MLKL and CaMKII Are Involved in RIPK3-Mediated Smooth Muscle Cell Necroptosis.

Receptor interacting protein kinase 3 (RIPK3)-mediated smooth muscle cell (SMC) necroptosis has been shown to contribute to the pathogenesis of abdominal aortic aneurysms (AAAs). However, the signaling steps downstream from RIPK3 during SMC necroptosis remain unknown. In this study, the roles of mixed lineage kinase domain-like pseudokinase (MLKL) and calcium/calmodulin-dependent protein kinase II (CaMKII) in SMC necroptosis were investigated. We found that both MLKL and CaMKII were phosphorylated in SMCs in a murine CaCl2-driven model of AAA and that Ripk3 deficiency reduced the phosphorylation of MLKL and CaMKII. In vitro, mouse aortic SMCs were treated with tumor necrosis factor α (TNFα) plus Z-VAD-FMK (zVAD) to induce necroptosis. Our data showed that both MLKL and CaMKII were phosphorylated after TNFα plus zVAD treatment in a time-dependent manner. SiRNA silencing of Mlkl-diminished cell death and administration of the CaMKII inhibitor myristoylated autocamtide-2-related inhibitory peptide (Myr-AIP) or siRNAs against Camk2d partially inhibited necroptosis. Moreover, knocking down Mlkl decreased CaMKII phosphorylation, but silencing Camk2d did not affect phosphorylation, oligomerization, or trafficking of MLKL. Together, our results indicate that both MLKL and CaMKII are involved in RIPK3-mediated SMC necroptosis, and that MLKL is likely upstream of CaMKII in this process.

CaMKIIδ is upregulated by pro-inflammatory cytokine IL-6 in a JAK/STAT3-dependent manner to promote angiogenesis.

Ca2+ /calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous serine threonine kinase with established roles in physiological and pathophysiological vascular remodeling. Based on our previous study demonstrating that CaMKIIδ promotes thrombin-induced endothelial permeability and recent reports that CaMKII may contribute to inflammatory remodeling in the heart, we investigated CaMKIIδ-dependent regulation of endothelial function downstream of an interleukin-6 (IL-6)/JAK/STAT3 signaling axis. Upon treatment with IL-6 and its soluble receptor (sIL-6r), CaMKIIδ expression is significantly induced in HUVEC. Using pharmacological inhibitors of JAK and siRNA targeting STAT3, we demonstrated that activation of STAT3 is sufficient to induce CaMKIIδ expression. Under these conditions, rather than promoting IL-6-induced permeability, we found that CaMKIIδ promotes endothelial cell migration as measured by live cell imaging of scratch wound closure and single-cell motility analysis. In a similar manner, endothelial cell proliferation was attenuated upon knockdown of CaMKIIδ as determined by growth curves, cell cycle analysis, and capacitance of cell-covered electrodes as measured by ECIS. Using inducible endothelial-specific STAT3 knockout mice, we demonstrate that STAT3 signaling promotes developmental angiogenesis in the neonatal mouse retina assessed at postnatal day 6. CaMKIIδ expression in retinal endothelium was attenuated in these animals as measured by qPCR. STAT3's effects on angiogenesis were phenocopied by the endothelial-specific knockout of CaMKIIδ, with significantly reduced vascular outgrowth and number of junctions in the developing P6 retina. For the first time, we demonstrate that transcriptional regulation of CaMKIIδ by STAT3 promotes endothelial motility, proliferation, and in vivo angiogenesis.

Interactive Roles of CaMKII/Ryanodine Receptor Signaling and Inflammation in Lung Diseases.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional protein kinase and has been recently recognized to play a vital role in pathological events in the pulmonary system. CaMKII has diverse downstream targets that promote vascular disease, asthma, and cancer, so improved understanding of CaMKII signaling has the potential to lead to new therapies for lung diseases. Multiple studies have demonstrated that CaMKII is involved in redox modulation of ryanodine receptors (RyRs). CaMKII can be directly activated by reactive oxygen species (ROS) which then regulates RyR activity, which is essential for Ca2+-dependent processes in lung diseases. Furthermore, both CaMKII and RyRs participate in the inflammation process. However, their role in the pulmonary physiology in response to ROS is still an ambiguous one. Because CaMKII and RyRs are important in pulmonary biology, cell survival, cell cycle control, and inflammation, it is possible that the relationship between ROS and CaMKII/RyRs signal complex will be necessary for understanding and treating lung diseases. Here, we review roles of CaMKII/RyRs in lung diseases to understand with how CaMKII/RyRs may act as a transduction signal to connect prooxidant conditions into specific downstream pathological effects that are relevant to rare and common forms of pulmonary disease.

High CO2 Downregulates Skeletal Muscle Protein Anabolism via AMP-activated Protein Kinase α2-mediated Depressed Ribosomal Biogenesis.

High CO2 retention, or hypercapnia, is associated with worse outcomes in patients with chronic pulmonary diseases. Skeletal muscle wasting is also an independent predictor of poor outcomes in patients with acute and chronic pulmonary diseases. Although previous evidence indicates that high CO2 accelerates skeletal muscle catabolism via AMPK (AMP-activated protein kinase)-FoxO3a-MuRF1 (E3-ubiquitin ligase muscle RING finger protein 1), little is known about the role of high CO2 in regulating skeletal muscle anabolism. In the present study, we investigated the potential role of high CO2 in attenuating skeletal muscle protein synthesis. We found that locomotor muscles from patients with chronic CO2 retention demonstrated depressed ribosomal gene expression in comparison with locomotor muscles from non-CO2-retaining individuals, and analysis of the muscle proteome of normo- and hypercapnic mice indicates reduction of important components of ribosomal structure and function. Indeed, mice chronically kept under a high-CO2 environment show evidence of skeletal muscle downregulation of ribosomal biogenesis and decreased protein synthesis as measured by the incorporation of puromycin into skeletal muscle. Hypercapnia did not regulate the mTOR pathway, and rapamycin-induced deactivation of mTOR did not cause a decrease in ribosomal gene expression. Loss-of-function studies in cultured myotubes showed that AMPKα2 regulates CO2-mediated reductions in ribosomal gene expression and protein synthesis. Although previous evidence has implicated TIF1A (transcription initiation factor-1α) and KDM2A (lysine-specific demethylase 2A) in AMPK-driven regulation of ribosomal gene expression, we found that these mediators were not required in the high CO2-induced depressed protein anabolism. Our research supports future studies targeting ribosomal biogenesis and protein synthesis to alleviate the effects of high CO2 on skeletal muscle turnover.

Thymine DNA glycosylase is a key regulator of CaMKIIγ expression and vascular smooth muscle phenotype.

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.

The Hemoglobin Homolog Cytoglobin in Smooth Muscle Inhibits Apoptosis and Regulates Vascular Remodeling.

OBJECTIVE: The role of hemoglobin and myoglobin in the cardiovascular system is well established, yet other globins in this context are poorly characterized. Here, we examined the expression and function of cytoglobin (CYGB) during vascular injury. APPROACH AND RESULTS: We characterized CYGB content in intact vessels and primary vascular smooth muscle (VSM) cells and used 2 different vascular injury models to examine the functional significance of CYGB in vivo. We found that CYGB was strongly expressed in medial arterial VSM and human veins. In vitro and in vivo studies indicated that CYGB was lost after VSM cell dedifferentiation. In the rat balloon angioplasty model, site-targeted delivery of adenovirus encoding shRNA specific for CYGB prevented its reexpression and decreased neointima formation. Similarly, 4 weeks after complete ligation of the left common carotid, Cygb knockout mice displayed little to no evidence of neointimal hyperplasia in contrast to their wild-type littermates. Mechanistic studies in the rat indicated that this was primarily associated with increased medial cell loss, terminal uridine nick-end labeling staining, and caspase-3 activation, all indicative of prolonged apoptosis. In vitro, CYGB could be reexpressed after VSM stimulation with cytokines and hypoxia and loss of CYGB sensitized human and rat aortic VSM cells to apoptosis. This was reversed after antioxidant treatment or NOS2 (nitric oxide synthase 2) inhibition. CONCLUSIONS: These results indicate that CYGB is expressed in vessels primarily in differentiated medial VSM cells where it regulates neointima formation and inhibits apoptosis after injury.

Dual Function for Mature Vascular Smooth Muscle Cells During Arteriovenous Fistula Remodeling.

BACKGROUND: The arteriovenous fistula (AVF) is the preferred form of hemodialysis access for patients with chronic kidney disease. However, AVFs are associated with significant problems including high incidence of both early and late failures, usually attributed to inadequate venous arterialization and neointimal hyperplasia, respectively. Understanding the cellular basis of venous remodeling in the setting of AVF could provide targets for improving AVF patency rates. METHODS AND RESULTS: A novel vascular smooth muscle cell (VSMC) lineage tracing reporter mouse, Myh11-Cre/ERT2-mTmG, was used to track mature VSMCs in a clinically relevant AVF mouse model created by a jugular vein branch end to carotid artery side anastomosis. Prior to AVF surgery, differentiated medial layer VSMCs were labeled with membrane green fluorescent protein (GFP) following tamoxifen induction. Four weeks after AVF surgery, we observed medial VSMC layer thickening in the middle region of the arterialized vein branch. This thickened medial VSMC layer was solely composed of differentiated VSMCs that were GFP+/MYH11+/Ki67-. Extensive neointimal hyperplasia occurred in the AVF region proximal to the anastomosis site. Dedifferentiated VSMCs (GFP+/MYH11-) were a major cellular component of the neointima. Examination of failed human AVF samples revealed that the processes of VSMC phenotypic modulation and intimal hyperplasia, as well as medial VSMC layer thickening, also occurred in human AVFs. CONCLUSIONS: We demonstrated a dual function for mature VSMCs in AVF remodeling, with differentiated VSMCs contributing to medial wall thickening towards venous maturation and dedifferentiated VSMCs contributing to neointimal hyperplasia. These results provide valuable insights into the mechanisms underlying venous adaptations during AVF remodeling.

MicroRNA-30 inhibits neointimal hyperplasia by targeting Ca(2+)/calmodulin-dependent protein kinase IIδ (CaMKIIδ).

The multifunctional Ca(2+)/calmodulin-dependent protein kinase II δ-isoform (CaMKIIδ) promotes vascular smooth muscle (VSM) proliferation, migration, and injury-induced vascular wall neointima formation. The objective of this study was to test if microRNA-30 (miR-30) family members are endogenous regulators of CaMKIIδ expression following vascular injury and whether ectopic expression of miR-30 can inhibit CaMKIIδ-dependent VSM cell function and neointimal VSM hyperplasia induced by vascular injury. The CaMKIIδ 3'UTR contains a consensus miR-30 binding sequence that is highly conserved across species. A significant decrease in miR-30 family members and increase in CaMKIIδ2 protein expression, with no change in CaMKIIδ mRNA expression, was observed in medial layers of VSM 7 days post-injury. In vitro, overexpression of miR-30c or miR-30e inhibited CaMKIIδ2 protein expression by ~50% in cultured rat aortic VSM cells, and inhibited VSM cell proliferation and migration. In vivo, lenti-viral delivery of miR-30c into injured rat carotid arteries prevented the injury-induced increase in CaMKIIδ2. Furthermore, neointima formation was dramatically inhibited by lenti-viral delivery of miR-30c in the injured medial smooth muscle. These studies define a novel mechanism for regulating CaMKIIδ expression in VSM and provide a new potential therapeutic strategy to reduce progression of vascular proliferative diseases, including atherosclerosis and restenosis.

Ca2+/calmodulin-dependent protein kinase II-γ (CaMKIIγ) negatively regulates vascular smooth muscle cell proliferation and vascular remodeling.

Vascular smooth muscle (VSM) expresses calcium/calmodulin-dependent protein kinase II (CaMKII)-δ and -γ isoforms. CaMKIIδ promotes VSM proliferation and vascular remodeling. We tested CaMKIIγ function in vascular remodeling after injury. CaMKIIγ protein decreased 90% 14 d after balloon injury in rat carotid artery. Intraluminal transduction of adenovirus encoding CaMKIIγC rescued expression to 35% of uninjured controls, inhibited neointima formation (>70%), inhibited VSM proliferation (>60%), and increased expression of the cell-cycle inhibitor p21 (>2-fold). Comparable doses of CaMKIIδ2 adenovirus had no effect. Similar dynamics in CaMKIIγ mRNA and protein expression were observed in ligated mouse carotid arteries, correlating closely with expression of VSM differentiation markers. Targeted deletion of CaMKIIγ in smooth muscle resulted in a 20-fold increase in neointimal area, with a 3-fold increase in the cell proliferation index, no change in apoptosis, and a 60% decrease in p21 expression. In cultured VSM, CaMKIIγ overexpression induced p53 mRNA (1.7 fold) and protein (1.8-fold) expression; induced the p53 target gene p21 (3-fold); decreased VSM cell proliferation (>50%); and had no effect on expression of apoptosis markers. We conclude that regulated CaMKII isoform composition is an important determinant of the injury-induced vasculoproliferative response and that CaMKIIγ and -δ isoforms have nonequivalent, opposing functions.

A small molecule PAI-1 functional inhibitor attenuates neointimal hyperplasia and vascular smooth muscle cell survival by promoting PAI-1 cleavage.

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of urokinase-and tissue-type plasminogen activators (uPA and tPA), is an injury-response gene implicated in the development of tissue fibrosis and cardiovascular disease. PAI-1 mRNA and protein levels were elevated in the balloon catheter-injured carotid and in the vascular smooth muscle cell (VSMC)-enriched neointima of ligated arteries. PAI-1/uPA complex formation and PAI-1 antiproteolytic activity can be inhibited, via proteolytic cleavage, by the small molecule antagonist tiplaxtinin which effectively increased the VSMC apoptotic index in vitro and attenuated carotid artery neointimal formation in vivo. In contrast to the active full-length serine protease inhibitor (SERPIN), elastase-cleaved PAI-1 (similar to tiplaxtinin) also promoted VSMC apoptosis in vitro and similarly reduced neointimal formation in vivo. The mechanism through which cleaved PAI-1 (CL-PAI-1) stimulates apoptosis appears to involve the TNF-α family member TWEAK (TNF-α weak inducer of apoptosis) and it's cognate receptor, fibroblast growth factor (FGF)-inducible 14 (FN14). CL-PAI-1 sensitizes cells to TWEAK-stimulated apoptosis while full-length PAI-1 did not, presumably due to its ability to down-regulate FN14 in a low density lipoprotein receptor-related protein 1 (LRP1)-dependent mechanism. It appears that prolonged exposure of VSMCs to CL-PAI-1 induces apoptosis by augmenting TWEAK/FN14 pro-apoptotic signaling. This work identifies a critical, anti-stenotic, role for a functionally-inactive (at least with regard to its protease inhibitory function) cleaved SERPIN. Therapies that promote the conversion of full-length to cleaved PAI-1 may have translational implications.

CaMKIIδ-dependent inhibition of cAMP-response element-binding protein activity in vascular smooth muscle.

One transcription factor mediator of Ca(2+)-signals is cAMP response element-binding protein (CREB). CREB expression and/or activity negatively correlates with vascular smooth muscle (VSM) cell proliferation and migration. Multifunctional Ca(2+)/calmodulin-dependent protein kinases, including CaMKII, have been demonstrated to regulate CREB activity through both positive and negative phosphorylation events in vitro, but the function of CaMKII as a proximal regulator of CREB in intact cell systems, including VSM, is not clear. In this study, we used gain- and loss-of-function approaches to determine the function of CaMKIIδ in regulating CREB phosphorylation, localization, and activity in VSM. Overexpression of constitutively active CaMKIIδ specifically increased CREB phosphorylation on Ser(142) and silencing CaMKIIδ expression by siRNA or blocking endogenous CaMKII activity with KN93 abolished thrombin- or ionomycin-induced CREB phosphorylation on Ser(142) without affecting Ser(133) phosphorylation. CREB-Ser(142) phosphorylation correlated with transient nucleocytoplasmic translocation of CREB. Thrombin-induced CREB promoter activity, CREB binding to Sik1 and Rgs2 promoters, and Sik1/Rgs2 transcription were enhanced by a kinase-negative CaMKIIδ2 (K43A) mutant and inhibited by a constitutively active (T287D) mutant. Taken together, these studies establish negative regulation of CREB activity by endogenous CaMKIIδ-dependent CREB-Ser(142) phosphorylation and suggest a potential mechanism for CaMKIIδ/CREB signaling in modulating proliferation and migration in VSM cells.

Vascular smooth muscle cell motility is mediated by a physical and functional interaction of Ca2+/calmodulin-dependent protein kinase IIδ2 and Fyn.

In vascular smooth muscle (VSM) cells, Ca(2+)/calmodulin-dependent protein kinase IIδ2 (CaMKIIδ2) activates non-receptor tyrosine kinases and EGF receptor, with a Src family kinase as a required intermediate. siRNA-mediated suppression of Fyn, a Src family kinase, inhibited VSM cell motility. Simultaneous suppression of both Fyn and CaMKIIδ2 was non-additive, suggesting coordinated regulation of cell motility. Confocal immunofluorescence microscopy indicated that CaMKIIδ2 and Fyn selectively (compared with Src) co-localized with the Golgi in quiescent cultured VSM cells. Stimulation with PDGF resulted in a rapid (<5 min) partial redistribution and co-localization of both kinases in peripheral membrane regions. Furthermore, CaMKIIδ2 and Fyn selectively (compared with Src) co-immunoprecipitated, suggesting a physical interaction in a signaling complex. Stimulation of VSM cells with ionomycin, a calcium ionophore, resulted in activation of CaMKIIδ2 and Fyn and disruption of the complex. Pretreatment with KN-93, a pharmacological inhibitor of CaMKII, prevented activation-dependent disruption of CaMKIIδ2 and Fyn, implicating CaMKIIδ2 as an upstream mediator of Fyn. Overexpression of constitutively active CaMKII resulted in the dephosphorylation of Fyn at Tyr-527, which is required for Fyn activation. Taken together, these data demonstrate a dynamic interaction between CaMKIIδ2 and Fyn in VSM cells and indicate a mechanism by which CaMKIIδ2 and Fyn may coordinately regulate VSM cell motility.

NADPH oxidase 4 is required for interleukin-1ß-mediated activation of protein kinase Cδ and downstream activation of c-jun N-terminal kinase signaling in smooth muscle.

Reactive oxygen species (ROS) are generated in the vascular wall upon stimulation by proinflammatory cytokines and are important mediators of diverse cellular responses that occur as a result of vascular injury. Members of the NADPH oxidase (NOX) family of proteins have been identified in vascular smooth muscle (VSM) cells as important sources of ROS. In this study, we tested the hypothesis that NOX4 is a proximal mediator of IL-1ß-dependent activation of PKCδ and increases IL-1ß-stimulated c-Jun kinase (JNK) signaling in primary rat aortic VSM cells. We found that stimulation of VSM cells with IL-1ß increased PKCδ activity and intracellular ROS generation. SiRNA silencing of NOX4 but not NOX1 ablated the IL-1ß-dependent increase in ROS production. Pharmacological inhibition of PKCδ activity as well as siRNA depletion of PKCδ or NOX4 blocked the IL-1ß-dependent activation of JNK. Further studies showed that the IL-1ß-dependent upregulation of inducible NO synthase expression was inhibited through JNK inhibition and NOX4 silencing. Taken together, these results indicate that IL-1ß-dependent activation of PKCδ is modulated by NOX4-derived ROS. Our study positions PKCδ as an important redox-sensitive mediator of IL-1ß-dependent signaling and downstream activation of inflammatory mediators in VSM cells.

MEF2 is regulated by CaMKIIδ2 and a HDAC4-HDAC5 heterodimer in vascular smooth muscle cells.

VSMCs (vascular smooth muscle cells) dedifferentiate from the contractile to the 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 demonstrated previously that balloon catheter injury of rat carotid arteries resulted in increased expression of CaMKII (Ca(2+)/calmodulin-dependent protein kinase) IIδ(2) in the medial wall and the expanding neointima [House and Singer (2008) Arterioscler. Thromb. Vasc. Biol. 28, 441-447]. These findings led us to hypothesize that increased expression of CaMKIIδ(2) is a positive mediator of synthetic VSMCs. HDAC (histone deacetylase) 4 and HDAC5 function as transcriptional co-repressors and are regulated in a CaMKII-dependent manner. In the present paper, we report that endogenous HDAC4 and HDAC5 in VSMCs are activated in a Ca(2+)- and CaMKIIδ(2)-dependent manner. We show further that AngII (angiotensin II)- and PDGF (platelet-derived growth factor)-dependent phosphorylation of HDAC4 and HDAC5 is reduced when CaMKIIδ(2) expression is suppressed or CaMKIIδ(2) activity is attenuated. The transcriptional activator MEF2 (myocyte-enhancer factor 2) is an important determinant of VSMC phenotype and is regulated in an HDAC-dependent manner. In the present paper, we report that stimulation of VSMCs with ionomycin or AngII potentiates MEF2's ability to bind DNA and increases the expression of established MEF2 target genes Nur77 (nuclear receptor 77) (NR4A1) and MCP1 (monocyte chemotactic protein 1) (CCL2). Suppression of CaMKIIδ(2) attenuates increased MEF2 DNA-binding activity and up-regulation of Nur77 and MCP1. Finally, we show that HDAC5 is regulated by HDAC4 in VSMCs. Suppression of HDAC4 expression and activity prevents AngII- and PDGF-dependent phosphorylation of HDAC5. Taken together, these results illustrate a mechanism by which CaMKIIδ(2) mediates MEF2-dependent gene transcription in VSMCs through regulation of HDAC4 and HDAC5.

Calcium/Calmodulin-dependent protein kinase II delta 6 (CaMKIIdelta6) and RhoA involvement in thrombin-induced endothelial barrier dysfunction.

Multiple Ca(2+) release and entry mechanisms and potential cytoskeletal targets have been implicated in vascular endothelial barrier dysfunction; however, the immediate downstream effectors of Ca(2+) signals in the regulation of endothelial permeability still remain unclear. In the present study, we evaluated the contribution of multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) as a mediator of thrombin-stimulated increases in human umbilical vein endothelial cell (HUVEC) monolayer permeability. For the first time, we identified the CaMKIIdelta(6) isoform as the predominant CaMKII isoform expressed in endothelium. As little as 2.5 nM thrombin maximally increased CaMKIIdelta(6) activation assessed by Thr(287) autophosphorylation. Electroporation of siRNA targeting endogenous CaMKIIdelta (siCaMKIIdelta) suppressed expression of the kinase by >80% and significantly inhibited 2.5 nM thrombin-induced increases in monolayer permeability assessed by electrical cell-substrate impedance sensing (ECIS). siCaMKIIdelta inhibited 2.5 nM thrombin-induced activation of RhoA, but had no effect on thrombin-induced ERK1/2 activation. Although Rho kinase inhibition strongly suppressed thrombin-induced HUVEC hyperpermeability, inhibiting ERK1/2 activation had no effect. In contrast to previous reports, these results indicate that thrombin-induced ERK1/2 activation in endothelial cells is not mediated by CaMKII and is not involved in endothelial barrier hyperpermeability. Instead, CaMKIIdelta(6) mediates thrombin-induced HUVEC barrier dysfunction through RhoA/Rho kinase as downstream intermediates. Moreover, the relative contribution of the CaMKIIdelta(6)/RhoA pathway(s) diminished with increasing thrombin stimulation, indicating recruitment of alternative signaling pathways mediating endothelial barrier dysfunction, dependent upon thrombin concentration.

Interplay between calcium and reactive oxygen/nitrogen species: an essential paradigm for vascular smooth muscle signaling.

Signaling cascades initiated or regulated by calcium (Ca(2+)), reactive oxygen (ROS), and nitrogen (RNS) species are essential to diverse physiological and pathological processes in vascular smooth muscle. Stimuli-induced changes in intracellular Ca(2+) regulate the activity of primary ROS and RNS, producing enzymes including NADPH oxidases (Nox) and nitric oxide synthases (NOS). At the same time, alteration in intracellular ROS and RNS production reciprocates through redox-based post-translational modifications altering Ca(2+) signaling networks. These may include Ca(2+) pumps such as sarcoplasmic endoplasmic reticulum Ca(2+)-ATPase (SERCA), voltage-gated channels, transient receptor potential canonical (TRPC), melastatin2 (TRPM2), and ankyrin1 (TRPA1) channels, store operated Ca(2+) channels such as Orai1/stromal interaction molecule 1 (STIM1), and Ca(2+) effectors such as Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In this review, we summarize and highlight current experimental evidence supporting the idea that cross-talk between Ca(2+) and ROS/RNS may represent a well-integrated signaling network in vascular smooth muscle.

CaM kinase II delta2-dependent regulation of vascular smooth muscle cell polarization and migration.

Previous studies indicate involvement of the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) in vascular smooth muscle (VSM) cell migration. In the present study, molecular loss-of-function studies were used specifically to assess the role of the predominant CaMKII delta2 isoform on VSM cell migration using a scratch wound healing assay. Targeted CaMKII delta2 knockdown using siRNA or inhibition of activity by overexpressing a kinase-negative mutant resulted in attenuation of VSM cell migration. Temporal and spatial assessments of kinase autophosphorylation indicated rapid and transient activation in response to wounding, in addition to a sustained activation in the leading edge of migrating and spreading cells. Furthermore, siRNA-mediated suppression of CaMKII delta2 resulted in the inhibition of wound-induced Rac activation and Golgi reorganization, and disruption of leading edge morphology, indicating an important function for CaMKII delta2 in regulating VSM cell polarization. Numerous previous reports link activation of CaMKII to ERK1/2 signaling in VSM. Wound-induced ERK1/2 activation was also found to be dependent on CaMKII; however, ERK activity did not account for effects of CaMKII in regulating Golgi polarization, indicating alternative mechanisms by which CaMKII affects the complex events involved in cell migration. Wounding a VSM cell monolayer results in CaMKII delta2 activation, which positively regulates VSM cell polarization and downstream signaling, including Rac and ERK1/2 activation, leading to cell migration.

Regulation of smooth muscle by inducible nitric oxide synthase and NADPH oxidase in vascular proliferative diseases.

Inflammation plays a critical role in promoting smooth muscle migration and proliferation during vascular diseases such as postangioplasty restenosis and atherosclerosis. Another common feature of many vascular diseases is the contribution of reactive oxygen (ROS) and reactive nitrogen (RNS) species to vascular injury. Primary sources of ROS and RNS in smooth muscle are several isoforms of NADPH oxidase (Nox) and the cytokine-regulated inducible nitric oxide (NO) synthase (iNOS). One important example of the interaction between NO and ROS is the reaction of NO with superoxide to yield peroxynitrite, which may contribute to the pathogenesis of hypertension. In this review, we discuss the literature that supports an alternate possibility: Nox-derived ROS modulate NO bioavailability by altering the expression of iNOS. We highlight data showing coexpression of iNOS and Nox in vascular smooth muscle demonstrating the functional consequences of iNOS and Nox during vascular injury. We describe the relevant literature demonstrating that the mitogen-activated protein kinases are important modulators of proinflammatory cytokine-dependent expression of iNOS. A central hypothesis discussed is that ROS-dependent regulation of the serine/threonine kinase protein kinase Cdelta is essential to understanding how Nox may regulate signaling pathways leading to iNOS expression. Overall, the integration of nonphagocytic NADPH oxidase with cytokine signaling in general and in vascular smooth muscle in particular is poorly understood and merits further investigation.

Calcium/calmodulin-dependent protein kinase II-delta isoform regulation of vascular smooth muscle cell proliferation.

There is accumulating evidence that Ca(2+)-dependent signaling pathways regulate proliferation and migration of vascular smooth muscle (VSM) cells, contributing to the intimal accumulation of VSM that is a hallmark of many vascular diseases. In this study we investigated the role of the multifunctional serine/threonine kinase, calmodulin (CaM)-dependent protein kinase II (CaMKII), as a mediator of Ca(2+) signals regulating VSM cell proliferation. Differentiated VSM cells acutely isolated from rat aortic media express primarily CaMKIIgamma gene products, whereas passaged primary cultures of de-differentiated VSM cells express primarily CaMKIIdelta(2), a splice variant of the delta gene. Experiments examining the time course of CaMKII isoform modulation revealed the process was rapid in onset following initial dispersion and primary culture of aortic VSM with a significant increase in CaMKIIdelta(2) protein and a significant decrease in CaMKIIgamma protein within 30 h, coinciding with the onset of DNA synthesis and cell proliferation. Attenuating the initial upregulation of CaMKIIdelta(2) in primary cultured cells using small-interfering RNA (siRNA) resulted in decreased serum-stimulated DNA synthesis and cell proliferation in primary culture. In passaged VSM cells, suppression of CaMKIIdelta(2) activity by overexpression of a kinase-negative mutant, or suppression of endogenous CaMKII content using multiple siRNAs, significantly attenuated serum-stimulated DNA synthesis and cell proliferation. Cell cycle analysis following either inhibitory approach indicated decreased proportion of cells in G1, an increase in proportion of cells in G2/M, and an increase in polyploidy, corresponding with accumulation of multinucleated cells. These results indicate that CaMKIIdelta(2) is specifically induced during modulation of VSM cells to the synthetic phenotypic and is a positive regulator of serum-stimulated proliferation.

Mitochondrial ROS-PKCepsilon signaling axis is uniquely involved in hypoxic increase in [Ca2+]i in pulmonary artery smooth muscle cells.

The molecular mechanisms underlying hypoxic responses in pulmonary and systemic arteries remain obscure. Here we for the first time report that acute hypoxia significantly increased total PKC and PKCepsilon activity in pulmonary, but not mesenteric arteries, while these two tissues showed comparable PKCepsilon protein expression and activation by the PKC activator phorbol 12-myristate 13-acetate. Hypoxia induced an increase in intracellular reactive oxygen species (ROS) generation in isolated pulmonary artery smooth muscle cells (PASMCs), but not in mesenteric artery SMCs. Inhibition of mitochondrial ROS generation with rotenone, myxothiazol, or glutathione peroxidase-1 overexpression prevented hypoxia-induced increases in total PKC and PKCepsilon activity in pulmonary arteries. The inhibitory effects of rotenone were reversed by exogenous hydrogen peroxide. A PKCepsilon translocation peptide inhibitor or PKCepsilon gene deletion decreased hypoxic increase in [Ca(2+)](i) in PASMCs, whereas the conventional PKC inhibitor GO6976 had no effect. These data suggest that acute hypoxia may specifically increase mitochondrial ROS generation, which subsequently activates PKC, particularly PKCepsilon, contributing to hypoxia-induced increase in [Ca(2+)](i) and contraction in PASMCs.