Rationale: Neointimal hyperplasia is characterized by excessive accumulation of vascular smooth muscle cells (SMCs) leading to occlusive disorders, such as atherosclerosis and stenosis. Blood vessel injury increases growth factor secretion and matrix synthesis, which promotes SMC proliferation and neointimal hyperplasia via FAK (focal adhesion kinase). Objective: To understand the mechanism of FAK action in SMC proliferation and neointimal hyperplasia. Methods and Results: Using combined pharmacological FAK catalytic inhibition (VS-4718) and SMC-specific FAK kinase-dead (Myh11-Cre-ER T2 ) mouse models, we report that FAK regulates SMC proliferation and neointimal hyperplasia in part by governing GATA4- (GATA-binding protein 4) cyclin D1 signaling. Inhibition of FAK catalytic activity facilitates FAK nuclear localization, which is required for proteasome-mediated GATA4 degradation in the cytoplasm. Chromatin immunoprecipitation identified GATA4 binding to the mouse cyclin D1 promoter, and loss of GATA4-mediated cyclin D1 transcription diminished SMC proliferation. Stimulation with platelet-derived growth factor or serum activated FAK and redistributed FAK from the nucleus to cytoplasm, leading to concomitant increase in GATA4 protein and cyclin D1 expression. In a femoral artery wire injury model, increased neointimal hyperplasia was observed in parallel with elevated FAK activity, GATA4 and cyclin D1 expression following injury in control mice, but not in VS-4718-treated and SMC-specific FAK kinase-dead mice. Finally, lentiviral shGATA4 knockdown in the wire injury significantly reduced cyclin D1 expression, SMC proliferation, and neointimal hyperplasia compared with control mice. Conclusions: Nuclear enrichment of FAK by inhibition of FAK catalytic activity during vessel injury blocks SMC proliferation and neointimal hyperplasia through regulation of GATA4-mediated cyclin D1 transcription.

Homeostatic control of vascular smooth muscle cell (VSMC) differentiation is critical for contractile activity and regulation of blood flow. Recently, we reported that precontracted blood vessels are relaxed and the phenotype of VSMC is regulated from a synthetic to contractile state by glucose-6-phosphate dehydrogenase (G6PD) inhibition. In the current study, we investigated whether the increase in the expression of VSMC contractile proteins by inhibition and knockdown of G6PD is mediated through a protein kinase G (PKG)-dependent pathway and whether it regulates blood pressure. We found that the expression of VSMC-restricted contractile proteins, myocardin (MYOCD), and miR-1 and miR-143 are increased by G6PD inhibition or knockdown. Importantly, RNA-sequence analysis of aortic tissue from G6PD-deficient mice revealed uniform increases in VSMC-restricted genes, particularly those regulated by the MYOCD-serum response factor (SRF) switch. Conversely, expression of Krüppel-like factor 4 (KLF4) is decreased by G6PD inhibition. Interestingly, the G6PD inhibition-induced expression of miR-1 and contractile proteins was blocked by Rp-β-phenyl-1,N2-etheno-8-bromo-guanosine-3',5'-cyclic monophosphorothioate, a PKG inhibitor. On the other hand, MYOCD and miR-143 levels are increased by G6PD inhibition through a PKG-independent manner. Furthermore, blood pressure was lower in the G6PD-deficient compared with wild-type mice. Therefore, our results suggest that the expression of VSMC contractile proteins induced by G6PD inhibition occurs via PKG1α-dependent and -independent pathways.

The H+-translocating ATPase located on vacuolar membranes of Neurospora crassa was partially puri- fied by solubilization in two detergents, Triton X-100 and N-hexadecyl-N,N-dimethyl-3-ammonio-l-propanesulfonate, followed by centrifugation on sucrose density gradients. Two polypeptides of Mr =70,000 and t62,000 consistently migrated with activity, along with several minor bands of lower molec- ular weight. Radioactively labeled inhibitors of ATPase activ- ity, N-(l4C)ethylmaleimide and 7-chloro-4-nitro(14C)benzo-2- oxa-l,3-diazole, labeled the Mr =70,000 polypeptide; this labeling was reduced in the presence of ATP. N,N'-(l4C)di- cyclohexylcarbodiimide labeled a polypeptide of Myr 15,000. Estimation of the functional size of the vacuolar membrane ATPase by radiation inactivation gave a value of Mr 5.2 x 10s, 10-15% larger than the mitochondrial ATPase. The Neurospo- ra vacuolar ATPase showed no crossreactivity with anti- serum to plasma membrane or mitochondrial ATPase but strongly crossreacted with antiserum against a polypeptide of Mr ?70,000 associated with the tonoplast ATPase of corn coleoptiles. These results suggest that fungal and plant vacuolar ATPases may be large multisubunit complexes, somewhat similar to, but immunologically distinct from, known FoFi ATPases.

The low-voltage-activated T-type Ca(2+) channels play an important role in mediating the cellular responses to altered oxygen tension. Among three T-type channel isoforms, α1G, α1H, and α1I, only α1H was found to be upregulated under hypoxia. However, mechanisms underlying such hypoxia-dependent isoform-specific gene regulation remain incompletely understood. We, therefore, studied the hypoxia-dependent transcriptional regulation of α1G and α1H gene promoters with the aim to identify the functional hypoxia-response elements (HREs). In rat pulmonary artery smooth muscle cells (PASMCs) and pheochromocytoma (PC12) cells after hypoxia (3% O2) exposure, we observed a prominent increase in α1H mRNA at 12 h along with a significant rise in α1H-mediated T-type current at 24 and 48 h. We then cloned two promoter fragments from the 5'-flanking regions of rat α1G and α1H gene, 2,000 and 3,076 bp, respectively, and inserted these fragments into a luciferase reporter vector. Transient transfection of PASMCs and PC12 cells with these recombinant constructs and subsequent luciferase assay revealed a significant increase in luciferase activity from the reporter containing the α1H, but not α1G, promoter fragment under hypoxia. Using serial deletion and point mutation analysis strategies, we identified a functional HRE at site -1,173cacgc-1,169 within the α1H promoter region. Furthermore, an electrophoretic mobility shift assay using this site as a DNA probe demonstrated an increased binding activity to nuclear protein extracts from the cells after hypoxia exposure. Taken together, these findings indicate that hypoxia-induced α1H upregulation involves binding of hypoxia-inducible factor to an HRE within the α1H promoter region.

Objective— Transient, repetitive occlusion stimulates coronary collateral growth (CCG) in normal animals. Vascular smooth muscle cells (VSMCs) switch to synthetic phenotype early in CCG, then return to contractile phenotype. CCG is impaired in the metabolic syndrome. We determined whether impaired CCG was attributable to aberrant VSMC phenotypic modulation by miR-145–mediated mechanisms, and whether restoration of physiological miR-145 levels in metabolic syndrome (JCR rat) improved CCG. Approach and Results— CCG was stimulated by transient, repetitive left anterior descending artery occlusion and evaluated after 9 days by coronary blood flow measurements (microspheres). miR-145 was delivered to JCR VSMCs via adenoviral vector (miR-145-Adv). In JCR rats, miR-145 was decreased late in CCG (≈2-fold day 6; ≈4-fold day 9 versus SD), which correlated with decreased expression of smooth muscle-specific contractile proteins (≈5-fold day 6; ≈10-fold day 9 versus SD), indicative of VSMCs’ failure to return to the contractile phenotype late in CCG. miR-145 expression in JCR rats (miR-145-Adv) on days 6 to 9 of CCG completely restored VSMCs contractile phenotype and CCG (collateral/normal zone flow ratio was 0.93±0.09 JCR+miR-145-Adv versus 0.12±0.02 JCR versus 0.87±0.02 SD). Conclusions— Restoration of VSMC contractile phenotype through miR-145 delivery is a highly promising intervention for restoration of CCG in the metabolic syndrome.

In pulmonary microvascular endothelial cells (PMVECs), Ca2+ entry through the site‐specific, voltage‐gated α1G T‐type Ca2+ channel promotes vWF release and P‐selectin surface expression. The present study seeks to determine whether α1G directly interacts with endothelial nitric oxide synthase (NOS3) and controls its activation in PMVECs. Rat NOS3 was initially cloned and confirmed to be functioning in a heterologous expression system via activation of cGMP‐dependent protein kinase that ultimately phosphorylates vasodilator‐stimulated phosphoprotein on serine239. In PMVECs over‐expressing rat NOS3 loaded with a nitric oxide (NO)‐sensitive fluorophore, DAF‐2, thrombin (2 units/mL) stimulated NO generation assessed by epifluorescence microscopy. This effect was abolished by pharmacologic T‐type Ca2+ channel blockade using mibefradil and potentiated in cells co‐expressing NOS3 and α1G. Three NOS3 deletion mutants (a.a. sequence D1: 1–450, D2: 440–860, and D3: 850–1,200) were generated and their transient expression in PMVECs revealed that D2 domain attenuated thrombin‐stimulated NO generation. Similarly, five α1G cytosolic fragments (a.a. sequence F1: 1–80, F2: 396–743, F3: 965–1272, F4: 1,538–1,610, and F5: 1,855–2,377) were cloned and expressed in PMVECs via retroviral infection. Thrombin‐stimulated NO generation was remarkably reduced in PMVECs individually expressing each of these fragments. Taken together, these data suggest a physical and functional interaction between α1G and NOS3, i.e., through binding of α1G cytosolic domains to NOS3 D2 domain, α1G controls NOS3 activation in PMVECs. Supported by HL66299.

E‐cadherin expression plays a predominant role in the development and homeostasis of epithelial architecture. Down‐regulation of transmembrane protein E‐cadherin is often considered as a marker for cancer malignancy and metastasis. In this study, the effect of hypoxia on E‐cadherin expression in different cancer cell lines was analyzed. We hypothesized that down‐regulation of E‐cadherin under hypoxia would lead to transmigration of its intracellular C‐terminal fragment to the nucleus and result in activation of ß‐catenin/ TCF pathway. Treatment of A549 lung cancer, SW480 and SW620 colon cancer, T47D breast cancer, and HEK293 cells with hypoxia, 1% oxygen for periods of two, three, or four days, resulted in a significant decrease in E‐cadherin expression, Under hypoxic conditions, E‐cadherin is highly degraded resulting in the generation of multiple bands of different molecular weights depending on cells and involved proteinases. This decrease in E‐cadherin is concomitant to an increase in Snail and Slug expression, well‐known E‐cadherin suppressors. Similarly, over‐expression of Snail and Slug in cancer cells resulted in an increase in TCF activity quantified by a luciferase assays. Finally, transient transfection of cancer cells with cloned soluble C‐terminal E‐cadherin resulted in high TCF activity compared to the control. These results suggest that in normoxia, E‐cadherin neutralizes ß‐catenin and prevent it from inducing nuclear TCF activity. However, following E‐cadherin degradation, C‐terminal E‐cadherin binds ß‐catenin and translocates to the nucleus to activate TCF.

The β‐catenin signaling pathway plays an essential role in the development and in diseases such as cancer. Cyclic GMP‐dependent protein kinase (PKG) has been reported to be implicated in β‐catenin signaling. However, the precisemechanism by which PKG modulates β‐catenin pathway isnot completely understood since most of cancer cells loose endogenous PKG expression. The present study was undertaken to determine the effects of ectopic overexpression of PKG on colon cancercell lines. PKG levelswere manipulated in human colon cancer cells by stable transfectionof full‐length PKGcDNA (PKG‐Iβ, PKG‐II) and β‐catenin levels as well as TCF‐luciferase activities were analyzed. In an electromobility shift assay (EMSA), PKG‐I and PKG‐II expression in SW480 colon cancer cells resulted in an increase of TCF, CREB, Ap1, and NF‐kβ binding but not Ap2 or Oct1. Both PKG isoforms stimulation with 8pCPTcGMP induced phosphorylation of VASP (serine239), GSK3β (serine9), and CREB (serine133), while Sox9 phosphorylation (serine181) was only achieved by activated PKG‐II isoform. The results also show that while the overexpression of PKG did not alter total β‐catenin levels, nuclear β‐catenin was slightly increased. The nuclear β‐catenin was concomitant to an increase of TCF‐luciferase activity and binding in PKG overexpressing cells compared to control. These results show that reintroducing PKG (PKG‐Iβ, PKG‐II) in cancer cells affects β‐catenin/TCF pathway and other signals. We are currently exploring the possible contribution of these changes in cell migration, proliferation, and new vessels formation.

Chronic hypoxia attenuates soluble guanylate cyclase-induced vasorelaxation in serotonin (5-HT)-contracted ovine carotid arteries. Because protein kinase G (PKG) mediates many effects of soluble guanylate cyclase activation through phosphorylation of multiple kinase targets in vascular smooth muscle, we tested the hypothesis that chronic hypoxia reduces the ability of PKG to phosphorylate its target proteins, which attenuates the ability of PKG to induce vasorelaxation. We also tested the hypothesis that hypoxia attenuates PKG expression and/or activity. Arteries from normoxic and chronically hypoxic (altitude of 3,820 m for 110 days) fetal and adult sheep were denuded of endothelium and equilibrated with 95% O2-5% CO2 in the presence of nitro-l-arginine methyl ester (l-NAME) and N(G)-nitro-l-arginine (l-NNA) to inhibit residual endothelial nitric oxide synthase. Concentration-response relations for 5-HT were determined in the presence of prazosin to minimize activation of α-adrenergic receptors. The PKG activator 8-(p-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (8-pCTP-cGMP) reduced agonist binding affinity of the 5-HT receptor in a concentration-dependent manner that was attenuated by hypoxia. Expression and activity of PKG-I was not significantly affected by chronic hypoxia in either fetal or adult arteries, although PKG-I abundance was greater in fetal arteries. Pretreatment with the large conductance calcium-sensitive potassium channel (BK) inhibitor iberiotoxin attenuated the vasorelaxation induced by 8-pCPT-cGMP in normoxic but not chronically hypoxic arteries. These results support the hypothesis that hypoxia attenuates the vasorelaxant effects of PKG through suppression of the ability of PKG to activate large conductance calcium-sensitive potassium channels in arterial smooth muscle. The results also reveal that this hypoxic effect is greater in fetal than adult arteries and that chronic maternal hypoxia can profoundly affect fetal vascular function.

Persistent hypoxic pulmonary vasoconstriction (HPV) plays a significant role in the pathogenesis of pulmonary hypertension, which is an emerging clinical problem around the world. We recently showed that hypoxia-induced activation of glucose-6-phosphate dehydrogenase (Glc-6-PD) in pulmonary artery smooth muscle links metabolic changes within smooth muscle cells to HPV and that inhibition of Glc-6PD reduces acute HPV. Here, we demonstrate that exposing pulmonary arterial rings to hypoxia (20-30 Torr) for 12 h in vitro significantly (P < 0.05) reduces (by 30-50%) SM22α and smooth muscle myosin heavy chain expression and evokes HPV. Glc-6-PD activity was also elevated in hypoxic pulmonary arteries. Inhibition of Glc-6-PD activity prevented the hypoxia-induced reduction in SM22α expression and inhibited HPV by 80-90% (P < 0.05). Furthermore, Glc-6-PD and protein kinase G (PKG) formed a complex in pulmonary artery, and Glc-6-PD inhibition increased PKG-mediated phosphorylation of VASP (p-VASP). In turn, increasing PKG activity upregulated SM22α expression and attenuated HPV evoked by Glc-6-PD inhibition. Increasing passive tension (from 0.8 to 3.0 g) in hypoxic arteries for 12 h reduced Glc-6-PD, increased p-VASP and SM22α levels, and inhibited HPV. The present findings indicate that increases in Glc-6-PD activity influence PKG activity and smooth muscle cell phenotype proteins, all of which affect pulmonary artery contractility and remodeling.

Degradation and resynthesis of the extracellular matrix (ECM) are essential during tissue remodeling. Expansion of the vascular intima in atherosclerosis and restenosis following injury is dependent upon smooth muscle cell (SMC) proliferation and migration. The migration of SMC from media to intima critically depends on degradation of ECM protein by matrix metalloproteinases (MMPs). MMP inhibitors and eNOS gene transfer have been shown to inhibit SMC migration in vitro and neointima formation in vivo. Nitric oxide (NO) and cyclic-GMP have been implicated in the inhibition of VSMC migration. But, there are few studies addressing the role of NO signaling pathways on the expression of MMPs. Here we reported the involvement of cyclic-GMP-dependent protein kinase (PKG) (an important mediator of NO and cGMP signaling pathway in VSMC) on MMP-2 expression in rat aortic SMC. The goal of the present study was to gain insight into the possible involvement of PKG on MMP-2 in rat aortic SMC. MMP-2 protein and mRNA level and activity were downregulated in PKG-expressing cells as compared to PKG-deficient cells. In addition, the secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2) was increased in PKG-expressing cells as compared to PKG-deficient cells. PKG-specific membrane permeable peptide inhibitor (DT-2) reverses the process. Interestingly, little or no changes of MMP-9 were observed throughout the study. Taken together our data suggest the possible role of PKG in the suppression of MMP-2.

The ability of the endothelium to produce nitric oxide, which induces generation of cyclic guanosine monophosphate (cGMP) that activates cGMP-dependent protein kinase (PKG-I), in vascular smooth muscle cells (VSMCs), is essential for the maintenance of vascular homeostasis. Yet, disturbance of this nitric oxide/cGMP/PKG-I pathway has been shown to play an important role in many cardiovascular diseases. In the last two decades, in vitro and in vivo models of vascular injury have shown that PKG-I is suppressed following nitric oxide, cGMP, cytokine, and growth factor stimulation. The molecular basis for these changes in PKG-I expression is still poorly understood, and they are likely to be mediated by a number of processes, including changes in gene transcription, mRNA stability, protein synthesis, or protein degradation. Emerging studies have begun to define mechanisms responsible for changes in PKG-I expression and have identified cis- and trans-acting regulatory elements, with a plausible role being attributed to post-translational control of PKG-I protein levels. This review will focus mainly on recent advances in understanding of the regulation of PKG-I expression in VSMCs, with an emphasis on the physiological and pathological significance of PKG-I down-regulation in VSMCs in certain circumstances.

We previously identified glucose-6-phosphate dehydrogenase (G6PD) as a regulator of vascular smooth muscle contraction. In this study, we tested our hypothesis that G6PD activated by KCl via a phosphatase and tensin homologue deleted on chromosome 10 (PTEN)-protein kinase C (PKC) pathway increases vascular smooth muscle contraction and that inhibition of G6PD relaxes smooth muscle by decreasing intracellular Ca 2+ ([Ca 2+ ] i ) and Ca 2+ sensitivity to the myofilament. Here we show that G6PD is activated by membrane depolarization via PKC and PTEN pathway and that G6PD inhibition decreases intracellular free calcium ([Ca 2+ ] i ) in vascular smooth muscle cells and thus arterial contractility. In bovine coronary artery (CA), KCl (30 mmol/l) increased PKC activity and doubled G6PD V max without affecting K m . KCl-induced PKC and G6PD activation was inhibited by bisperoxo(pyridine-2-carboxyl)oxovanadate (Bpv; 10 μmol/l), a PTEN inhibitor, which also inhibited ( P < 0.05) KCl-induced CA contraction. The G6PD blockers 6-aminonicotinamide (6AN; 1 mmol/l) and epiandrosterone (EPI; 100 μmol/l) inhibited KCl-induced increases in G6PD activity, [Ca 2+ ] i , Ca 2+ -dependent myosin light chain (MLC) phosphorylation, and contraction. Relaxation of precontracted CA by 6AN and EPI was not blocked by calnoxin (10 μmol/l), a plasma membrane Ca 2+ ATPase inhibitor or by lowering extracellular Na + , which inhibits the Na + /Ca 2+ exchanger (NCX), but cyclopiazonic acid (200 μmol/l), a sarcoplasmic reticulum Ca 2+ ATPase inhibitor, reduced ( P < 0.05) 6AN- and EPI-induced relaxation. 6AN also attenuated phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at Ser855, a site phosphorylated by Rho kinase, inhibition of which reduced ( P < 0.05) KCl-induced CA contraction and 6AN-induced relaxation. By contrast, 6AN increased ( P < 0.05) vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser239, indicating that inhibition of G6PD increases PKA or PKG activity. Inhibition of PKG by RT-8-Br-PET-cGMPs (100 nmol/l) diminished 6AN-evoked VASP phosphorylation ( P < 0.05), but RT-8-Br-PET-cGMPs increased 6AN-induced relaxation. These findings suggest G6PD inhibition relaxes CA by decreasing Ca 2+ influx, increasing Ca 2+ sequestration, and inhibiting Rho kinase but not by increasing Ca 2+ extrusion or activating PKG.

The type-I cGMP-dependent protein kinase (PKG-I) expression regulation is not yet completely understood. In this study, we examined the role of 3'-untranslated region (3'UTR)-PKG-I messenger RNA (mRNA) in the control of PKG-I expression in vascular smooth muscle cells (VSMCs). Using a 3'-rapid amplification of cDNA ends (RACE) for the amplification of complementary DNA (cDNA) ends, we generated and cloned a 1.2-kb-3'UTR mRNA PKG-I in pGL3 control vector downstream of the luciferase reporter gene. Serial deletions and functional studies revealed that among the deleted constructs, only the 1.2-kb-3'UTR PKG-I mRNA possesses the highest activity in transfected VSMC. Kinetic luciferase assays in the presence of actinomycin D showed that this construct stabilizes luciferase activity compared to the control vector. Sequence analysis of 3'UTR-PKG-I mRNA revealed the existence of four AU-rich regions (AU1 through AU4) in addition to a potential poly(A) site. Different riboprobes were generated either by 5'-end-labelling of designed ribonucleotides, containing individual AU-rich regions or by in vitro transcription assay using cloned 1.2-kb cDNA as a template. RNA-electrophoretic mobility shift assay (EMSA) and ultra-violet cross-linking (UV-CL) assays showed that AU1, AU3, AU4 and 1.2-kb probes were able to retard cytosolic and nuclear proteins. Taken together, these data suggest that PKG-I expression is subjected to post-transcriptional regulation in VSMC through the 3'UTR of its mRNA.

The transcriptional activator β-catenin is a key mediator of the canonical Wnt signaling pathway. β-catenin itself does not bind DNA but functions via interaction with T-cell factor (TCF)/lymphoid-enhancing factor (LEF) transcription factors. Thus, in the case of active Wnt signaling, β-catenin, in cooperation with TCF/LEF proteins family, activates the expression of a wide variety of genes. To date, the list of established β-catenin interacting targets is far from complete. In this study, we aimed to establish the interaction between β-catenin and transcription factors that might affect TCF activity. We took advantage of EMSA, using TCF as a probe, to screen oligonucleotides known to bind specific transcription factors that might dislodge or antagonize β-catenin/TCF binding. We found that Sox9 and KLF4 antagonize β-catenin/TCF binding in HEK293, A549, SW480, and T47D cells. This inhibition of TCF binding was concentration-dependent and correlated to the in vitro TCF-luciferase functional assays. Overexpression of Sox9 and KLF4 transcription factors in cancer cells shows a concentration-dependent reduction of TCF-luciferase as well as the TCF-binding activities. In addition, we demonstrated that both Sox9 and KLF4 interact with β-catenin in an immunoprecipitation assay and reduce its binding to TCF4. Together, these results demonstrate that Sox9 and KLF4 transcription factors antagonize β-catenin/TCF in cancer cells.