Increased retinoid signaling in vascular smooth muscle cells by proinflammatory cytokines

Retinoids have been shown to modulate inflammation and the immune response in many cell types including macrophages, endothelial cells, and vascular smooth muscle cells. However, present knowledge of whether inflammatory mediators modulate vitamin A status in these cells is limited. To identify the role of inflammation on retinoid metabolism in vascular smooth muscle cells, the cells were exposed to a combination of proinflammatory cytokines: interleukin-1beta, interferon-gamma, and lipopolysaccharides. Without stimulation with proinflammatory cytokines, vascular smooth muscle cells expressed retinol dehydrogenases-2 and 5 mRNA detected by RT-PCR. Stimulation with the combination of cytokines induced a substantial increase of retinol dehydrogenase-5 mRNA. This was associated with increased production of ligands for retinoic acid receptors, when assayed in a retinoic acid receptor-dependent luciferase reporter system. Our results demonstrate that inflammatory mediators activate the retinoid metabolic pathway in vascular smooth muscle cells, which potentially may modulate the inflammatory response in the vascular wall.     

Pulmonary arterial smooth muscle cells modulate cytokine- and LPS-induced cytotoxicity in endothelial cells

Cytokines and lipopolysaccharide (LPS) are known to be injurious to vascular endothelial cells (ECs), but the influence of adjacent vascular smooth muscle cells (SMCs) on this injury is unknown. Exposure of cultured rat (RPMECs) or human (HPMECs) pulmonary microvascular ECs on tissue culture plastic to a mixture of cytokines (interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma) and LPS (cytomix) resulted in a significant increase in (51)Cr release to 35-40%. When unstimulated RPMECs were cocultured with cytomix-pretreated rat pulmonary microvascular SMCs (RPMSMCs) there was an increase in (51)Cr release to 8.4%, which was nitric oxide dependent. However, when RPMECs or HPMECs were stimulated in direct contact with their respective SMCs, rather than a further increase in cytomix-induced injury (e.g., >35-40%), (51)Cr release decreased to <10%. This cytoprotection was fully reproduced with fixed RPMSMCs, and partially reproduced by plating HPMECs on gelatin. These data show that the direct toxicity of a cytokine and endotoxin mixture on cultured ECs can be reduced by contact with vascular smooth muscle.     

Possible involvement of tyrosine kinase in the LPS-Promoted initiation of l-arginine-induced relaxation of rat aorta mediated by induction of no synthase

Tyrosine kinase inhibitors herbimycin A, genistein and erbstatin analog prevented endotoxin (LPS)-promoted initiation of l-arginine (Arg)-induced relaxations and cGMP formation in rat thoracic aorta, which appear to be mediated by nitric oxide synthase expressed by LPS in the vascular smooth muscle. Similarly, interleukin-1β (IL-1β) triggered initiation of Arg-induced relaxation of the arteries. In addition, in the aortic smooth muscle cells cultured in the presence of Arg, LPS- or IL-1β-triggered accumulation of nitrite was suppressed by the tyrosine kinase inhibitors. These results suggest that tyrosine kinase is involved in the LPS- and IL-1β-promoted induction of nitric oxide synthase in the vascular smooth muscle, which in turn mediates production of NO from added Arg, thus stimulating formation of cGMP and causing relaxation. Alternatively, it is possible that LPS acts indirectly through cytokines such as IL-1β.     

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.     

Vascular mechanisms of increased arterial pressure in preeclampsia: lessons from animal models

Normal pregnancy is associated with reductions in total vascular resistance and arterial pressure possibly due to enhanced endothelium-dependent vascular relaxation and decreased vascular reactivity to vasoconstrictor agonists. These beneficial hemodynamic and vascular changes do not occur in women who develop preeclampsia; instead, severe increases in vascular resistance and arterial pressure are observed. Although preeclampsia represents a major cause of maternal and fetal morbidity and mortality, the vascular and cellular mechanisms underlying this disorder have not been clearly identified. Studies in hypertensive pregnant women and experimental animal models suggested that reduction in uteroplacental perfusion pressure and the ensuing placental ischemia/hypoxia during late pregnancy may trigger the release of placental factors that initiate a cascade of cellular and molecular events leading to endothelial and vascular smooth muscle cell dysfunction and thereby increased vascular resistance and arterial pressure. The reduction in uterine perfusion pressure and the ensuing placental ischemia are possibly caused by inadequate cytotrophoblast invasion of the uterine spiral arteries. Placental ischemia may promote the release of a variety of biologically active factors, including cytokines such as tumor necrosis factor-alpha and reactive oxygen species. Threshold increases in the plasma levels of placental factors may lead to endothelial cell dysfunction, alterations in the release of vasodilator substances such as nitric oxide (NO), prostacyclin (PGI(2)), and endothelium-derived hyperpolarizing factor, and thereby reductions of the NO-cGMP, PGI(2)-cAMP, and hyperpolarizing factor vascular relaxation pathways. The placental factors may also increase the release of or the vascular reactivity to endothelium-derived contracting factors such as endothelin, thromboxane, and ANG II. These contracting factors could increase intracellular Ca(2+) concentrations ([Ca(2+)](i)) and stimulate Ca(2+)-dependent contraction pathways in vascular smooth muscle. The contracting factors could also increase the activity of vascular protein kinases such as protein kinase C, leading to increased myofilament force sensitivity to [Ca(2+)](i) and enhancement of smooth muscle contraction. The decreased endothelium-dependent mechanisms of vascular relaxation and the enhanced mechanisms of vascular smooth muscle contraction represent plausible causes of the increased vascular resistance and arterial pressure associated with preeclampsia.     

The role of nitric oxide in motoneuron spike activity and muscarinic-evoked changes in cGMP in the CNS of larval Manduca sexta

Nitric oxide and muscarinic agonists both stimulate motoneuron spike activity and cGMP production in the central nervous system of larval Manduca sexta. The possible role of nitric oxide in mediating muscarinic changes in excitability was examined by measuring cGMP accumulation and proleg motoneuron activity while blocking or mimicking the production of nitric oxide. All the muscarinic-induced changes in cGMP are blocked by the nitric oxide-synthase inhibitor, nitro-l-arginine, an effect that is partially prevented by co-incubation with arginine. Action potential blockage with tetrodotoxin revealed that muscarinic increases in cGMP production have both spike-dependent and spike-independent mechanisms. Furthermore, nitric oxide donors can increase proleg motoneuron activity and this stimulation is blocked by 1H-{1,2,4}oxadiazolo{4, 3-a}quinoxalin-1-one suggesting that it is mediated by a nitric oxide-sensitive guanylyl cyclase. In contrast, nitro-l-arginine and a variety of other nitric oxide-synthase inhibitors and nitric oxide scavengers have no significant effect on muscarinic stimulation of motoneuron activity. Therefore, although a nitric oxide sensitive guanylyl cyclase is capable of elevating spike activity and muscarinic agonists can increase cGMP, this mechanism is not necessary for the normal muscarinic increase in excitability. It is concluded that muscarinic receptors are coupled to nitric oxide and cGMP production in neurons other than those controlling the prolegs. Accepted: 22 July 1999     

Regulation of the nitric oxide synthase-nitric oxide-cGMP pathway in rat mesenteric endothelial cells.

Most of the available data on the nitric oxide (NO) pathway in the vasculature is derived from studies performed with cells isolated from conduit arteries. We investigated the expression and regulation of components of the NO synthase (NOS)-NO-cGMP pathway in endothelial cells from the mesenteric vascular bed. Basally, or in response to bradykinin, cultured mesenteric endothelial cells (MEC) do not release NO and do not express endothelial NOS protein. MEC treated with cytokines, but not untreated cells, express inducible NOS (iNOS) mRNA and protein, increase nitrite release, and stimulate cGMP accumulation in reporter smooth muscle cells. Pretreatment of MEC with genistein abolished the cytokine-induced iNOS expression. On the other hand, exposure of MEC to the microtubule depolymerizing agent colchicine did not affect the cytokine-induced increase in nitrite formation and iNOS protein expression, whereas it inhibited the induction of iNOS in smooth muscle cells. Collectively, our findings demonstrate that MEC do not express endothelial NOS but respond to inflammatory stimuli by expressing iNOS, a process that is blocked by tyrosine kinase inhibition but not by microtubule depolymerization.     

Induction of nitric oxide synthase by lipoteichoic acid from Staphylococcus aureus in vascular smooth muscle cells.

Inducible vascular nitric oxide synthase accounts for the contractile impairment observed in endotoxemia. We provide evidence that lipoteichoic acid (LTA) from Staphylococcus aureus, a micro-organism without endotoxin, also induces nitric oxide synthase. Our study demonstrates that on endothelium-free rings of rat aorta. LTA-like lipopolysaccharide induces a loss of contractility restored by Methylene blue and NG-nitro-L-arginine-methyl ester (LNAME). Moreover in cultured vascular smooth muscle cells, LTA produces a dose-dependent increase in intracellular cyclic GMP which is antagonized by LNAME and prevented by dexamethasone.     

High glucose induced rat aorta vascular smooth muscle cell oxidative injury: involvement of protein tyrosine nitration

The alteration and further damage of vascular smooth muscle function have been implicated in the development of vascular complications and diabetes. Little is known about protein tyrosine nitration in vascular smooth muscle cell injury induced by high glucose. In this article, vascular smooth muscle cell was exposed to 30 and 40 mM high glucose for 72 h, and then the cell injury in vascular smooth muscle cell induced by high glucose was studied. It was found that high glucose stimulated vascular smooth muscle cell injury in a dose-dependent manner, including decreasing intracellular and extracellular glutathione contents, increasing malondialdehyde and intracellular reactive oxygen species content, increasing the production of nitric oxide (increased nitrite content in cell and medium), as well as increasing protein tyrosine nitration. By comparing protein tyrosine nitration induced by high glucose conditions and extrinsic factors (hemin–nitrite–glucose oxidase system and 3-morpholinosydnonimine), it may be speculated that protein is nitrated selectively, and specific protein tyrosine nitration is involved in diabetic vascular complications.     

Vascular smooth muscle-derived relaxing factor (MDRF) and its close similarity to nitric oxide

The principal finding in this study is that vascular smooth muscle generates a labile relaxing factor that possesses pharmacological and chemical properties that are similar to those of authentic nitric oxide. MDRF was generated by perfusion of endothelium-denuded bovine pulmonary artery as assessed by bioassay. In addition, endothelium-denuded arterial rings that were incubated at 37 degrees C for 24 hr to lower endogenous L-arginine levels relaxed in response to L-arginine but not D-arginine. Freshly mounted, endothelium-denuded arterial rings were not relaxed by L-arginine but did relax in response to the dipeptide L-arginyl-L-alanine. Relaxant responses were accompanied by increases in smooth muscle levels of cyclic GMP and nitrite, and were inhibited by oxyhemoglobin, methylene blue, and NG-nitro-L-arginine. NG-Nitro-L-arginine also caused endothelium-independent contractile responses. Thus, a relaxing factor with the properties of nitric oxide can be generated from vascular smooth muscle.     

Regulation of adrenomedullin secretion from cultured cells.

Characterization of immunoreactive adrenomedullin (AM) secreted from cultured human vascular smooth muscle cells and 7 other cells indicates that AM is synthesized and secreted from all cultured cells we surveyed. The secretion rate of AM measured ranges from 0.001-6.83 fmol/10(5) cells/h, and endothelial cells, vascular smooth muscle cells and fibroblasts generally secrete AM at high rates. Based on the results of regulation of AM secretion from vascular wall cells, fibroblasts, macrophages and other cells measured in this and previous studies, AM secretion is found to be generally stimulated by inflammatory cytokines, lipopolysaccharide (LPS) and hormones. Especially, vascular smooth muscle cells and fibroblasts elicited uniform and strong stimulatory responses of AM secretion to tumor necrosis factor (TNF), interleukin-1 (IL-1), LPS and glucocorticoid, but endothelial cells did not elicit such prominent responses. AM secretion of monocyte-macrophage was mainly regulated by the degree of differentiation into macrophage and activation by LPS and inflammatory cytokines including interferon-gamma. The other examined cells showed weaker responses to LPS and IL-1. Although cultured cells may have been transformed as compared with those in the tissue, these data indicate that AM is widely synthesized and secreted from most of the cells in the body and functions as a local factor regulating inflammation and related reactions in addition to as a potent vasodilator. The responses of AM secretion to LPS and inflammatory cytokines suggest that fibroblasts, vascular smooth muscle cells and macrophage are the major sources of AM in the septic shock.     

Release of nitric oxide from human vascular smooth muscle cells

It has recently been shown that nitric oxide (NO) or a labile NO-releasing compound is produced in endothelial cells. In the present study we measured the NO-release from human vascular smooth muscle cells in culture. The cells released an average 2.2 x 10(-9) moles nitric oxide per 10(8) cells in ten minutes with a large variation between different cell lines and passages without stimulators. The NO-release was markedly reduced by the inhibitor of NO-formation NG-momomethyl-L-arginine (L-NMMA, 10(-5) M) to 3% of the control levels (p less than 0.02, n = 4), but unaffected by acetylcholine, bradykinin or endothelin -1, -2 or -3. In a microperfusion system the compound released from human vascular smooth muscle cells caused profound relaxation of isolated rat mesenteric resistance arteries. Thus, human vascular smooth muscle cells in culture produce and release biologically active NO from L-arginine.     

Cellular actions of testosterone in vascular cells: Mechanism independent of aromatization to estradiol

In this work we investigated the role of testosterone on cellular processes involved in vascular disease, and whether these effects depend on its local conversion to estradiol. Cultures of rat aortic endothelial and smooth muscle cells in vitro treated with physiological concentrations of testosterone were employed. Testosterone rapidly increased endothelial nitric oxide production. To evaluate whether this non genomic action was dependent on testosterone aromatization we used an aromatase inhibitor. Anastrozole compound did not modify the fast increase in nitric oxide production elicited by testosterone. The hormonal effect was completely blocked by an androgen receptor antagonist (flutamide); meanwhile it wasn′t modified by the presence of an estrogen receptor antagonist (ICI182780).The possibility of intracellular estradiol synthesis was ruled out when no differences were found in estradiol measurements performed in culture incubation medium from control and testosterone treated cells. The 5α-reductase inhibitor finasteride partially suppressed the enhancement in nitric oxide production, suggesting that the effect of testosterone was partially due to dihydrotestosterone conversion. Testosterone stimulated muscle cell proliferation independent of local conversion to estradiol. When cellular events that play key roles in vascular disease development were analyzed, testosterone prevented monocyte adhesion to endothelial cells induced by a proinflammatory stimulus (bacterial lipopolysaccharides), and prompted muscle cell migration in presence of a cell motility inducer. In summary, testosterone modulates vascular behavior through its direct action on vascular cells independent of aromatization to estradiol. The cellular actions exhibited by the steroid varied whether cells were under basal or inflammatory conditions.     

Defective smooth muscle regulation in cGMP kinase I-deficient mice.

Regulation of smooth muscle contractility is essential for many important biological processes such as tissue perfusion, cardiovascular haemostasis and gastrointestinal motility. While an increase in calcium initiates smooth muscle contraction, relaxation can be induced by cGMP or cAMP. cGMP-dependent protein kinase I (cGKI) has been suggested as a major mediator of the relaxant effects of both nucleotides. To study the biological role of cGKI and its postulated cross-activation by cAMP, we inactivated the gene coding for cGKI in mice. Loss of cGKI abolishes nitric oxide (NO)/cGMP-dependent relaxation of smooth muscle, resulting in severe vascular and intestinal dysfunctions. However, cGKI-deficient smooth muscle responded normally to cAMP, indicating that cAMP and cGMP signal via independent pathways, with cGKI being the specific mediator of the NO/cGMP effects in murine smooth muscle.     

Cyclooxygenase-2 induction by bradykinin in aortic vascular smooth muscle cells

Vascular smooth muscle cell proliferation and migration play an important role in the pathophysiology of several vascular diseases, including atherosclerosis. Prostaglandins that have been implicated in this process are synthesized by two isoforms of cyclooxygenase (COX), with the expression of the regulated COX-2 isoform increased in atherosclerotic plaques. Bradykinin (BK), a vasoactive peptide increased in inflammation, induces the formation of prostaglandins through specific receptor activation. We hypothesized that BK plays an important role in the regulation of COX-2, contributing to the increase in production of prostaglandins in vascular smooth muscle cells. Herein we examined the signaling pathways that participate in the BK regulation of COX-2 protein levels in primary cultured aortic vascular smooth muscle cells. We observed an increase in COX-2 protein levels induced by BK that was maximal at 24 h. This increase was blocked by a B2 kinin receptor antagonist but not a B1 receptor antagonist, suggesting that the B2 receptor is involved in this pathway. In addition, we conclude that the activation of mitogen-activated protein kinases p42/p44, protein kinase C, and nitric oxide synthase is necessary for the increase in COX-2 levels induced by BK because either of the specific inhibitors for these enzymes blocked the effect of BK. Using a similar approach, we further demonstrated that reactive oxygen species and cAMP were not mediators on this pathway. These results suggest that BK activates several intracellular pathways that act in combination to increase COX-2 protein levels. This study suggests a role for BK on the evolution of the atheromatous plaque by virtue of controlling the levels of COX-2.     

Signaling across myoendothelial gap junctions--fact or fiction?

Gap junctions interconnect vascular cells homocellularly, thereby allowing the spread of signals along the vessel wall, which serve to coordinate vessel behavior. In addition, gap junctions provide heterocellular coupling between endothelial and vascular smooth muscle cells, creating so-called myoendothelial gap junctions (MEGJs). Endothelial cells control vascular tone by the release of factors that relax vascular smooth muscle. Endothelial factors include nitric oxide, prostaglandins, and an additional dilator principle, which acts by smooth muscle hyperpolarization and is therefore named endothelium-derived hyperpolarizing factor (EDHF). Whether this principle indeed relies on a factor or on intact MEGJs, which allow direct current transfer from endothelial to smooth muscle cells, has recently been questioned. Careful studies revealed the presence of vascular cell projections that make contact through the internal elastic lamina, exhibit the typical GJ morphology, and express connexins in many vessels. The functional study of the physiological role of MEGJs is confined by the difficulty of selectively blocking these channels. However, in different vessels studied in vitro, the dilation related to EDHF was sensitive to experimental interventions that block MEGJs more or less specifically. Additionally, bidirectional electrical coupling between endothelial and smooth muscle cells was demonstrated in isolated small vessels. In marked contrast, similar approaches used in conjunction with intravital microscopy, which allows examination of vascular behavior in the intact animal, did not verify electrical or dye-coupling in different models investigated. The discrepancy between in vitro and in vivo investigations may be due to size and origin of the vessels studied using these distinct experimental approaches. Additionally, MEGJ coupling is possibly tightly controlled in vivo by yet unknown mechanisms that prevent unrestricted direct signaling between endothelial and smooth muscle cells.     

Insulin regulates its own delivery to skeletal muscle by feed-forward actions on the vasculature

Insulin, at physiological concentrations, regulates the volume of microvasculature perfused within skeletal and cardiac muscle. It can also, by relaxing the larger resistance vessels, increase total muscle blood flow. Both of these effects require endothelial cell nitric oxide generation and smooth muscle cell relaxation, and each could increase delivery of insulin and nutrients to muscle. The capillary microvasculature possesses the greatest endothelial surface area of the body. Yet, whether insulin acts on the capillary endothelial cell is not known. Here, we review insulin's actions at each of three levels of the arterial vasculature as well as recent data suggesting that insulin can regulate a vesicular transport system within the endothelial cell. This latter action, if it occurs at the capillary level, could enhance insulin delivery to muscle interstitium and thereby complement insulin's actions on arteriolar endothelium to increase insulin delivery. We also review work that suggests that this action of insulin on vesicle transport depends on endothelial cell nitric oxide generation and that insulin's ability to regulate this vesicular transport system is impaired by inflammatory cytokines that provoke insulin resistance.     

Nitric oxide stimulated vascular smooth muscle cells undergo apoptosis induced in part by arachidonic acid derived eicosanoids

The role of eicosanoids in atherogenesis has not been thoroughly explained. This is partly due to the numerous eicosanoids and the variable effects that each has on different systems. Apoptosis of vascular smooth muscle cells has been shown to play a role in the atherosclerotic disease leading to lesion formation and further destabilization of the formed lesion. In this study, we have investigated the role of arachidonic acid derived eicosanoids in nitric oxide (NO)-stimulated vascular smooth muscle cells. We have shown previously that the nitric oxide (NO)-induced apoptosis of vascular smooth muscle cells was accompanied by arachidonic acid release via cytoplasmic phospholipase A(2) (cPLA(2)) activation. Also, arachidonic acid, but not oleic acid, induced apoptosis of these cells at low concentrations (5-10 microM). Our results revealed that the cPLA(2) specific inhibitor, arachidonyl trifluoromethyl ketone (AACOCF(3)), blocked NO-induced eicosanoid production, while the presence of arachidonic acid enhanced the ability of the cells to make prostaglandin E(2) (PGE(2)). Also, inhibitors of the cyclo-oxygenase (Cox) enzymes, such as N-[2-cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398), a specific Cox-2 inhibitor, or indomethacin, a non-specific Cox inhibitor, blocked NO-induced PGE(2) production and apoptosis of vascular smooth muscle cells to the same extent, indicating that apoptosis might be induced by a Cox-2 metabolic product. In addition to these observations, the eicosanoids investigated, namely, PGE(2), PGI(2) LTB(4), and PGJ(2), showed different effects on vascular smooth muscle cells. Both PGJ(2) and LTB(4) decreased the percentage of viable cells and induced apoptosis of vascular smooth muscle cells, while PGE(2) and PGI(2) had no effect on cell viability and failed to induce apoptosis. These data suggest that eicosanoids, such as PGJ(2), but not PGE(2) or PGI(2), are involved in NO-induced apoptosis of vascular smooth muscle cells and that the eicosanoid synthesis pathways might be utilized for vascular therapeutic strategies.