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(Circulation. 2003;107:1539.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Insulin-Stimulated Cyclic Guanosine Monophosphate Inhibits Vascular Smooth Muscle Cell Migration by Inhibiting Ca/Calmodulin-Dependent Protein Kinase II

Sui Zhang, PhD; Yu Yang, MS; Bruce C. Kone, MD; Julius C. Allen, PhD; Andrew M. Kahn, MD

From the Division of Renal Diseases and Hypertension, Department of Medicine, the University of Texas Health Science Center, Houston (A.M.K., Y.Y., B.C.K., S.Z.), and Department of Medicine, Baylor College of Medicine (J.C.A.), Houston, Tex.

Correspondence to Andrew M. Kahn, MD, University of Texas Medical School, 6431 Fannin, MSB 4.107, Houston, TX 77030. E-mail Andrew.M.Kahn{at}uth.tmc.edu

	Abstract

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Background— Insulin resistance is associated with vascular disease. Physiological concentrations of insulin inhibit cultured vascular smooth muscle cell (VSMC) migration in the presence of nitric oxide, and the failure to do so in insulin-resistant states may aggravate vascular disease. We sought to determine the molecular mechanisms by which insulin inhibits VSMC migration.

Methods and Results— Insulin at 1 nmol/L stimulated cGMP production in cultured rat VSMCs that were induced to express inducible nitric oxide synthase (iNOS). VSMC migration was measured in a wound-closure assay, and the platelet-derived growth factor-AB (PDGF-AB)-stimulated component of VSMC migration after wounding was inhibited by insulin, 8-Br-cGMP, and 1-[N-0-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a selective inhibitor of calcium/calmodulin-dependent protein kinase II (CaM kinase II). Wounding alone or incubating cells with only PDGF-AB stimulated CaM kinase II activity in an insulin- and 8-Br-cGMP-inhibitable manner. Transfecting VSMCs with a constitutively active CaM kinase II mutant blocked the inhibition by insulin of both wound-induced and wound plus PDGF-AB-induced VSMC migration. High intracellular Ca²⁺ ([Ca]_i)-stimulated CaM kinase II activity was inhibited by 8-Br-cGMP by an okadaic acid-sensitive mechanism.

Conclusions— We conclude that in cultured rat VSMCs expressing iNOS, insulin, via stimulation of cGMP production, inhibits both wound alone-induced and the PDGF-AB-stimulated component of VSMC migration by inhibiting CaM kinase II activity. cGMP inhibits CaM kinase II at a post-[Ca]_i step by a protein phosphatase-dependent mechanism.

Key Words: insulin • muscle, smooth • migration • calcium • kinases

	Introduction

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Migration of vascular smooth muscle cells (VSMCs) from the arterial media across the internal elastic membrane into the neointima is an important process in the pathogenesis of atherosclerosis and restenosis after balloon catheter angioplasty.^1,2 Increased atherosclerosis and restenosis occur in patients with insulin-resistant states,^3–5 raising the possibility that a functional lack of insulin contributes to increased vascular disease in humans. This hypothesis is consistent with our previous report, which demonstrated that if cultured canine VSMCs had been induced to express inducible nitric oxide synthase (iNOS), insulin stimulated cGMP production, which in turn inhibited cell migration in a wound closure assay.⁶ Others have reported that cGMP inhibits VSMC migration, but the mechanisms are unknown.^7,8

The mechanisms by which wounding stimulates migration of cultured VSMCs have not been determined. When the cells adjacent to the wound are exposed to exogenous stimulators of VSMC migration, such as platelet-derived growth factor (PDGF) or endothelin, the rate of cell migration into the wound is increased.⁹ In the present study, we wished to examine the mechanism for the inhibition by insulin of VSMC migration after wounding.

PDGF is a well-known stimulator of VSMC migration, and it has been demonstrated that migration of rat VSMCs toward an increasing concentration gradient of PDGF is dependent on activation of the multifunctional calcium/calmodulin-dependent protein kinase II (CaM kinase II).^10,11 PDGF induces an intracellular Ca²⁺ ([Ca]_i) transient in VSMCs.¹² The resulting Ca²⁺/calmodulin complex binds to the regulatory domain of CaM kinase II, causing autophosphorylation of threonine 286, which renders the enzyme partially active after [Ca]_i falls (autonomous activity).^12,13 It is not known whether this enzyme is involved in wound alone-stimulated VSMC migration. In the present study, we tested whether insulin inhibits wound alone-induced migration and the PDGF-stimulated component of VSMC migration after wounding by inhibiting autonomous CaM kinase II activity.

	Methods

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Cell Culture
Male Sprague-Dawley rats (Harlan, Indianapolis, Ind) were killed with intraperitoneal pentobarbital sodium, and the thoracic aortas were dissected free. Endothelia and adventitia were stripped away; the media of the arteries were minced, incubated at 37°C in a solution containing elastase (type V, Sigma) and collagenase (type I, Worthington Biochemical); and cultured VSMCs were prepared as previously described.⁶ Cultured cells of passages 3 to 10 were used in experiments. Because a permissive amount of NO is necessary for insulin to stimulate cGMP production in cultured dog and rat VSMCs (Kahn et al¹⁴ and data not shown), most experiments were performed with cells that had been induced to express iNOS as previously described.⁶

cGMP Assay
Dishes of VSMCs were incubated for 30 minutes at 37°C with the desired agents in physiological salt solution containing (in mmol/L) 136 NaCl, 4 KCl, 5 glucose, 1.8 CaCl₂, 0.8 MgSO₄, 10 HEPES-Tris, pH 7.4, plus 0.1% BSA and 0.5 mmol/L isobutylmethylxanthine. The cGMP content of cells was determined by radioimmunoassay as previously described.⁶

Cell Migration
Dishes of cells were washed twice with PBS, and a wound was made through the monolayer with a stylet, producing a cell-free area 0.3 mm wide. One milliliter of serum-free DMEM was placed in the dish, and an initial and subsequent video images were taken. The percentage of initial wound area that had closed at 9 hours was taken as an index of migration, as previously described.⁶

CaM Kinase II Activity
CaM kinase II activity was assessed by measuring the phosphorylation of the specific substrate autocamtide III by [-³²P]ATP as described previously.¹⁵ Autonomous CaM kinase II activity was determined in the presence of 1 mmol/L EGTA and absence of Ca²⁺ and calmodulin. Values for autonomous activity were usually expressed as a percentage of total activity. In each experiment, total activities were not statistically different among the different experimental perturbations, except when cells were stably transfected with a constitutively active CaM kinase II mutant. In that case (Figure 4), the absolute values of autonomous CaM kinase II activities are presented.

View larger version (34K): [in this window] [in a new window]   Figure 4. Effect of insulin and PDGF-AB on CaM kinase II activity and migration of VSMCs transfected with a constitutively active CaM kinase II mutant. Dishes of VSMCs were stably transfected with a constitutively active (CA) mutant for CaM kinase II or vector alone, induced to express iNOS and (A) cells treated with insulin (Ins) and/or PDGF-AB as in Figure 3A. Data presented are mean±SEM of absolute values for autonomous CaM kinase II activities 3 minutes after adding PDGF-AB or vehicle. n=4. *P<0.05 vs vector. **P<0.05 vs vector+PDGF. +P=NS vs CA+PDGF. B, Identically transfected cells were exposed to insulin or vehicle and wounded as in Figure 1. Data presented are mean±SEM of percentage of initial wound area that had closed at 9 hours. n=3. *P<0.05 vs vector, +P=NS vs CA. C, Identically transfected cells were exposed to insulin and/or PDGF-AB and wounded as in Figure 1. Data presented are mean±SEM of percentage of initial wound area that had closed at 9 hours. n=3. *P<0.05 vs vector+PDGF. +P=NS vs CA+PDGF.

View larger version (26K): [in this window] [in a new window]   Figure 3. Effect of PDGF-AB, KN-62, and cGMP on VSMC migration and CaM kinase II activity. A, Dishes of VSMCs were wounded and incubated in presence and absence (Control) of 10 ng/mL PDGF-AB and/or 10 µmol/L K-N62 and/or 50 µmol/L 8-Br-cGMP. Percentage of initial wound area that had closed at 9 hours was calculated and expressed as mean±SEM from 4 separate experiments. *P<0.05 vs control. B, Dishes of VSMCs were preincubated with or without (Control) 10 µmol/L KN-62 or 50 µmol/L 8-Br-cGMP for 30 minutes and then incubated with 10 ng/mL PDGF-AB for 3 additional minutes. Autonomous CaM kinase II activity was calculated as a percentage of total activity and expressed as mean±SEM from 4 separate experiments. Total CaM kinase II activity averaged 3.7 pmol/mg protein per minute. *P<0.05 vs control. +P<0.05 vs PDGF.

View larger version (15K): [in this window] [in a new window]   Figure 1. Effects of insulin and PDGF-AB on VSMC migration. Dishes of VSMCs expressing iNOS were scored with a 0.3-mm linear wound and incubated for 9 hours in absence (control) and presence of 1 nmol/L insulin and/or 10 ng/mL PDGF-AB. Images were taken immediately after wounding and 3, 6, and 9 hours thereafter. Percentage of initial wound area that remained cell-free at subsequent time points was calculated. Data are expressed as mean±SEM from 4 separate experiments. *P<0.05 vs control.

Stable Transfection With Constitutively Active CaM Kinase II Mutant
Subconfluent cultured rat VSMCs (passage 5) were stably transfected as described previously¹⁶ with the plasmids pRC/CMV (as vector control) or with pRC/CMV CACKIIT287D, which contains a constitutively active mutant of CaM kinase II¹⁷ (a gift from Dr. Michael Crow, National Institute on Aging, National Institutes of Health). The cells were incubated with DMEM plus 10% FCS containing G418 (Life Technologies) until individual resistant colonies emerged. They were isolated with sterile cotton buds, cultured in G418 medium, and frozen after 1 to 3 passages, as described previously.¹⁶

Western Blotting
Cells were lysed and protein concentrations measured and adjusted for uniform loading. Eighty to 120 µg protein was loaded in each lane, separated by 10% SDS-PAGE, and transferred electrophoretically onto polyvinylidene difluoride membranes. Subsequent probing of the blots was performed with anti-active CaM kinase II (phosphorylated at thr 286) (Promega) at 1:5000 dilution. Immunoreactive bands were detected with horseradish peroxidase-conjugated secondary antibody and visualized on enhanced chemiluminescence film (Hyperfilm, Amersham) as described previously.⁶ The membrane was stripped, reprobed with anti-ß-actin antibody (Sigma) at 1:2000 dilution, and immunoactive bands were detected as described above.

Bovine insulin, N^G-monomethyl-L-arginine, PDGF-AB, lipopolysaccharide (Escherichia coli 026:B6), recombinant interleukin-1ß, 8-Br-cGMP, leupeptin, pepstatin, aprotinin, and okadaic acid were obtained from Sigma; 1-H-1[1,2,4]oxadiazolo-[4,3-1]quinoxalin-1-one (ODQ) from Tocris Cookson, 1-[N-0-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) from Alexis, autocamtide III and calmodulin from Calbiochem, and [-³²P]ATP from Amersham. Protein was measured by the method of Bradford. Statistical analysis was performed on paired data by use of Student’s t test and ANOVA with multiple comparisons using the Newman-Keuls test. Statistical significance was taken as a value of P<0.05.

	Results

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Effects of Insulin and cGMP on VSMC Migration
To determine whether migration of cultured rat VSMCs expressing iNOS was inhibited by insulin, confluent rat VSMCs were scored by a linear wound in the presence and absence of 10 ng/mL PDGF-AB and/or 1 nmol/L insulin, and closure of the wound (migration) was assayed over a period of 9 hours. Figure 1 shows that after a linear wound, without PDGF-AB, VSMC migration occurred, and PDGF-AB further stimulated migration at 9 hours. Insulin 1 nmol/L inhibited wound-induced migration, and insulin decreased the PDGF-AB-stimulated component of migration at 9 hours from 15.9±2.0% to 6.7±0.7% (n=4, P<0.05). When VSMCs expressing iNOS were incubated for 30 minutes with 1 nmol/L insulin, the basal cGMP level (which averaged 177 fmol/mg protein) was stimulated to 238±14% (P<0.05, n=4). 8-Br-cGMP also inhibited wound alone-induced migration and the PDGF-AB-stimulated component of VSMC migration (see Figure 3A). The insulin-inhibited component of wound plus PDGF-AB-stimulated migration of cultured rat VSMCs was inhibited 86±9% (mean±SEM) by 10 µmol/L ODQ, an inhibitor-soluble guanylate cyclase (n=4, P<0.05).

Effect of Insulin on VSMC CaM Kinase II Activity
To determine whether the inhibition by insulin of PDGF-AB-stimulated rat VSMC migration was dependent on the hormone’s putative inhibition of CaM kinase II activity, we first determined whether insulin inhibits PDGF-AB-stimulated enzyme activity. Cells expressing iNOS were preincubated for 30 minutes with or without insulin, and a time course of PDGF-AB-stimulated CaM kinase II activity was measured. As shown in Figure 2A, PDGF-AB stimulated CaM kinase II activity in a time-dependent fashion. Similar results have been reported for PDGF-BB-stimulated CaM kinase II activity in cultured rat VSMCs.¹⁰ Figure 2A also shows that insulin inhibited PDGF-AB-stimulated activity. 8-Br-cGMP also inhibited PDGF-AB-stimulated CaM kinase II activity (see Figure 3B). A wound alone stimulates migration in an insulin-inhibitable manner in VSMCs expressing iNOS (Figure 1). Figure 2B shows that wounding a confluent dish of cells expressing iNOS with 50 parallel linear wounds stimulated CaM kinase II activity 5-fold after 3 minutes and that insulin or 8-Br-cGMP blocked stimulation of enzyme activity.

View larger version (22K): [in this window] [in a new window]   Figure 2. Inhibition of PDGF-AB- or wound-stimulated autonomous CaM Kinase II activity. A, Dishes of VSMCs expressing iNOS were preincubated with or without 1 nmol/L insulin for 30 minutes and then incubated with or without 10 ng/mL PDGF-AB in absence or continued presence of insulin over a time course of 45 minutes. Autonomous CaM kinase II activity as a percentage of total activity was calculated for each time point and expressed as mean±SEM from 3 separate experiments. Total CaM kinase II activity averaged 2.4 pmol/mg protein per minute. *P<0.05 vs control. +P<0.05 vs PDGF. B, Dishes of VSMCs expressing iNOS were preincubated for 30 minutes without (Control, Wound) or with 1 nmol/L insulin (Ins) or 50 µmol/L 8-Br-cGMP. Cells were left intact (Control) or scored with 50 parallel 0.3-mm wounds. Reaction was stopped 3 minutes after wounding, and autonomous CaM kinase II activity was calculated as a percentage of total activity and expressed as mean±SEM from 5 separate experiments. Total CaM kinase II activity averaged 2.3 pmol/mg protein per minute. *P<0.05 vs control.

Effect of KN-62 and cGMP on VSMC CaM Kinase II Activity and Migration
Figure 3A shows that KN-62, an inhibitor of CaM kinase II activity, inhibited both wound alone-induced migration and the PDGF-AB-stimulated component of VSMC migration. Figure 3A also shows that 8-Br-cGMP inhibited both wound alone-induced migration and the PDGF-AB-stimulated component of VSMC migration. Figure 3B confirms that KN-62 inhibited PDGF-AB-stimulated CaM kinase II activity and shows that 8-Br-cGMP inhibited PDGF-AB-stimulated CaM kinase II activity, as was the case for wound-induced CaM kinase II activity (Figure 2B).

Effect of Transfection With a Constitutively Active CaM Kinase II Mutant on the Inhibition by Insulin of VSMC CaM Kinase II Activity and Migration
The data presented thus far are consistent with the hypothesis that insulin inhibits CaM kinase II activity and thereby inhibits wound alone-induced and wound plus PDGF-AB-induced VSMC migration. To test this hypothesis further, we stably transfected cultured rat VSMCs with a constitutively active CaM kinase II mutant and then induced them to express iNOS to determine whether insulin then failed to inhibit CaM kinase II activity and migration. Figure 4A shows that absolute autonomous CaM kinase II activity in those transfected cells in the absence of any form of stimulation was 10 times higher than cells stably transfected with vector alone, and PDGF-AB did not stimulate enzyme activity further. As also shown, insulin did not inhibit CaM kinase II activity in cells transfected with the constitutively active gene in the presence of PDGF-AB. Figure 4B shows that wound-induced migration of cells transfected with vector alone was inhibited by insulin, that wound-induced migration of cells transfected with constitutively active CaM kinase II was greater than in the vector control cells, and importantly, that insulin had no effect on migration of cells transfected with the constitutively active mutant. All these findings were also obtained for wound plus PDGF-AB-induced migration (Figure 4C). These data demonstrate that insulin cannot inhibit wound alone-induced migration and the PDGF-AB-stimulated component of VSMC migration if CaM kinase II activity cannot be suppressed.

Effect of cGMP on Ionomycin-Stimulated VSMC CaM Kinase II Activity and Threonine 286 Phosphorylation
The inhibition by 8-Br-cGMP of PDGF-AB-stimulated CaM kinase II activity might be a result of attenuation of the PDGF-AB-induced [Ca]_i transient. To test this, [Ca]_i was increased with ionomycin. We have shown previously that ionomycin clamps [Ca]_i at a high level in cultured VSMCs despite the presence of insulin and cGMP.¹⁸ As shown in Figure 5A, ionomycin increased autonomous activity 3-fold at 3 minutes, and preincubating cells with 8-Br-cGMP entirely abrogated that stimulation. This indicated that cGMP blocks Ca²⁺/calmodulin-induced autophosphorylation of CaM kinase II at threonine 286 and/or causes dephosphorylation at that site at a post-[Ca]_i step. As also shown in Figure 5A, preincubating cells with the protein phosphatase inhibitor okadaic acid inhibited block by 8-Br-cGMP of ionomycin-stimulated autonomous CaM kinase II activity. The 8-Br-cGMP-inhibited component of ionomycin-stimulated autonomous activity was inhibited 45±6% (mean±SEM) by 100 nmol/L okadaic acid (n=4, P<0.05). These data suggest that cGMP inhibits autonomous CaM kinase II activity at a post-[Ca]_i step, at least in part, by a protein phosphatase-dependent mechanism. The immunoblot in Figure 5B shows that ionomycin stimulated phosphorylation of CaM kinase II at threonine 286 at 3 minutes, similar to the findings of others,¹⁹ and this effect was inhibited by 8-Br-cGMP. The ß-actin content of cells was the same in all groups, confirming uniform loading. These phosphorylation data confirm the functional data, which support the notion that cGMP inhibits autonomous CaM kinase II activity at a post-[Ca]_i step.

View larger version (40K): [in this window] [in a new window]   Figure 5. Effect of cGMP on ionomycin-stimulated CaM kinase II activity and threonine 286 phosphorylation. A, Dishes of VSMCs were preincubated for 30 minutes without or with 50 µmol/L 8-Br-cGMP and/or 100 nmol/L okadaic acid (OA) and incubated with 2.5 µmol/L ionomycin for 3 additional minutes. Autonomous CaM kinase II activity was calculated as a percentage of total activity and expressed as mean±SEM from 4 separate experiments. Total CaM kinase II activity averaged 1.9 pmol/mg protein per minute. *P<0.05 vs control. +P<0.05 vs ionomycin+OA. B, Dishes of VSMCs were preincubated without or with 50 µmol/L 8-Br-cGMP for 30 minutes and incubated with or without 2.5 µmol/L ionomycin for an additional 3 minutes. Equal amounts of protein from cell lysates were subjected to SDS-PAGE and immunoblotted with anti-phosphorylated (thr 286) CaM kinase II and anti-ß-actin antibodies.

	Discussion

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Atherosclerosis and restenosis of arteries after balloon angioplasty involve migration of VSMCs from the media into the neointima.^1,2 These events are exacerbated in insulin-resistant states.^3–5 It is controversial whether increased plasma insulin concentrations resulting from insulin resistance contributes to vascular pathology.^20,21 Another view is that insulin actually ameliorates vascular disease and that the functional lack of insulin in insulin-resistant states contributes to deleterious VSMC behavior.²⁰ In keeping with this hypothesis, it has been reported that insulin infusion reduces forearm vascular resistance in humans and that this effect is attenuated in insulin-resistant states.^22–24 In addition, we have shown that insulin can inhibit normal cultured canine VSMC contraction and migration.^6,25 The present study was undertaken to determine whether a physiological concentration of insulin affected cultured rat VSMC migration, and if so, how.

We reported previously that 1 nmol/L insulin stimulated cGMP production in cultured canine VSMCs that had been induced to express iNOS.⁶ Insulin did not stimulate iNOS activity¹⁴ but required the permissive presence of NO, derived from iNOS or an exogenous source, to stimulate guanylate cyclase activity.¹⁴ We reported that insulin inhibited wound-induced cultured canine VSMC migration via the stimulation by the hormone of cGMP production.⁶ In the present study, we sought to determine whether the same was true for cultured rat VSMCs and whether the known increased rate of VSMC migration caused by adding PDGF-AB to a dish of freshly wounded cells was also inhibited by insulin and cGMP. In the present study, we demonstrated that 1 nmol/L insulin stimulated cGMP production in cultured rat VSMCs expressing iNOS and that insulin, via increasing cGMP, inhibited both wound alone-induced migration and the PDGF-AB-stimulated component of migration of these cells. Insulin 1 nmol/L (130 µU/mL) is a physiological concentration in rats,²⁶ dogs,²⁷ and humans.²⁸ We have previously performed insulin dose-response studies in cultured VSMCs expressing iNOS using similar concentrations of insulin.²⁵

We found that PDGF-AB or linear wounds, which alone initiate cell migration, stimulate CaM kinase II activity. PDGF-AB-stimulated CaM kinase II activity and wound alone-stimulated enzyme activity was blocked by insulin or cGMP. These data link insulin- or cGMP-inhibited VSMC CaM kinase II activity and insulin- or cGMP-inhibited migration. We demonstrated that the inhibition by insulin of wound-alone-induced migration and wound plus PDGF-AB-stimulated migration is mediated by the inhibition by the hormone of CaM kinase II activity by showing that insulin did not affect migration of cells stably transfected with a constitutively active CaM kinase II mutant.

The present data show that cGMP blocks stimulation of autonomous CaM kinase II activity at a post-[Ca]_i step and indicate that this effect is mediated, at least in part, by a protein phosphatase-dependent mechanism. It is possible that cGMP activates a protein phosphatase that dephosphorylates the enzyme at threonine 286, thus reducing autonomous activity. It has been reported that cGMP stimulates protein phosphatase activities in VSMCs and other cell types.^29–31 The finding that insulin did not inhibit CaM kinase II activity in cells transfected with the constitutively active CaM kinase II mutant is in keeping with this conclusion, because this mutant does not require phosphorylation to confer autonomous activity.¹⁷

The present study may have clinical relevance. VSMCs in atherosclerotic lesions or adjacent to areas subjected to balloon injury acquire iNOS.^32,33 We show that insulin, by stimulating cGMP production, inhibits CaM kinase II and thus migration in VSMCs with iNOS. The failure of insulin to do so in vivo in insulin-resistant states may underlie, at least in part, the vascular disease seen in those clinical conditions. Strategies to selectively inhibit arterial CaM kinase II in insulin-resistant states might offer clinical benefit.

	Acknowledgments

 
These studies were supported by grants HL-50660 (Dr Kahn), HL-24585 (Dr Allen), and DK-50745 (Dr Kone) from the National Institutes of Health and Grant-in-Aid 0150013N from the American Heart Association (Dr Kahn).

	Footnotes

 
Guest Editor for this article was Marschall S. Runge, MD, PhD, University of North Carolina.

Received July 12, 2002; revision received November 5, 2002; accepted December 4, 2002.

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