Murine Vascular Smooth Muscle Cells Research Articles

Nitric oxide regulates vascular calcification by interfering with TGF-β signalling

Vascular calcification often occurs with advancing age, atherosclerosis, and metabolic disorders such as diabetes mellitus and end-stage renal disease. Vascular calcification is associated with cardiovascular events and increased mortality. Nitric oxide (NO) is crucial for maintaining vascular function, but little is known about how NO affects vascular calcification. The aim of this study was to examine the effect of NO on vascular calcification. In this study, we examined the inhibitory effects of NO on calcification of murine vascular smooth muscle cells (VSMCs) in vitro. We measured calcium concentration, alizarin red staining, and alkaline phosphatase activity to examine the effect of NO on calcification of VSMCs and differentiation of VSMCs into osteoblastic cells. We also determined gene expression and levels of phosphorylation of Smad2/3 by RT-PCR and western blotting. NO inhibited calcification of VSMCs and differentiation of VSMCs into osteoblastic cells. An inhibitor of cyclic guanosine monophosphate (cGMP)-dependent protein kinase restored the inhibition by NO of osteoblastic differentiation and calcification of VSMCs. NO inhibited transforming growth factor-beta (TGF-beta)-induced phosphorylation of Smad2/3 and expression of TGF-beta-induced genes such as plasminogen activator inhibitor-1. In addition, NO inhibited expression of the TGF-beta receptor ALK5. Our data show that NO prevents differentiation of VSMCs into osteoblastic cells by inhibiting TGF-beta signalling through a cGMP-dependent pathway. Our findings suggest that NO may play a beneficial role in atherogenesis in part by limiting vascular calcification.

Molecular mechanisms of the antiproliferative effect of beraprost, a prostacyclin agonist, in murine vascular smooth muscle cells

Prostacyclin (PGI2) has been shown to inhibit proliferation in vascular smooth muscle cells. To clarify the underlying molecular mechanism, we investigated the vasoprotection of beraprost (a PGI2 agonist) both in vivo and in vitro. Beraprost eliminated increases in proliferation of rat aortic smooth muscle cells (RASMCs) by 12-O-tetradecanoylphorbol 13-acetate, and enhanced the peroxisome proliferator-activated receptor-delta (PPARdelta) and inducible nitric oxide synthetase (iNOS) expressions, which were associated with the antiproliferative action of beraprost according to inhibition experiments by [(3)H]thymidine incorporation. Additionally, elimination of iNOS activity by PPARdelta antagonists suggested that iNOS is the downstream target of PPARdelta. Furthermore, beraprost increased both consensus PPARdelta-responsive element (PPRE)-driven luciferase activity and the binding activity of the PPARdelta to the putative PPRE in the iNOS promoter; nevertheless, it was abolished by PPARdelta antagonists. Deletion of PPRE (-1,349/-1,330) in the iNOS promoter region (-1,359/+2) strongly reduced promoter-driven activity, representing a novel mechanism of iNOS induction by beraprost. Consistent with this, PPARdelta and the concomitant iNOS induction by beraprost were also evident in vivo. Beraprost-mediated protection in a murine model of balloon angioplasty was significantly attenuated by 13S-HODE, a PPARdelta antagonist. Taken together, the results suggest that the causal relationship between PPARdelta and iNOS contributes to the vasoprotective action of beraprost in RASMCs.

Activation of the cGMP/cGKI signalling pathway in primary murine VSMCs accelerates growth

The endogenous signaling molecule nitric oxide (NO) exerts many of its actions via the cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase type I (cGKI) pathway. Recently, the analysis of an in vivo mouse model for atherosclerosis suggested that activation of cGKI in vascular smooth muscle cells (VSMCs) promotes the phenotypic modulation of medial VSMCs and, thus, vascular lesion formation. In contrast, many in vitro studies demonstrate an anti-proliferative role for cGKI. In the current study, primary and subcultured wild-type and cGKI-deficient VSMCs in response to the membrane permeable cGMP analogue 8-Br-cGMP were compared. In agreement with the common view an anti-proliferative effect of cGKI was found in repeatedly passaged VSMCs (P5–11). In contrast, the analysis of primary VSMCs revealed that activation of cGKI in primary VSMCs strongly promotes growth. Analysing proliferation, apoptosis, cytoskeletal dynamics and various signaling pathways indicated that an increase in cell adhesion is a major mechanism for cGKI-mediated growth in primary VSMCs. This pro-adhesive effect of cGKI might be mediated via an inhibition of Rho kinase (ROCK) and enhanced integrin signaling. These processes might contribute to inhibit anoikis, the programmed cell death induced by the loss of cell/matrix interactions. from 3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications Dresden, Germany. 15–17 June 2007

Structure–function properties of the apoE-dependent COX-2 pathway in vascular smooth muscle cells

Apolipoprotein (apoE) E is a multifunctional protein that plays a critical role in atherogenesis, in part by regulating the intimal proliferation of vascular smooth muscle cells. Recently, a novel cyclooxygenase (COX)-2 pathway was shown to contribute to the anti-proliferative action of human apoE3 in vascular smooth muscle cells (VSMC). Here, we provide insight into the structure–function properties by which apoE mediates these effects. ApoE3 is most effective in promoting COX-2 expression as a lipid-free protein and is less active after lipidation. Alterations in the stability of the helix bundle N-terminal domain of apoE that contains the binding site for the low density lipoprotein (LDL) receptor and heparin do not affect the up-regulation of the COX-2 pathway. In addition, the apoE2, 3, and 4 isoforms are all capable of up-regulating the COX-2 pathway. Finally, the effect of apoE on COX-2 was found to be independent of expression on the VSMC surface of the LDL receptor and heparan sulfate proteoglycans (HSPG). In summary, our data indicates that apoE, particularly in the lipid-free state, can up-regulate COX-2 in murine vascular smooth muscle cells apparently independently of binding to the LDLR, LRP or HSPG.

Role of cysteinyl leukotrienes in the proliferation and the migration of murine vascular smooth muscle cells in vivo and in vitro

Objective Cysteinyl leukotrienes (Cys-LTs) are proinflammatory lipid mediators generated from arachidonic acid through 5-lipoxygenase (5-LO). It was reported that the 5-LO pathway is associated with vascular smooth muscle cell (VSMC) proliferation and migration; however, the relationship between Cys-LTs and VSMC proliferation and migration remains unclear. Methods: We used a mouse (C57/BL6J) model of femoral artery wire injury, and compared neointimal formation between controls and animals treated with the Cys-LT receptor antagonist, Montelukast (3 mg/kg/day). The effects of Cys-LTs with or without Montelukast on mouse aortic VSMC proliferation and migration were also investigated. Results After wire injury, neointimal hyperplasia was observed, and immunohistochemical analysis demonstrated expression of the Cys-LT receptors (Cys-LT1 and Cys-LT2) and α-smooth muscle actin in the injured arterial walls. RT-PCR analysis revealed that transcription was not altered during the observation period (before, 1, 2 and 4 weeks after injury). Administration of Montelukast significantly inhibited neointimal formation 4 weeks after injury (intima/media ratio; control group 1.94±0.56 versus Montelukast-treated group 0.94±0.26, n =11, P <0.001). In an in vitro study, LTD4, but not LTC4 and LTE4 significantly stimulated VSMC proliferation (1000 nM), and this effect was inhibited by Montelukast (10 μM). LTC4, LTD4 and LTE4 significantly stimulated VSMC migration (1000 nM), and this effect was inhibited by Montelukast (10 μM). Conclusions Our data suggest that Cys-LTs are involved in VSMC proliferation and migration, and a Cys-LT receptor antagonist may have beneficial effects on vascular remodeling after mechanical injury.

Cellular Mechanism Through Which Parathyroid Hormone–Related Protein Induces Proliferation in Arterial Smooth Muscle Cells

Parathyroid hormone-related protein (PTHrP) is present in vascular smooth muscle (VSM), is markedly upregulated in response to arterial injury, is essential for normal VSM proliferation, and also markedly accentuates neointima formation following rat carotid angioplasty. PTHrP contains a nuclear localization signal (NLS) through which it enters the nucleus and leads to marked increases in retinoblastoma protein (pRb) phosphorylation and cell cycle progression. Our goal was to define key cell cycle molecules upstream of pRb that mediate cell cycle acceleration induced by PTHrP. The cyclin D/cdk-4,-6 system and its upstream regulators, the inhibitory kinases (INKs), are not appreciably influenced by PTHrP. In striking contrast, cyclin E/cdk-2 kinase activity is markedly increased by PTHrP, and this is a result of a specific, marked, PTHrP-induced proteasomal degradation of p27(kip1). Adenoviral restoration of p27(kip1) fully reverses PTHrP-induced cell cycle progression, indicating that PTHrP mediates its cell cycle acceleration in VSM via p27(kip1). In confirmation, adenoviral delivery of PTHrP to murine primary vascular smooth muscle cells (VSMCs) significantly decreases p27(kip1) expression and accelerates cell cycle progression. p27(kip1) is well known to be a central cell cycle regulatory molecule involved in both normal and pathological VSM proliferation and is a target of widely used drug-eluting stents. The current observations define a novel "PTHrP/p27(kip1) pathway" in the arterial wall and suggest that this pathway is important in normal arterial biology and a potential target for therapeutic manipulation of the arterial response to injury.

Exposure to modeled microgravity induces metabolic idleness in malignant human MCF-7 and normal murine VSMC cells

We investigated the effect of modeled microgravity (MMG) on normal vascular smooth muscle cells (VSMC) and neoplastic human breast cancer cells (MCF-7). In both cell types, MMG induced partial arrest in G 2M and increased p14-3-3, HSP70, HSP60 and p21 expression. Cells synchronized by 24 h starvation reentered the normal cycle within 24 h if released in complete medium and exposed to normal gravity, but not if exposed to MMG. Similarly, MMG prevented VSMC and MCF-7 cells from overcoming growth arrest and re-synthesizing DNA. This study shows that cells adjust their metabolic rate in response to MMG.

Heat-shock response is associated with decreased production of interleukin-6 in murine aortic vascular smooth muscle cells

Heat shock has been known to change cellular responses to noxious stimuli by inducing heat-shock proteins (Hsps). We hypothesized that a heat-shock response modulates cytokine production in murine aortic vascular smooth muscle cells (VSMCs). VSMCs were exposed to 44 degrees C for 15-60 min, and subjected to interleukin-1beta (IL-1beta) or tumor necrosis factor alpha (TNFalpha), which induced interleukin-6 (IL-6) production. Expression of Hsps was examined with immunoblots, immunocytochemistry, or enzyme-linked immunosorbent assay (ELISA), and that of IL-6 with reverse transcription-polymerase chain reaction (RT-PCR) or ELISA. Heat shock (44 degrees C for 45 min) induced Hsp72 in VSMCs at 4 h and elicited its maximal expression at 8 h after the end of heat shock. Treatment with IL-1beta increased IL-6 transcription in VSMCs up to 24 h in an incubation time-dependent manner. Treatment with IL-1beta or TNFalpha caused a concentration-dependent increase in IL-6 production in culture medium, which was attenuated by heat shock. Although treatment with Hsp72 or Hsp60 alone did not significantly affect basal IL-6 release into culture medium statistically, cotreatment with IL-1beta and Hsp72, but not Hsp60 or boiled Hsp72, decreased IL-1beta-induced IL-6 production in culture medium. Introduction of Hsp72, but not Hsp60, into VSMCs decreased IL-1beta-induced IL-6 production in culture medium. These results indicate that the heat-shock response transcriptionally attenuated production of IL-6 in murine aortic VSMCs.

Induction, differentiation, and remodeling of blood vessels after transplantation of Bcl-2-transduced endothelial cells

Implants of collagen-fibronectin gels containing Bcl-2-transduced human umbilical vein endothelial cells (Bcl-2-HUVECs) induce the formation of human endothelial cell (EC)/murine vascular smooth muscle cell (VSMC) chimeric vessels in immunodeficient mice. Microfil casting of the vasculature 60 d after implantation reveals highly branched microvascular networks within the implants that connect with and induce remodeling of conduit vessels arising from the abdominal wall circulation. Approximately 85% of vessels within the implants are lined by Bcl-2-positive human ECs expressing VEGFR1, VEGFR2, and Tie-2, but not integrin alpha(v)beta(3). The human ECs are seated on a well formed human laminin/collagen IV-positive basement membrane, and are surrounded by mouse VSMCs expressing SM-alpha actin, SM myosin, SM22alpha, and calponin, all markers of contractile function. Transmission electron microscopy identified well formed EC-EC junctions, chimeric arterioles with concentric layers of contractile VSMC, chimeric capillaries surrounded by pericytes, and chimeric venules. Bcl-2-HUVEC-lined vessels retain 70-kDa FITC-dextran, but not 3-kDa dextran; local histamine rapidly induces leak of 70-kDa FITC-dextran or India ink. As in skin, TNF induces E-selectin and vascular cell adhesion molecule 1 only on venular ECs, whereas intercellular adhesion molecule-1 is up-regulated on all human ECs. Bcl-2-HUVEC implants are able to engraft within and increase perfusion of ischemic mouse gastrocnemius muscle after femoral artery ligation. These studies show that cultured Bcl-2-HUVECs can differentiate into arterial, venular, and capillary-like ECs when implanted in vivo, and induce arteriogenic remodeling of the local mouse vessels. Our results support the utility of differentiated EC transplantation to treat tissue ischemia.

The use of temperature–composition combinatorial libraries to study the effects of biodegradable polymer blend surfaces on vascular cells

Controlling cellular and physiological responses such as adhesion, proliferation and migration is a highly desirable feature of engineered scaffolds. One important application would be the design of tissue engineered vascular grafts that regulate cell adhesion and growth. We utilized temperature–composition combinatorial polymer libraries to investigate the effects of surfaces of blended poly( d, l-lactic- co-glycolic acid) (PLGA) and poly( ε-caprolactone) (PCL) on murine vascular smooth muscle cells (SMC). In this manner, SMCs were exposed to ∼1000 distinguishable surfaces in a single experiment, allowing the discovery of optimal polymer compositions and processing conditions. SMC adhesion, aggregation, proliferation, and protein production were highest in regions with mid- to high-PCL concentrations and high annealing temperatures. These regions exhibited increased surface roughness, increased microscale PLGA-rich matrix stiffness, and significant change of bulk PCL-rich crystallinity relative to other library regions. This study revealed a previously unknown processing temperature and blending composition for two well-known polymers that optimized SMC interactions.

Unraveling gene-gene interactions regulated by ligands of the aryl hydrocarbon receptor.

The co-expression of genes coupled to additive probabilistic relationships was used to identify gene sets predictive of the complex biological interactions regulated by ligands of the aryl hydrocarbon receptor ((Italic)Ahr(/Italic)). To maximize the number of possible gene-gene combinations, data sets from murine embryonic kidney, fetal heart, and vascular smooth muscle cells challenged (Italic)in vitro(/Italic) with ligands of the (Italic)Ahr(/Italic) were used to create predictor/training data sets. Biologically relevant gene predictor sets were calculated for (Italic)Ahr(/Italic), cytochrome P450 1B1, insulin-like growth factor-binding protein-5, lysyl oxidase, and osteopontin. Transcript levels were categorized into ternary expressions and target genes selected from the data set and tested for all possible combinations using three gene sets as predictors of transitional level. The goodness of prediction for each set was quantified using a multivariate nonlinear coefficient of determination. Evidence is presented that predictor gene combinations can be effectively used to resolve gene-gene interactions regulated by (Italic)Ahr(/Italic) ligands. (Italic)Key words:(/Italic) aryl hydrocarbon receptor, bioinformatics, gene networks, genomics. (Italic)Environ Health Perspect (/Italic)112:403-412 (2004). [Online 14 January 2004]

Platelet-derived growth factor (PDGF)-induced chemotaxis does not require the G protein-coupled receptor S1P 1 in murine embryonic fibroblasts and vascular smooth muscle cells

Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, signals via G protein-coupled receptors (GPCR). The prototypical S1P receptor, S1P 1 (also known as EDG-1), a G i-linked receptor, is critical for vascular maturation during development. Recent work suggested that platelet-derived growth factor (PDGF)-induced cell migration required the S1P 1 receptor, representing a novel mechanism for cross-talk between receptor tyrosine kinases and GPCRs. Since both S1P and PDGF are implicated in vascular smooth muscle cell (VSMC) pathobiology and development, we investigated this issue in rat VSMC and in embryonic fibroblasts derived from S1P 1 null mice. Our data suggest that the S1P 1 receptor is critical for S1P-induced, G i-dependent migration but not for PDGF-BB-induced, receptor tyrosine kinase-dependent chemotaxis in VSMC. In addition, lack of S1P 1 receptor in mouse embryonic fibroblasts did not significantly affect PDGF-induced cell migration. These data question the generality of the concept that S1P 1 GPCR is a critical downstream component of PDGF-induced chemotaxis.

Effect of Perilla frutescens on nitric oxide production and DNA synthesis in cultured murine vascular smooth muscle cells.

The effects of perilla (Perilla frutescens, Labiatae) on murine cultured vascular smooth muscle cells (VSMC) were investigated. The water extract of perilla leaves induced nitric oxide (NO) production of VSMC and this effect was synergistically augmented when combined with interferon (IFN)-gamma or tumour necrosis factor (TNF)-alpha, while the perilla extract significantly inhibited NO production induced by IFN-gamma combined with lipopolysaccharide (LPS). Northern blot analysis revealed that these effects of the perilla extract paralleled mRNA expression of inducible nitric oxide synthase. However, the perilla extract significantly inhibited platelet derived growth factor (PDGF) or TNF-alpha-induced VSMC proliferation measured as DNA synthesis. The inhibitory effect of the perilla extract on TNF-alpha-induced VSMC proliferation was significantly suppressed by N(G)-monomethyl-L-arginine, a non-specific nitric synthase inhibitor, suggesting that this effect was partially mediated by NO production as an autocrine/paracrine factor. The present findings suggest that perilla would be useful for the prevention of vascular diseases such as arteriosclerosis.

Constitutive and inducible expression of Cyp1a1 and Cyp1b1 in vascular smooth muscle cells: role of the Ahr bHLH/PAS transcription factor.

Ahr is a ligand-activated bHLH/PAS transcription factor involved in cytochrome P450 (CYP) gene regulation and murine susceptibility to atherogenic stimuli. The present studies were conducted to examine constitutive and inducible expression of Cyp1a1 and Cyp1b1 in vascular smooth muscle cells (VSMCs) from Ahr(+/+) and Ahr(-/-) mice. Cyp1a1 mRNA was not expressed constitutively in VSMCs irrespective of Ahr phenotype. Although Cyp1a1 was inducible in Ahr(+/+) by 3 micromol/L benzo(a)pyrene, a known hydrocarbon inducer, the protein was uninducible. In contrast, Cyp1b1 mRNA and protein were expressed under constitutive and inducible conditions irrespective of Ahr phenotype or growth status. CYP-encoded aryl hydrocarbon hydroxylase activity was higher in Ahr(-/-) VSMCs under constitutive conditions and induced by benzo(a)pyrene in Ahr(+/+) and Ahr(-/-) VSMCs. CYP expression was influenced by mitogenic status, because randomly cycling cells consistently exhibited higher levels than growth-arrested counterparts. Actinomycin D (2 microgram/mL) or cycloheximide (10 micromol/L) did not inhibit constitutive or hydrocarbon-inducible aryl hydrocarbon hydroxylase activity in VSMCs. These data indicate that in murine VSMCs, expression of Cyp1al and Cyp1b1 is differentially influenced by Ahr phenotype and mitogenic status, with patterns that may dictate inherent susceptibility to atherogenic stimuli.

Lipopolysaccharide can activate BK channels of arterial smooth muscle in the absence of iNOS expression

The role of inducible nitric oxide synthase (iNOS) in the acute activation of large-conductance, Ca 2+-dependent K + channels (BK channels) by Escherichia coli endotoxin (lipopolysaccharide, LPS) was studied in murine vascular smooth muscle cells. Confocal laser scanning microscopy and patch clamp recordings were utilised. Within 2 h of donor rat sacrifice, iNOS-like immunoreactivity could be detected in cerebrovascular smooth muscle cells (CVSMCs) enzymatically dispersed from rat cerebral arteries. This staining was absent in cells fixed immediately after donor rat sacrifice. LPS was then applied to the cytoplasmic face of inside-out membrane patches excised from rat CVSMCs within 2–4 h of donor rat sacrifice. It was found that LPS (10–100 μg/ml) rapidly and reversibly increased the open probability of BK channels in these patches. This LPS response was not altered in the presence of the non-isoform specific NOS inhibitor N ω-nitro- l-arginine. LPS responses were then compared in aortic smooth muscle (ASMC) BK channels derived from wild-type and iNOS-knockout (iNOS-KO) mice. LPS activated BK channels in inside-out patches of ASMC membrane derived from both wild-type and iNOS-knockout mice. These studies establish that LPS can activate BK channels by a mechanism quite independent of the well-established pathway mediated by iNOS in vascular smooth muscle cells.

Sorting of Murine Vascular Smooth Muscle Cells during Wound Healing in the Chicken Chorioallantoic Membrane

The vascular wall is built up of a heterogeneous population of smooth muscle cells, which exhibit not only morphological distinctions but also important differences in the composition of their structural and contractile proteins. “Epithelioid” smooth muscle cells correspond to an intimal-like type and display features associated with immaturity, whereas “spindle-shaped” cells closely resemble the more typical medial smooth muscle population. We have investigated the integration of these two cell types into the vascular architecture of an in vivo wound-healing model. Stably transfected with the β-galactosidase gene, intima- and media-like cells were injected intravenously into the chicken chorioallantoic membrane, within which superficial foci of granulation tissue had been created by thermal or chemical injury. At 24 to 72 h after injection, cells had homed in on the lesion sites and were observed in juxtaposition to the endothelial lining of the capillaries. They began to deposit laminin, thereby indicating an impending role in the formation of the vascular wall. Intima- and media-like smooth muscle cells did not differ in their capacity to associate with capillaries, and, in so doing, their biochemical lineage characteristics became indistinguishable from one another. However, intima-like cells also penetrated the adventitial and medial layers of arteries. These findings reveal vascular smooth muscle cells to possess an extraordinary degree of plasticity, being able to adapt flexibly to changes in functional demands.

CGMP accumulation and gene expression of soluble guanylate cyclase in human vascular tissue.

Gene expression of soluble guanylate cyclase (sGC) and cGMP accumulation in response to sodium nitroprusside (SNP) were studied in cultured human vascular cells and freshly harvested vascular tissue. As revealed by reverse transcriptase-polymerase chain reaction, cultured smooth muscle and endothelial cells, as well as freshly isolated human vascular tissue, express mRNA for the alpha 3 and beta 3 subunits but not for the alpha 2 and beta 3 subunits is evident even in the absence of increased cGMP accumulation in response to SNP. cGMP accumulation in human cells cultured from different vascular beds typically increased two- to five-fold (maximum of 11.4-fold) over baseline following stimulation with 100 microM SNP. Bovine, murine, canine, and avian vascular smooth muscle cells accumulated similar or lower amounts of cGMP than human cells, whereas porcine, rat, and rabbit smooth muscle cells accumulated greater amounts of cGMP. In freshly harvested human vessels, cGMP accumulation in response to SNP was found to increase fifteen-fold over baseline. In contrast to the SNP-induced cGMP accumulation, cGMP levels in response to particulate guanylate cyclase activator atriopeptin II were equal to or greater in cultured human cells than in fresh human vascular tissue. We conclude that human vascular cells (fresh and cultured) express the mRNA for both a large (alpha 3) and a small (beta 3) sGC subunit and that fresh human cells are more sensitive to SNP stimulation.

Oxidant Stress Enhances Adenylyl Cyclase Activation

The generation of oxygen-derived free radicals has been implicated in the disordered vascular regulation of inflammation and reperfusion. In the vasculature, oxygen-derived free radicals are vasodilatory. The mechanisms underlying this effect remain unclear. To examine the cellular processes involved, we studied the effects of hydrogen peroxide (H2O2) on adenylyl cyclase activity in A10 cells, a murine vascular smooth muscle cell line. Pretreatment with H2O2 caused a dose-dependent enhancement of forskolin-stimulated adenylyl cyclase activity (ED50, 44 mumol/L to a maximum of 166% of control activity; n = 4). This enhancement was attenuated by iron chelation with deferoxamine and by the intracellular hydroxyl scavenger dimethylthiourea and mimicked by preincubation with purine/xanthine oxidase either alone or in the presence of superoxide dismutase. The effects of H2O2 were completely blocked by the tyrosine kinase inhibitors genistein and tyrphostin A9 but not by its inactive analogue tyrphostin A1 (H2O2 alone, 149 +/- 13%; H2O2 + tyrphostin A9, 100 +/- 9%; H2O2 + tyrphostin A1, 171 +/- 21%; n = 4). H2O2 comparably enhanced adenylyl cyclase activity stimulated by isoproterenol (166 +/- 17% of control, n = 5) and sodium fluoride (177 +/- 18% of control, n = 5). Thus oxygen-derived free radicals enhance adenylyl cyclase activation, probably via tyrosine kinase-mediated effects on the catalytic subunit of adenylyl cyclase. Sensitization of adenylyl cyclase activation may be an important mechanism by which free radicals modulate hormone-mediated vasodilation.

T Cell—Vascular Smooth Muscle Cell Interactions: Antigen-Specific Activation and Cell Cycle Blockade of T Helper Clones by Cloned Vascular Smooth Muscle Cells

Histological observations have demonstrated the presence of T lymphocytes in atherosclerotic plaques, often in close association with vascular smooth muscle cells (VSMC). We have examined the interaction occurring between cloned murine VSMC and histocompatibitity-matched, antigen-specific Th1 and Th2 cell lines. Incubation of either Th1 or Th2 cells with antigen-pulsed VSMC resulted in the formation of T cell-VSMC conjugates accompanied by morphological changes in both cell types. This interaction resulted in an antigen-dependent activation of IL-2 receptor expression by the Th cells, demonstrating the ability of cloned VSMC to process and present antigen through the exogenous pathway. However, although the T cells were activated to express IL-2 receptors by antigen-pulsed VSMC, they were unable to progress through cell cycle. The secretion of an inhibitory mediator by VSMC was suggested by the observations that (1) fixation of the VSMC's eliminated the inhibitory signal and (2) the supernatants of IFNγ-primed VSMC displayed similar inhibitory activity. The inhibitory effect could not be abrogated with indomethacin or an inhibitor of the generation of reactive nitrogen intermediates, indicating that prostaglandin synthesis and/or nitric oxide production are not solely responsible for the inhibition of proliferation. Flow cytometric cell cycle analysis revealed that VSMC delivered signals resulting in a late G1 blockade of T cell cycle progression. Mitogen responses of purified primary T cells are also dramatically inhibited by IFNγ-treated VSMC, despite significant IL-2 production. Our data depict a complex and intimate T cell-VSMC interaction and suggest that mutual activation events may occur.

Release of the cytokines colony-stimulating factor-1, granulocyte-macrophage colony-stimulating factor, and IL-6 by cloned murine vascular smooth muscle cells.

Vascular smooth muscle cells were cultured from the mesenteric arteries of MRL lpr/lpr, MRL +/+, CBA/J, or C3H/HeJ mice and evaluated for their ability to synthesize a range of cytokines. Vascular smooth muscle cells of MRL +/+, MRL lpr/lpr, and CBA/J origin released biologically significant amounts of CSF-1 and IL-6 and relatively low but detectable amounts of granulocyte macrophage-CSF (GM-CSF) but not IL-2, IL-3, or IL-4. Vascular smooth muscle cells of C3H/HeJ origin produced lower amounts of CSF-1 and IL-6, and GM-CSF was barely detectable. Production of these cytokines did not require the exogenous growth factors present in FCS and occurred, although at lower levels, in serum-free medium supplemented with insulin, transferrin, and albumin. Cloned lines of MRL +/+ vascular smooth muscle cells, with electron microscopic and immunochemical properties of vascular smooth muscle cells, produced CSF-1, IL-6, and GM-CSF, establishing that vascular smooth muscle cells were a direct source of CSF-1, IL-6, and GM-CSF. These observations highlight the need for experiments to directly address the question of whether vascular smooth muscle cells constitutively produce these cytokines under physiologic conditions in vivo and suggest that vascular smooth muscle cells may participate actively in inflammation by releasing cytokines that are active on lympho-hemopoietic and other cells.