Blocking Interferon β Stimulates Vascular Smooth Muscle Cell Proliferation and Arteriogenesis

Increased interferon (IFN)-β signaling in patients with insufficient coronary collateralization and an inhibitory effect of IFNβ on collateral artery growth in mice have been reported. The mechanisms of IFNβ-induced inhibition of arteriogenesis are unknown. In stimulated monocytes from patients with chronic total coronary artery occlusion and decreased arteriogenic response, whole genome expression analysis showed increased expression of IFNβ-regulated genes. Immunohistochemically, the IFNβ receptor was localized in the vascular media of murine collateral arteries. Treatment of vascular smooth muscle cells (VSMC) with IFNβ resulted in an attenuated proliferation, cell-cycle arrest, and increased expression of cyclin-dependent kinase inhibitor-1A (p21). The growth inhibitory effect of IFNβ was attenuated by inhibition of p21 by RNA interference. IFNβ-treated THP1 monocytes showed enhanced apoptosis. Subsequently, we tested if collateral artery growth can be stimulated by inhibition of IFNβ-signaling. RNA interference of the IFNβ receptor-1 (IFNAR1) increased VSMC proliferation, cell cycle progression, and reduced p21 gene expression. IFNβ signaling and FAS and TRAIL expression were attenuated in monocytes from IFNAR1^−/− mice, indicating reduced monocyte apoptosis. Hindlimb perfusion restoration 1 week after femoral artery ligation was improved in IFNAR1^−/− mice compared with wild-type mice as assessed by infusion of fluorescent microspheres. These results demonstrate that IFNβ inhibits collateral artery growth and VSMC proliferation through p21-dependent cell cycle arrest and induction of monocyte apoptosis. Inhibition of IFNβ stimulates VSMC proliferation and collateral artery growth.     

Rhinovirus and dsRNA Induce RIG-I-Like Receptors and Expression of Interferon β and λ1 in Human Bronchial Smooth Muscle Cells

Rhinovirus (RV) infections cause exacerbations and development of severe asthma highlighting the importance of antiviral interferon (IFN) defence by airway cells. Little is known about bronchial smooth muscle cell (BSMC) production of IFNs and whether BSMCs have dsRNA-sensing receptors besides TLR3. dsRNA is a rhinoviral replication intermediate and necrotic cell effect mimic that mediates innate immune responses in bronchial epithelial cells. We have explored dsRNA-evoked IFN-β and IFN-λ₁ production in human BSMCs and potential involvement of TLR3 and RIG-I-like receptors (RLRs). Primary BSMCs were stimulated with 0.1–10 µg/ml dsRNA, 0.1–1 µg/ml dsRNA in complex with the transfection agent LyoVec (dsRNA/LyoVec; selectively activating cytosolic RLRs) or infected with 0.05–0.5 MOI RV1B. Both dsRNA stimuli evoked early (3 h), concentration-dependent IFN-β and IFN-λ₁ mRNA expression, which with dsRNA/LyoVec was much greater, and with dsRNA was much less, after 24 h. The effects were inhibited by dexamethasone. Further, dsRNA and dsRNA/LyoVec concentration-dependently upregulated RIG-I and MDA5 mRNA and protein. dsRNA and particularly dsRNA/LyoVec caused IFN-β and IFN-λ₁ protein production (24 h). dsRNA- but not dsRNA/LyoVec-induced IFN expression was partly inhibited by chloroquine that suppresses endosomal TLR3 activation. RV1B dose-dependently increased BSMC expression of RIG-I, MDA5, IFN-β, and IFN-λ₁ mRNA. We suggest that BSMCs express functional RLRs and that both RLRs and TLR3 are involved in viral stimulus-induced BSMC expression of IFN-β and IFN-λ₁.     

Effects of Pyridoxal 5′-Phosphate on Synaptic Transmission in Smooth Muscle

We studied the influence of the vitamin B₆ form most extensively distributed in the organism, pyridoxal 5-phosphate (PyrP), on neuromuscular transmission in the smooth muscle of the circular layer of the guinea pig distal colon and of the ileum and an initial segment of the jejunum of humans. Application of 10^-10 to 10^-3 M PyrP reversibly and in a dose-dependent manner decreased the amplitude of non-cholinergic non-adrenergic inhibitory synaptic potentials (ISP) and increased their duration. Under the influence of 10^-8 to 10^-4 M PyrP, both the amplitude and duration of ATP- and noradrenaline-induced hyperpolarizations increased. Application of 10^-4 M PyrP completely suppressed the sensitivity of smooth muscle cells to noradrenaline, but a hyperpolarizing effect of exogenous ATP was preserved. The PyrP-induced amplitude decrease and prolongation of ISP were preserved in the presence of 10^-4 M hexonium (a ganglioblocker), 5 · 10^-7 M apamin (a blocker of Ca²⁺-dependent K⁺ channels of small conductance), 10^-5 M verapamil (a blocker of L-type Ca²⁺ channels), and 10^-4 M N-nitro-L-arginine (a blocker of NO-synthase). It seems probable that a decrease in the ISP amplitude is related to a presynaptic PyrP effect. Under conditions of PyrP-induced suppression of non-cholinergic non-adrenergic inhibition, non-cholinergic short-latency excitatory synaptic potentials could be recorded in smooth muscle. Thus, PyrP is an effective modulator of synaptic transmission in smooth muscle of the gastrointestinal tract of mammals.     

Cytochemical Detection of a Senescence-Associated β-Galactosidase in Endothelial and Smooth Muscle Cells from Human and Rabbit Blood Vessels

A β-galactosidase activity has recently been used as a histochemical marker of replicative senescence in human fibroblasts and keratinocytes. To establish whether this marker could be used to detect senescence of vascular cells, we have investigated its presence in cultures of serially passaged human umbilical vein endothelial cells and rabbit aortic smooth muscle cells. β-Galactosidase activity was detected by light microscopy using the chromogenic substrate 5-bromo-4-chloro-3-indolyl β- -galactopyranoside. In endothelial cell cultures, lysosomal β-galactosidase activity, which is detected at pH 4.0, was present in all cells regardless of their replicative age. In contrast, senescence-associated β-galactosidase activity, which is detected at pH 6.0, was absent in the majority of cells in early passage cultures (<15 cumulative population doublings), but was present in a large proportion of cells (up to 62%) in late passage cultures (>30 cumulative population doublings); in intermediate passage cultures (15–30 cumulative population doublings) it was found in fewer than 15% of the cells. The increase in the percentage of senescence-associated β-galactosidase-positive cells correlated with a decrease in the cell density at confluence and with a marked increase in cell size. Counterstaining with an antibody directed against the endothelial cell marker CD31 showed that senescent cells retained the expression of this antigen. Senescence-associated β-galactosidase was also detected in serially passaged, but not in primary explant cultures of rabbit aortic vascular smooth muscle cells. The presence of senescence-associated β-galactosidase in cultured vascular smooth muscle cells and endothelial cells suggests that this marker could be used to study the role of cellular senescence in vascular disease.     

Tungstate-Targeting of BKαβ1 Channels Tunes ERK Phosphorylation and Cell Proliferation in Human Vascular Smooth Muscle

Despite the substantial knowledge on the antidiabetic, antiobesity and antihypertensive actions of tungstate, information on its primary target/s is scarce. Tungstate activates both the ERK1/2 pathway and the vascular voltage- and Ca²⁺-dependent large-conductance BKαβ₁ potassium channel, which modulates vascular smooth muscle cell (VSMC) proliferation and function, respectively. Here, we have assessed the possible involvement of BKαβ₁ channels in the tungstate-induced ERK phosphorylation and its relevance for VSMC proliferation. Western blot analysis in HEK cell lines showed that expression of vascular BKαβ₁ channels potentiates the tungstate-induced ERK1/2 phosphorylation in a G_i/o protein-dependent manner. Tungstate activated BKαβ₁ channels upstream of G proteins as channel activation was not altered by the inhibition of G proteins with GDPβS or pertussis toxin. Moreover, analysis of G_i/o protein activation measuring the FRET among heterologously expressed G_i protein subunits suggested that tungstate-targeting of BKαβ₁ channels promotes G protein activation. Single channel recordings on VSMCs from wild-type and β₁-knockout mice indicated that the presence of the regulatory β₁ subunit was essential for the tungstate-mediated activation of BK channels in VSMCs. Moreover, the specific BK channel blocker iberiotoxin lowered tungstate-induced ERK phosphorylation by 55% and partially reverted (by 51%) the tungstate-produced reduction of platelet-derived growth factor (PDGF)-induced proliferation in human VSMCs. Our observations indicate that tungstate-targeting of BKαβ₁ channels promotes activation of PTX-sensitive G_i proteins to enhance the tungstate-induced phosphorylation of ERK, and inhibits PDGF-stimulated cell proliferation in human vascular smooth muscle.     

Role of Vascular Smooth Muscle PPARγ in Regulating AT1 Receptor Signaling and Angiotensin II-Dependent Hypertension

Peroxisome proliferator activated receptor γ (PPARγ) has been reported to play a protective role in the vasculature; however, the underlying mechanisms involved are not entirely known. We previously showed that vascular smooth muscle-specific overexpression of a dominant negative human PPARγ mutation in mice (S-P467L) leads to enhanced myogenic tone and increased angiotensin-II-dependent vasoconstriction. S-P467L mice also exhibit increased arterial blood pressure. Here we tested the hypotheses that a) mesenteric smooth muscle cells isolated from S-P467L mice exhibit enhanced angiotensin-II AT₁ receptor signaling, and b) the increased arterial pressure of S-P467L mice is angiotensin-II AT₁ receptor dependent. Phosphorylation of mitogen-activated protein/extracellular signal-regulated kinase (ERK1/2) was robustly increased in mesenteric artery smooth muscle cell cultures from S-P467L in response to angiotensin-II. The increase in ERK1/2 activation by angiotensin-II was blocked by losartan, a blocker of AT₁ receptors. Angiotensin-II-induced ERK1/2 activation was also blocked by Tempol, a scavenger of reactive oxygen species, and correlated with increased Nox4 protein expression. To investigate whether endogenous renin-angiotensin system activity contributes to the elevated arterial pressure in S-P467L, non-transgenic and S-P467L mice were treated with the AT₁ receptor blocker, losartan (30 mg/kg per day), for 14-days and arterial pressure was assessed by radiotelemetry. At baseline S-P467L mice showed a significant increase of systolic arterial pressure (142.0±10.2 vs 129.1±3.0 mmHg, p<0.05). Treatment with losartan lowered systolic arterial pressure in S-P467L (132.2±6.9 mmHg) to a level similar to untreated non-transgenic mice. Losartan also lowered arterial pressure in non-transgenic (113.0±3.9 mmHg) mice, such that there was no difference in the losartan-induced depressor response between groups (−13.53±1.39 in S-P467L vs −16.16±3.14 mmHg in non-transgenic). Our results suggest that interference with PPARγ in smooth muscle: a) causes enhanced angiotensin-II AT₁ receptor-mediated ERK1/2 activation in resistance vessels, b) and may elevate arterial pressure through both angiotensin-II AT₁ receptor-dependent and -independent mechanisms.     

PGC-1α Is a Key Regulator of Glucose-Induced Proliferation and Migration in Vascular Smooth Muscle Cells

Background

Atherosclerosis is a complex pathological condition caused by a number of mechanisms including the accelerated proliferation of vascular smooth muscle cells (VSMCs). Diabetes is likely to be an important risk factor for atherosclerosis, as hyperglycemia induces vascular smooth muscle cell (VSMC) proliferation and migration and may thus contribute to the formation of atherosclerotic lesions. This study was performed to investigate whether PGC-1α, a PPARγ coactivator and metabolic master regulator, plays a role in regulating VSMC proliferation and migration induced by high glucose.

Methodology/Principal Findings

PGC-1α mRNA levels are decreased in blood vessel media of STZ-treated diabetic rats. In cultured rat VSMCs, high glucose dose-dependently inhibits PGC-1α mRNA expression. Overexpression of PGC-1α either by infection with adenovirus, or by stimulation with palmitic acid, significantly reduces high glucose-induced VSMC proliferation and migration. In contrast, suppression of PGC-1α by siRNA mimics the effects of glucose on VSMCs. Finally, mechanistic studies suggest that PGC-1α-mediated inhibition of VSMC proliferation and migration is regulated through preventing ERK1/2 phosphorylation.

Conclusions/Significance

These results indicate that PGC-1α is a key regulator of high glucose-induced proliferation and migration in VSMCs, and suggest that elevation of PGC-1α in VSMC could be a useful strategy in preventing the development of diabetic atherosclerosis.     

Role of TGF-β1 and MAP Kinases in the Antiproliferative Effect of Aspirin in Human Vascular Smooth Muscle Cells

Background

We aimed to test the antiproliferative effect of acetylsalicylic acid (ASA) on vascular smooth muscle cells (VSMC) from bypass surgery patients and the role of transforming growth factor beta 1 (TGF-β1).

Methodology/Principal Findings

VSMC were isolated from remaining internal mammary artery from patients who underwent bypass surgery. Cell proliferation and DNA fragmentation were assessed by ELISA. Protein expression was assessed by Western blot. ASA inhibited BrdU incorporation at 2 mM. Anti-TGF-β1 was able to reverse this effect. ASA (2 mM) induced TGF-β1 secretion; however it was unable to induce Smad activation. ASA increased p38^MAPK phosphorylation in a TGF-β1-independent manner. Anti-CD105 (endoglin) was unable to reverse the antiproliferative effect of ASA. Pre-surgical serum levels of TGF-β1 in patients who took at antiplatelet doses ASA were assessed by ELISA and remained unchanged.

Conclusions/Significance

In vitro antiproliferative effects of aspirin (at antiinflammatory concentration) on human VSMC obtained from bypass patients are mediated by TGF-β1 and p38^MAPK. Pre-surgical serum levels of TGF- β1 from bypass patients who took aspirin at antiplatelet doses did not change.     

Angiotensin II Type I and Prostaglandin F2α Receptors Cooperatively Modulate Signaling in Vascular Smooth Muscle Cells

The angiotensin II type I (AT1R) and the prostaglandin F2α (PGF2α) F prostanoid (FP) receptors are both potent regulators of blood pressure. Physiological interplay between AT1R and FP has been described. Abdominal aortic ring contraction experiments revealed that PGF2α-dependent activation of FP potentiated angiotensin II-induced contraction, whereas FP antagonists had the opposite effect. Similarly, PGF2α-mediated vasoconstriction was symmetrically regulated by co-treatment with AT1R agonist and antagonist. The underlying canonical Gα_q signaling via production of inositol phosphates mediated by each receptor was also regulated by antagonists for the other receptor. However, binding to their respective agonists, regulation of receptor-mediated MAPK activation and vascular smooth muscle cell growth were differentially or asymmetrically regulated depending on how each of the two receptors were occupied by either agonist or antagonist. Physical interactions between these receptors have never been reported, and here we show that AT1R and FP form heterodimeric complexes in both HEK 293 and vascular smooth muscle cells. These findings imply that formation of the AT1R/FP dimer creates a novel allosteric signaling unit that shows symmetrical and asymmetrical signaling behavior, depending on the outcome measured. AT1R/FP dimers may thus be important in the regulation of blood pressure.     

Defective Resensitization in Human Airway Smooth Muscle Cells Evokes β-Adrenergic Receptor Dysfunction in Severe Asthma

β₂-adrenergic receptor (β₂AR) agonists (β₂-agonist) are the most commonly used therapy for acute relief in asthma, but chronic use of these bronchodilators paradoxically exacerbates airway hyper-responsiveness. Activation of βARs by β-agonist leads to desensitization (inactivation) by phosphorylation through G-protein coupled receptor kinases (GRKs) which mediate β-arrestin binding and βAR internalization. Resensitization occurs by dephosphorylation of the endosomal βARs which recycle back to the plasma membrane as agonist-ready receptors. To determine whether the loss in β-agonist response in asthma is due to altered βAR desensitization and/or resensitization, we used primary human airway smooth muscle cells (HASMCs) isolated from the lungs of non-asthmatic and fatal-asthmatic subjects. Asthmatic HASMCs have diminished adenylyl cyclase activity and cAMP response to β-agonist as compared to non-asthmatic HASMCs. Confocal microscopy showed significant accumulation of phosphorylated β₂ARs in asthmatic HASMCs. Systematic analysis of desensitization components including GRKs and β-arrestin showed no appreciable differences between asthmatic and non-asthmatic HASMCs. However, asthmatic HASMC showed significant increase in PI3Kγ activity and was associated with reduction in PP2A activity. Since reduction in PP2A activity could alter receptor resensitization, endosomal fractions were isolated to assess the agonist ready β₂ARs as a measure of resensitization. Despite significant accumulation of β₂ARs in the endosomes of asthmatic HASMCs, endosomal β₂ARs cannot robustly activate adenylyl cyclase. Furthermore, endosomes from asthmatic HASMCs are associated with significant increase in PI3Kγ and reduced PP2A activity that inhibits β₂AR resensitization. Our study shows that resensitization, a process considered to be a homeostasis maintaining passive process is inhibited in asthmatic HASMCs contributing to β₂AR dysfunction which may underlie asthma pathophysiology and loss in asthma control.     

Smooth Muscle α Actin (Acta2) and Myofibroblast Function during Hepatic Wound Healing

Smooth muscle α actin (Acta2) expression is largely restricted to smooth muscle cells, pericytes and specialized fibroblasts, known as myofibroblasts. Liver injury, associated with cirrhosis, induces transformation of resident hepatic stellate cells into liver specific myofibroblasts, also known as activated cells. Here, we have used in vitro and in vivo wound healing models to explore the functional role of Acta2 in this transformation. Acta2 was abundant in activated cells isolated from injured livers but was undetectable in quiescent cells isolated from normal livers. Both cellular motility and contraction were dramatically increased in injured liver cells, paralleled by an increase in Acta2 expression, when compared with quiescent cells. Inhibition of Acta2 using several different techniques had no effect on cytoplasmic actin isoform expression, but led to reduced cellular motility and contraction. Additionally, Acta2 knockdown was associated with a significant reduction in Erk1/2 phosphorylation compared to control cells. The data indicate that Acta2 is important specifically in myofibroblast cell motility and contraction and raise the possibility that the Acta2 cytoskeleton, beyond its structural importance in the cell, could be important in regulating signaling processes during wound healing in vivo.     

Dual Regulation of Myocardin Expression by Tumor Necrosis Factor-α in Vascular Smooth Muscle Cells

De-differentiation of vascular smooth muscle cells (VSMCs) plays a critical role in the development of atherosclerosis, a chronic inflammatory disease involving various cytokines such as tumor necrosis factor-α (TNFα). Myocardin is a co-factor of serum response factor (SRF) and is considered to be the master regulator of VSMC differentiation. It binds to SRF and regulates the expression of contractile proteins in VSMCs. Myocardin is also known to inhibit VSMC proliferation by inhibiting the NF-κB pathway, whereas TNFα is known to activate the NF-κB pathway in VSMCs. NF-κB activation has also been shown to inhibit myocardin expression and smooth muscle contractile marker genes. However, it is not definitively known whether TNFα regulates the expression and activity of myocardin in VSMCs. The current study aimed to investigate the role of TNFα in regulating myocardin and VSMC function. Our studies showed that TNFα down-regulated myocardin expression and activity in cultured VSMCs by activating the NF-κB pathway, resulting in decreased VSMC contractility and increased VSMC proliferation. Surprisingly, we also found that TNFα prevented myocardin mRNA degradation, and resulted in a further significant increase in myocardin expression and activity in differentiated VSMCs. Both the NF-κB and p44/42 MAPK pathways were involved in TNFα regulation of myocardin, which further increased the contractility of VSMCs. These differential effects of TNFα on myocardin seemingly depended on whether VSMCs were in a differentiated or de-differentiated state. Taken together, our results demonstrate that TNFα differentially regulates myocardin expression and activity, which may play a key role in regulating VSMC functions.     

Characteristics of an Adenylate Cyclase Coupled β-Adrenergic Receptor in a Smooth Muscle Tumor Cell Line

Abstract

We have shown that binding of ³H-dihydroalprenolol ([³H] DHA) to DDT₁ MF-2 cells and cell membranes was of high affinity, saturable, stereoselective and reversible. The [³H]DHA dissociation constants were 0.63 ± 0.15 nM (n=6) and 0.83 ± 0.04 nM (n=5) for intact cells and cell membranes, respectively, with a binding site concentration for cells of 27,300 ± 5,200 sites/ cell (n=6) and for membranes 468 ± 24 fmoles/mg protein (n=5). The order of agonist competition for the [³H]-DHA binding site of DDT₁ cell membranes was isoproterenol (K_i = 0.20 ± 0.07 μM) > epinephrine (K_i = 0.4 ± 0.2 μM) > norepinephrine (K_i = 66.5 ± 5.15 μM) consistent with a β₂-selective receptor interaction. Zinterol, a β₂-selective antagonist, (K_i = 0.05 ± 0.01 μM) was 18x more effective than metoprolol, a β₁-selective antagonist (K_i = 0.9 ± 0.1 μM), in competing for the DHA binding site. A nonlinear iterative curve fitting analysis of zinterol and metoprolol binding isotherms indicated that (p>0.05) DDT₁ cells possess a pure population of β₂-adrenergic receptors. Finally, we have shown that DDT₁ MF-2 cell β₂-adrenergic receptor is functionally coupled to adenylate cyclase via a G/F protein complex as demonstrated in part by a guanine nucleotide requirement for isoproterenol stimulation of adenylate cyclase activity. In addition, guanine nucleotide mediated a reduction in the affinities of isoproterenol and epinephrine for the [³H]DHA binding site.     

Contribution of Intestinal Smooth Muscle to Crohn’s Disease Fibrogenesis

Mesenchymal cells transdifferentiation and extracellular matrix deposition are involved in the fibrotic process of Crohn’s disease (CD). Mesenchymal smooth muscle cells (SMCs) de-differentiation, driven by Platelet-derived growth factor (PDGF) that counteracts Transforming growth factor (TGF-β) has been studied in vascular muscle. The role of SMCs in intestinal fibrogenesis is still not clearly elucidated. Aim of the study was to evaluate the possible myogenic contribution to CD fibrotic process through the comparative analysis of histological, morphometric and molecular alterations occurring in human smooth muscle. Full thickness specimens were obtained from CD (non-involved and stenotic tracts) and healthy (control) ileum. Tissues were processed for histological and immunohistochemical (IHC) analyses and SMCs were isolated from the muscularis propria for morphofunctional and molecular (qPCR) analyses. CD stenotic ileum showed a significant increased thickness of all layers compared to CD non-involved and control ileum. IHC revealed an overexpression of α-smooth muscle actin and collagens I-III throughout all intestinal layers only in stenotic tracts. The two growth factors, PDGF and TGF-β, showed a progressive increase in expression in the muscle layer from CD non-involved to stenotic tracts. Freshly isolated SMCs presented alterations in CD non-involved tracts that progressively increased in the stenotic tracts consisting in a statistical increase in mRNA encoding for PDGF-β and collagen III, paralleled to a decrease in TGF-β and Tribbles-like protein-3 mRNA, and altered morphofunctional parameters consisting in progressive decreases in cell length and contraction to acetylcholine. These findings indicate that intrinsic myogenic alterations occur in CD ileum, that they likely precede stricture formation, and might represent suitable new targets for anti-fibrotic interventions.Key words: Fibrosis, Crohn’s disease, ileal smooth muscle cells, stricture formation, PDGF, TGF-β