Testosterone Induces Vascular Smooth Muscle Cell Migration by NADPH Oxidase and c-Src-Dependent Pathways

Testosterone has been implicated in vascular remodeling associated with hypertension. Molecular mechanisms underlying this are elusive, but oxidative stress may be important. We hypothesized that testosterone stimulates generation of reactive oxygen species (ROS) and migration of vascular smooth muscle cells (VSMCs), with enhanced effects in cells from spontaneously hypertensive rats (SHRs). The mechanisms (genomic and nongenomic) whereby testosterone induces ROS generation and the role of c-Src, a regulator of redox-sensitive migration, were determined. VSMCs from male Wistar-Kyoto rats and SHRs were stimulated with testosterone (10(-7) mol/L, 0-120 minutes). Testosterone increased ROS generation, assessed by dihydroethidium fluorescence and lucigenin-enhanced chemiluminescence (30 minutes [SHR] and 60 minutes [both strains]). Flutamide (androgen receptor antagonist) and actinomycin D (gene transcription inhibitor) diminished ROS production (60 minutes). Testosterone increased Nox1 and Nox4 mRNA levels and p47phox protein expression, determined by real-time PCR and immunoblotting, respectively. Flutamide, actinomycin D, and cycloheximide (protein synthesis inhibitor) diminished testosterone effects on p47phox. c-Src phosphorylation was observed at 30 minutes (SHR) and 120 minutes (Wistar-Kyoto rat). Testosterone-induced ROS generation was repressed by 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine (c-Src inhibitor) in SHRs and reduced by apocynin (antioxidant/NADPH oxidase inhibitor) in both strains. Testosterone stimulated VSMCs migration, assessed by the wound healing technique, with greater effects in SHRs. Flutamide, apocynin, and 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day] pyrimidin-4-amine blocked testosterone-induced VSMCs migration in both strains. Our study demonstrates that testosterone induces VSMCs migration via NADPH oxidase-derived ROS and c-Src-dependent pathways by genomic and nongenomic mechanisms, which are differentially regulated in VSMCs from Wistar-Kyoto rats and SHRs. (Hypertension. 2012; 59: 1263-1271.). Online Data Supplement

Emerging role of angiotensin type 2 receptor (AT2R)/Akt/NO pathway in vascular smooth muscle cell in the hyperthyroidism

Hyperthyroidism is characterized by increased vascular relaxation and decreased vascular contraction and is associated with augmented levels of triiodothyronine (T3) that contribute to the diminished systemic vascular resistance found in this condition. T3 leads to augmented NO production via PI3K/Akt signaling pathway, which in turn causes vascular smooth muscle cell (VSMC) relaxation; however, the underlying mechanisms involved remain largely unknown. Evidence from human and animal studies demonstrates that the renin-angiotensin system (RAS) plays a crucial role in vascular function and also mediates some of cardiovascular effects found during hyperthyroidism. Thus, in this study, we hypothesized that type 2 angiotensin II receptor (AT2R), a key component of RAS vasodilatory actions, mediates T3 induced-decreased vascular contraction. Marked induction of AT2R expression was observed in aortas from T3-induced hyperthyroid rats (Hyper). These vessels showed decreased protein levels of the contractile apparatus: α-actin, calponin and phosphorylated myosin light chain (p-MLC). Vascular reactivity studies showed that denuded aortic rings from Hyper rats exhibited decreased maximal contractile response to angiotensin II (AngII), which was attenuated in aortic rings pre-incubated with an AT2R blocker. Further study showed that cultured VSMC stimulated with T3 (0.1 µmol/L) for 24 hours had increased AT2R gene and protein expression. Augmented NO levels and decreased p-MLC levels were found in VSMC stimulated with T3, both of which were reversed by a PI3K/Akt inhibitor and AT2R blocker. These findings indicate for the first time that the AT2R/Akt/NO pathway contributes to decreased contractile responses in rat aorta, promoted by T3, and this mechanism is independent from the endothelium.

Different vascular smooth muscle cell apoptosis in the human internal mammary artery and the saphenous vein. Implications for bypass graft disease

BACKGROUND: The remarkable patency of internal mammary artery (MA) grafts compared to saphenous vein (SV) grafts has been related to different biological properties of the two blood vessels. We examined whether proliferation and apoptosis of vascular smooth muscle cells (VSMC) from human coronary artery bypass vessels differ according to patency rates. METHODS AND RESULTS: Proliferation rates to serum or platelet-derived growth factor (PDGF)-BB were lower in VSMC from MA than SV. Surface expression of PDGF beta-receptor was slightly lower, while that of alpha-receptor was slightly higher in MA than SV. Cell cycle distribution, expression of cyclin E, cdk2, p21, p27, p57, and cdk2 kinase activity were identical in PDGF-BB-stimulated cells from MA and SV. However, apoptosis rates were higher in MA than SV determined by lactate dehydrogenase release, DNA fragmentation, and Hoechst 33258 staining. Moreover, caspase inhibitors (Z-VAD-fmk, Boc-D-fmk) abrogated the different proliferation rates of VSMC from MA versus SV. Western blotting and GSK3-beta kinase assay revealed lower Akt activity in VSMC from MA versus SV, while total Akt expression was identical. Adenoviral transduction of a constitutively active Akt mutant abrogated the different proliferation rates of VSMC from MA versus SV. CONCLUSIONS: Higher apoptosis rates due to lower Akt activity rather than different cell cycle regulation account for the lower proliferation of VSMC from MA as compared to SV. VSMC apoptosis may protect MA from bypass graft disease.

A genetically engineered, adherent smooth muscle cell lining for left ventricular assist devices

Due to the clinical success of left ventricular assist devices (LVADs) used for short term "bridge to transplant" and the limited availability of donor organs, heart assist devices are being considered for long term implantation as an alternative to heart transplantation. In an effort to improve biocompatibility, a nonthrombogenic cellular lining was developed from genetically engineered smooth muscle cells (GE-SMC) for the Thermocardiosystems Heartmate$\sp{\rm TM}$ LVAD. SMCs have been transduced with the genes for endothelial nitric oxide synthase (NOS III) and GTP cyclohydrolase (GTPCH) with subsequent stable expression of the NOS III protein via an Epstein Barr based DNA expression vector. Transduced SMCs produce nitric oxide at concentrations that reduce platelet deposition and smooth muscle cell proliferation when tested in vitro. In addition, the adhesive capabilities of GE-SMC linings were also examined, and optimized in physical environments mimicking typical in vivo LVAD operation. Preliminary investigations examining cell adhesion during constant shear stress exposure demonstrated an acute phase of cell loss corresponding to cytoskeletal F-actin rearrangement. Subsequently, an in vitro circulatory loop was designed to expose cell lined LVADs to in vivo operating conditions. Cumulative cell loss from cell lined LVADs was less than 10% after 24 hours of flow. Using a protocol for "preconditioning" the cell lining within the mock circulatory loop, the first implantation of an LVAD containing a genetically engineered SMC lining was successfully implemented in a bovine model. Results from this 24 hour study indicate that the flow-conditioned cellular lining remained intact with no evidence of thromboembolization and only minimal changes in coagulation studies. ^

MicroRNA miR-199a-5p regulates smooth muscle cell proliferation and morphology by targeting Wnt2 signaling pathway

MicroRNA miR-199a-5p impairs tight junction formation leading to increased urothelial permeability in bladder pain syndrome. Now using transcriptome analysis in urothelial TEU-2 cells we implicate it in the regulation of cell cycle, cytoskeleton remodeling, TGF and Wnt signaling pathways. MiR-199a-5p is highly expressed in the smooth muscle layer of the bladder and we altered its levels in bladder smooth muscle cells (SMC) to validate the pathway analysis. Inhibition of miR-199a-5p with antimiR increased SMC proliferation, reduced cell size and up-regulated miR-199a-5p targets, including Wnt2. Overexpression of Wnt2 protein or treating SMCs with recombinant Wnt2 closely mimicked the miR-199a-5p inhibition, whereas down-regulation of Wnt2 in antimiR-expressing SMCs with shRNA restored cell phenotype and proliferation rates. Overexpression of miR-199a-5p in the bladder SMCs significantly increased cell size and up-regulated SM22, SM alpha-actin and SM myosin heavy chain mRNA and protein levels. These changes, as well as increased expression of ACTG2, TGFB1I1, and CDKN1A were mediated by up-regulation of smooth muscle-specific transcriptional activator myocardin at mRNA and protein levels. Myocardin-related transcription factor (MRTF-A) downstream targets Id3 and MYL9 were also induced. Up-regulation of myocardin was accompanied by down-regulation of Wnt-dependent inhibitory Kruppel-like transcription factor 4 (KLF4) in miR-199a-5p overexpressing cells. In contrast, KLF4 was induced in antimiR-expressing cells following the activation of Wnt2 signaling, leading to repression of myocardin-dependent genes. MiR-199a-5p plays a critical role in the Wnt2-mediated regulation of proliferative and differentiation processes in the smooth muscle and may behave as a key modulator of smooth muscle hypertrophy, relevant for organ remodeling.

EFFECTS OF THE ACTA2 R258C MUTATION ON VASCULAR SMOOTH MUSCLE CELL PHENOTYPE AND PROPERTIES

Thoracic Aortic Aneurysms and Dissections (TAAD) are the fifteenth leading cause of death in the United States. About 15% of TAAD patients have family history of the disease. The most commonly mutated gene in these families is ACTA2, encoding smooth muscle-specific α-actin. ACTA2 missense mutations predispose individuals both to TAAD and to vascular occlusive disease of small, muscular arteries. Mice carrying an Acta2 R258C mutant transgene with a wildtype Acta2 promoter were generated and bred with Acta2-/- mice to decrease the wildtype: mutant Acta2 ratio. Acta2+/+ R258C TGmice have decreased aortic contractility without aortic disease. Acta2+/- R258C TG mice, however, have significant aortic dilatations by 12 weeks of age and a hyperproliferative response to injury. We characterized smooth muscle cells (SMCs) from bothmouse models under the hypothesis that mutant α-actin has a dominant negative effect, leading to impaired contractile filament formation/stability, improper focal adhesion maturation and increased proliferation. Explanted aortic SMCs from Acta2+/+ R258C TG mice are differentiated - they form intact filaments, express higher levels of contractile markers compared to wildtype SMCs and have predominantly nuclear Myocardin-Related Transcription Factor A (MRTF-A) localization. However, ultracentrifugation assays showed large unpolymerized actin fractions, suggesting that the filaments are brittle. In contrast, Acta2+/- R258C TG SMCs are less well-differentiated, with pools of unpolymerized actin, more cytoplasmic MRTF-A and decreased contractile protein expression compared to wildtype cells. Ultracentrifugation assays after treating Acta2+/- R258C TGSMCs with phalloidin showed actin filament fractions, indicating that mutant α-actin can polymerize into filaments. Both Acta2+/+ R258C TGand Acta2+/- R258C TGSMCs have larger and more peripheral focal adhesions compared to wildtype SMCs. Rac1 was more activated in Acta2+/+ R258C TGSMCs; both Rac1 and RhoA were less activated in Acta2+/- R258C TG SMCs, and FAK was more activated in both transgenic SMC lines compared to wildtype. Proliferation in both cell lines was significantly increased compared to wildtype cells and could be partially attenuated by inhibition of FAK or PDGFRβ. These data support a dominant negative effect of the Acta2 R258C mutation on the SMC phenotype, with increasing phenotypic severity when wildtype: mutant α-actin levels are decreased.

Binding of Insulin-like Growth Factor (IGF)–Binding Protein-5 to Smooth-Muscle Cell Extracellular Matrix Is a Major Determinant of the Cellular Response to IGF-I

Insulin-like growth factor–binding protein-5 (IGFBP-5) has been shown to bind to fibroblast extracellular matrix (ECM). Extracellular matrix binding of IGFBP-5 leads to a decrease in its affinity for insulin-like growth factor-I (IGF-I), which allows IGF-I to better equilibrate with IGF receptors. When the amount of IGFBP-5 that is bound to ECM is increased by exogenous addition, IGF-I’s effect on fibroblast growth is enhanced. In this study we identified the specific basic residues in IGFBP-5 that mediate its binding to porcine smooth-muscle cell (pSMC) ECM. An IGFBP-5 mutant containing alterations of basic residues at positions 211, 214, 217, and 218 had the greatest reduction in ECM binding, although three other mutants, R214A, R207A/K211N, and K202A/R206N/R207A, also had major decreases. In contrast, three other mutants, R201A/K202N/R206N/R208A, and K217N/R218A and K211N, had only minimal reductions in ECM binding. This suggested that residues R207 and R214 were the most important for binding, whereas alterations in K211 and R218, which align near them, had minimal effects. To determine the effect of a reduction in ECM binding on the cellular replication response to IGF-I, pSMCs were transfected with the mutant cDNAs that encoded the forms of IGFBPs with the greatest changes in ECM binding. The ECM content of IGFBP-5 from cultures expressing the K211N, R214A, R217A/R218A, and K202A/R206N/R207A mutants was reduced by 79.6 and 71.7%, respectively, compared with cells expressing the wild-type protein. In contrast, abundance of the R201A/K202N/R206N/R208A mutant was reduced by only 14%. Cells expressing the two mutants with reduced ECM binding had decreased DNA synthesis responses to IGF-I, but the cells expressing the R201A/K202N/R206N/R208A mutant responded well to IGF-I. The findings suggest that specific basic amino acids at positions 207 and 214 mediate the binding of IGFBP-5 to pSMC/ECM. Smooth-muscle cells that constitutively express the mutants that bind weakly to ECM are less responsive to IGF-I, suggesting that ECM binding of IGFBP-5 is an important variable that determines cellular responsiveness.

Role of the arginine-nitric oxide pathway in the regulation of vascular smooth muscle cell proliferation

The objective of this study was to elucidate the mechanisms by which nitric oxide (NO) inhibits rat aortic smooth muscle cell (RASMC) proliferation. Two products of the arginine-NO pathway interfere with cell growth by distinct mechanisms. NG-hydroxyarginine and NO appear to interfere with cell proliferation by inhibiting arginase and ornithine decarboxylase (ODC), respectively. S-nitroso-N-acetylpenicillamine, (Z)-1-[N-(2-aminoethyl)-N-(2-aminoethyl)-amino]-diazen-1-ium-1,2-diolate, and a nitroaspirin derivative (NCX 4016), each of which is a NO donor agent, inhibited RASMC growth at concentrations of 1–3 μM by cGMP-independent mechanisms. The cytostatic action of the NO donor agents as well as α-difluoromethylornithine (DFMO), a known ODC inhibitor, was prevented by addition of putrescine but not ornithine. These observations suggested that NO, like DFMO, may directly inhibit ODC. Experiments with purified, recombinant mammalian ODC revealed that NO inhibits ODC possibly by S-nitrosylation of the active site cysteine in ODC. DFMO, as well as the NO donor agents, interfered with cellular polyamine (putrescine, spermidine, spermine) production. Conversely, increasing the expression and catalytic activity of arginase I in RASMC either by transfection of cells with the arginase I gene or by induction of arginase I mRNA with IL-4 resulted in increased urea and polyamine production as well as cell proliferation. Finally, coculture of rat aortic endothelial cells, which had been pretreated with lipopolysaccharide plus a cytokine mixture to induce NO synthase and promote NO production, caused NO-dependent inhibition of target RASMC proliferation. This study confirms the inhibitory role of the arginine-NO pathway in vascular smooth muscle proliferation and indicates that one mechanism of action of NO is cGMP-independent and attributed to its capacity to inhibit ODC.

d-alpha-tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentrations, correlates with protein kinase C inhibition, and is independent of its antioxidant properties.

d-alpha-Tocopherol, but not d-beta-tocopherol, negatively regulates proliferation of vascular smooth muscle cells at physiological concentrations. d-alpha-Tocopherol inhibits protein kinase C (PKC) activity, whereas d-beta-tocopherol is ineffective. Furthermore d-beta-tocopherol prevents the inhibition of cell growth and of PKC activity caused by d-alpha-tocopherol. The negative regulation by d-alpha-tocopherol of PKC activity appears to be the cause and not the effect of smooth muscle cell growth inhibition. d-alpha-Tocopherol does not act by binding to PKC directly but presumably by preventing PKC activation. It is concluded that, in vascular smooth muscle cells, d-alpha-tocopherol acts specifically through a nonantioxidant mechanism and exerts a negative control on a signal transduction pathway regulating cell proliferation.

Rho and smooth muscle cell phenotype

Smooth muscle cell (SMC) phenotypic modulation from the mature ’contractile’ to a less differentiated ’synthetic’ phenotype involves not only altered expression but also a reorganisation of contractile and cytoskeletal proteins. Objective: To investigate the role of RhoA, a known regulator of the actin cytoskeleton, in SMC phenotypic regulation. Methods: Rho transcription (RT-PCR), expression (Western analysis) and activation (membrane translocation or Rho ’pull-down’ assay) was investigated in cultured rabbit aortic SMC during phenotypic modulation, and under the influence of known SM-regulatory proteins (thrombin, heparin and TGF- β). Rho’s effect on cell morphology was examined by transient transfection of ’synthetic’ state SMC with either constitutively active Rho (Val14RhoA) or its inhibitor, C3 transferase. Results: RhoA transcription was elevated in the first 3 days of primary culture, and protein expression peaked at 2 days post-confluence when SMC return to a more ’contractile’ state. However, RhoA showed augmented activation at three time-points in primary culture: the transition point when SMCs enter logarithmic growth and are highly motile, upon reaching quiescence, and when they return to a more ’contractile’ state. Thrombin, heparin and TGF-β activated RhoA in ’synthetic’ state SMCs. Transfection with Val14RhoA caused a dramatic decrease in SMC size and a reorganization of cytoskeletal proteins, reminiscent of the ’contractile’ phenotype. Specific inhibition of endogenous Rho by C3 transferase resulted in an almost complete loss of contractile proteins. Conclusion: These data indicate that Rho is an important determining factor of SMC functional state.

Potent suppression of vascular smooth muscle cell migration and human neointimal hyperplasia by KV1.3 channel blockers

Aim - The aim of the study was to determine the potential for KV1 potassium channel blockers as inhibitors of human neoinitimal hyperplasia. Methods and results - Blood vessels were obtained from patients or mice and studied in culture. Reverse transcriptasepolymerase chain reaction and immunocytochemistry were used to detect gene expression. Whole-cell patch-clamp, intracellular calcium measurement, cell migration assays, and organ culture were used to assess channel function.  KV1.3 was unique among the  KV1 channels in showing preserved and up-regulated expression when the vascular smooth muscle cells switched to the proliferating phenotype. There was strong expression in neointimal formations. Voltage-dependent potassium current in proliferating cells was sensitive to three different blockers of  KV1.3 channels. Calcium entry was also inhibited. All three blockers reduced vascular smooth muscle cell migration and the effects were non-additive. One of the blockers (margatoxin) was highly potent, suppressing cell migration with an IC of 85 pM. Two of the blockers were tested in organ-cultured human vein samples and both inhibited neointimal hyperplasia. Conclusion - KV1.3 potassium channels are functional in proliferating mouse and human vascular smooth muscle cells and have positive effects on cell migration. Blockers of the channels may be useful as inhibitors of neointimal hyperplasia and other unwanted vascular remodelling events. © 2010 The Author.

Investigating the Role of PDE4D1/2 in Vascular Smooth Muscle Cell Desensitization

Vascular smooth muscle cell (VSMC) behaviour and phenotypic modulation is critical to vessel repair following damage, and the progression of various cardiovascular diseases. The second messenger cyclic adenosine monophosphosphate (cAMP) plays a key role in VSMC function under the synthetic/activated phenotype, which is typically associated with unhealthy cell behaviour. Consequently, cAMP signaling is often targeted in attempts to impact several pathological diseases, including atherosclerosis, restenosis, and pulmonary arterial hypertension (PAH). The cyclic nucleotide phosphodiesterases (PDEs) catalyze hydrolysis of cAMP to an inactive form, and therefore directly regulate cAMP signaling. The PDE4D family dominates in synthetic VSMCs, and there is considerable interest in determining how distinct PDE4D isoforms affect cell function. Specifically, we are interested in the potential link between short isoforms of PDE4D and VSMC desensitization to pharmacological agents that impact cardiovascular disease via cAMP signaling. This study extends on previous work that assessed the expression of PDE4D splice variants in rat aortic VSMCs following prolonged challenge with cAMP-elevating agents. It was determined that PDE4D1 and PDE4D2 were uniquely expressed in synthetic VSMCs incubated with these agents, and that this upregulation impacted PDE activity and cAMP accumulation in these cells. Here, we report that PDE4D1 and PDE4D2 are markedly upregulated in synthetic human aortic smooth muscle cells (HASMCs) following prolonged challenge with cAMP-elevating agents. Using a combination of RNAi-based and pharmacological approaches, we establish that this upregulation is reflected in levels of cAMP PDE activity, and restricted to the cytosolic sub-cellular compartment. Our results suggest a role for localized PDE4D1 and PDE4D2 activity in regulating cAMP-mediated desensitization in HASMCs, and highlight their therapeutic potential in treating various cardiovascular diseases.