSM. Suppression of HDAC4 expression and activity prevents AngII- and PDGF- dependent phosphorylation of HDAC5. Taken together, these results illustrate a mechanism by which CaMKIIδ2 mediates MEF2-dependent gene transcription in VSM cells through regulation of HDAC4 and HDAC5.

Multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a multigene family with isoform-specific regulation of vascular smooth muscle (VSM) functions. In previous studies, we found that vascular injury resulted in VSM dedifferentiation and reduced expression of the CaMKIIγ isoform in medial wall VSM. Smooth muscle knockout of CaMKIIγ enhanced injury-induced VSM neointimal hyperplasia, whereas CaMKIIγ overexpression inhibited VSM proliferation and neointimal formation. In this study, we evaluated DNA cytosine methylation/demethylation as a mechanism for regulating CaMKII isoform expression in VSM. Inhibition of cytosine methylation with 5-Aza-2′-deoxycytidine significantly upregulated CaMKIIγ expression in cultured VSM cells and inhibited CaMKIIγ downregulation in organ-cultured aorta ex vivo. With the use of methylated cytosine immunoprecipitation, the rat Camk2g promoter was found hypomethylated in differentiated VSM, whereas injury- or cell culture-induced VSM dedifferentiation coincided with Camk2g promoter methylation and decreased expression. We report for the first time that VSM cell phenotype switching is accompanied by marked induction of thymine DNA glycosylase (TDG) protein and mRNA expression in injured arteries in vivo and in cultured VSM synthetic phenotype cells. Silencing Tdg in VSM promoted expression of CaMKIIγ and differentiation markers, including myocardin, and inhibited VSM cell proliferation and injury-induced neointima formation. This study indicates that CaMKIIγ expression in VSM is regulated by cytosine methylation/demethylation and that TDG is an important determinant of this process and, more broadly, VSM phenotype switching and function. NEW & NOTEWORTHY Expression of the calcium calmodulin-dependent protein kinase II-γ isoform (CaMKIIγ) is associated with differentiated vascular smooth muscle (VSM) and negatively regulates proliferation in VSM synthetic phenotype (VSMSyn) cells. This study demonstrates that thymine DNA glycosylase (TDG) plays a key role in regulating CaMKIIγ expression in VSM through promoter cytosine methylation/demethylation. TDG expression is strongly induced in VSMSyn cells and plays key roles in negatively regulating CaMKIIγ expression and more broadly VSM phenotype switching.

Airway smooth muscle (ASM) is a key target cell in allergen-induced asthma known to contribute to airway hyperresponsiveness (AHR) and chronic airway remodeling. Changes in ASM calcium homeostasis have been shown to contribute to AHR although the mechanisms and Ca2+ signal effectors are incompletely understood. In the present study we tested the function of ASM multifunctional protein kinase Ca2+/calmodulin-dependent kinase II (CaMKII) isoforms CaMKIIδ and CaMKIIγ in allergen-induced AHR and airway remodeling in vivo. Using a murine model of atopic asthma we demonstrate CaMKIIδ protein is up-regulated in ASM derived from ovalbumin (OVA)-treated animals compared to controls. A genetic approach to conditionally knockout smooth muscle CaMKIIδ and CaMKIIγ in separate Cre-loxp systems was validated, and using this loss-of function approach, the function of these CaMKII isoforms was tested in ovalbumin (OVA)-induced airway remodeling and AHR. OVA treatment in control mice had no effect on ASM remodeling in this model of AHR and CaMKIIδ knockouts had no independent effects on ASM content. However, at 1day post final OVA challenge, OVA-induced AHR were eliminated in the CaMKIIδ knockouts. OVA-induced peribronchial inflammation and bronchoalveolar lavage fluid (BALF) levels of the Th2 cytokine IL-13 were significantly decreased in the CaMKIIδ knockouts. Unexpectedly, we found increased peribronchial eosinophils in the smooth muscle CaMKIIδ knockouts compared to control animals at 1 day post final challenge, suggesting that lack of ASM CaMKIIδ delays the progression of AHR rather than inhibiting it. Indeed, when AHR was determined at 7 day post final OVA challenge, CaMKIIδ knockouts showed robust AHR while AHR was fully resolved in OVA-challenged control mice. These in vivo studies demonstrate a role for smooth muscle CaMKIIδ in promoting airway inflammation and AHR and suggest a complex signaling role for CaMKIIδ in regulating ASM function. These studies confirm the diverse roles of ASM cells as immune effectors that control AHR and calls for further studies into CaMKIIδ-mediated signaling in ASM cells during disease.

Vascular smooth muscle is well known for phenotype plasticity and upon injury or in response to disease it modulates from a contractile phenotype to a sythetic phenotype that contributes to vascular remodeling. We have demonstrated that increased expression of CaMKII is associated with modulation of VSM to the synthetic phenotype but there is very little direct evidence indicating a function for the kinase in regulating VSM cell gene transcription. Herein, we report that the transcriptional co‐repressors HDAC4/5 are phosphorylated in a CaMKII‐dependent manner . Furthermore, effective suppression of CaMKII expression resulted in decreased HDAC5 phosphorylation in response to PDGF and AngII. Nur77 is an immediate response gene shown to be regulated in a Mef2/HDAC‐dependent manner. Our initial studies in cultured VSM indicate robust activation of nur77 in response to PDGF. Inhibition or suppression of CaMKII resulted in the decrease of Nur77 expression. Taken together, these results lead us to hypothesize that Ca2+ signaling, via CaMKII, regulates VSM gene transcription and may be a determinant of VSM phenotype.