Catecholamine-mediated constrictor effects of aldosterone on vascular smooth muscle

The possibility that the corticosteroid hormone, aldosterone, might possess direct vasoconstrictor properties was examined in the isolated central ear artery of the New Zealand white rabbit. Aldosterone alone produced only minimal contractile effects in the arterial segments; but following pretreatment of the tissue with desipramine (10^?7M), a blocker of neuronal uptake of norepinephrine, aldosterone concentrations of 10^?6M, 10^?5M, and 10^?4M produced stepwise contractile responses of 0.16 ± 0.03 (SE)g, 0.48 ± .04g, and 1.31 ± 0.06g. The possible involvement of norepinephrine in this action of aldosterone was tested in a series of tissues stored for 2 days at 2°C in Krebsbicarbonate medium so as to deplete endogenous catecholamine stores. Treatment with desipramine followed by aldosterone (10^?4M) now produced an average contraction of only 0.1 ± 0.06g; but if the labile neuronal tissue stores of norepinephrine in these tissues were then replenished by exposure to norepinephrine 10^?7M, contractions of 1.2 ± 0.3g now occurred when desipramine and aldosterone were added. To examine whether aldosterone's action might be due to blockade of extraneuronal norepinephrine uptake (uptake-2), ³H-norepinephrine was added to ear artery tissues exposed to desipramine with or without aldosterone: a significant (P<0.005) decrease of 25% in ³H-norepinephrine uptake occurred in the tissues treated with aldosterone. In further studies, the contractile effects of aldosterone could be prevented by pretreatment with prazosin (10^?7M) or phentolamine (10^?7M); if added after the aldosterone, each of these alpha-blockers completely reversed the contractile responses. Although the physiological relevance of these findings is yet to be fully defined, these studies indicate that the $\text{in}$$\text{vitro}$ contractile effects of aldosterone are dependent upon its inhibition of extraneuronal uptake of endogenous norepinephrine; it is likely that the resulting increase in extracellular norepinephrine concentration then produces vasoconstriction by stimulation of post-synaptic alpha-adrenergic receptors.     

Rapid actions of aldosterone: lymphocytes, vascular smooth muscle and endothelial cells.

The genomic theory of steroid action has been the unquestioned dogma for the explanation of steroid effects over the past four decades. Despite early observations on rapid steroid effects being clearly incompatible with this theory, only recently has nongenomic steroid action been recognized more widely and led to a critical reappraisal of unsolved questions about this dogma. Evidence for nongenomic steroid effects come from all fields of steroid research now, and mechanisms of agonist action are studied with regard to membrane receptors and second messengers involved. A prominent example of a receptor/effector-cascade for nongenomic steroid effects has been described for rapid aldosterone effects in various cell types, including lymphocytes, cultured vascular smooth muscle, and endothelial cells involving nonclassical membrane receptors with a high affinity for aldosterone, but not for cortisol, and phosphoinositide turnover. As another important second messenger, [Ca2+]i is consistently increased by aldosterone within 1-2 min. In vascular smooth muscle cells, calcium is released from perinuclear stores, while in endothelial cells a predominant increase of subplasmalemmal calcium is seen. Effects are half maximal at physiological concentrations of free aldosterone (0.1 nmol/L), while cortisol is inactive up to 0.1 micromol/L; the classical mineralocorticoid antagonist canrenone is ineffective in blocking the action of aldosterone. The data show that intracellular signaling for nongenomic aldosterone effects also involves calcium, but pathways of cell activation may vary between different cell types. Future research will have to target the cloning of the first membrane receptor for steroids, and the evaluation of the clinical relevance of these rapid steroid effects.     

Anti-atherosclerotic effects of sirolimus on human vascular smooth muscle cells

Sirolimus is a potent immunosuppressive agent and has an anti-atherosclerotic effect through its anti-proliferative property. The present study was undertaken to investigate the effect of sirolimus on intracellular cholesterol homeostasis in human vascular smooth muscle cells (VSMCs) in the presence of inflammatory cytokine IL-1 beta. We explored the effect of sirolimus on the lipid accumulation of VSMCs in the presence of IL-1 beta, using Oil Red O staining and quantitative measurement of intracellular cholesterol. The effect of sirolimus on the gene and protein expression of lipoprotein receptors and ATP binding cassettes (ABCA1 and ABCG1) was examined by real-time PCR and Western blotting, respectively. Furthermore, the effect of sirolimus on cholesterol efflux from VSMCs in the presence or absence of IL-1 beta was also investigated using [(3)H] cholesterol efflux. Finally, we examined the effect of sirolimus on the production of inflammatory cytokines in VSMCs using ELISA. Sirolimus reduced intracellular lipid accumulation in VSMCs mediated by IL-1 beta possibly due to the reduction of expression of low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) receptors. Sirolimus increased cholesterol efflux from VSMCs and overrode the suppression of cholesterol efflux induced by IL-1 beta. Sirolimus also increased ABCA1 and ABCG1 genes expression, even in the presence of IL-1 beta. We further confirmed that sirolimus inhibited mRNA and protein expression of inflammatory cytokines IL-6, tumor necrosis factor-alpha, IL-8, and monocyte chemoattractant protein-1. Inhibition of lipid uptake together with increasing cholesterol efflux and the inhibition of inflammatory cytokines are all important aspects of the anti-atherosclerotic effects of sirolimus on VSMCs.     

Cross-talk between aldosterone and angiotensin signaling in vascular smooth muscle cells

In hypertension or other forms of cardiovascular disease, the chronic activation of the renin-angiotensin-aldosterone system (RAAS) leads to dysfunction of the vasculature, including, increased vascular tone, inflammation, fibrosis and thrombosis. Cross-talk between the main mediators of the RAAS, aldosterone and angiotensin (Ang) II, participates in the development of this vascular dysfunction. Recent studies have highlighted the molecular mechanisms supporting this cross-talk in vascular smooth muscle cells (VSMCs). Some of the signaling pathways activated by the Ang II type 1 receptor (AT₁R) are dependent on the mineralocorticoid receptor (MR) and vice versa. VSMC signaling pathways involved in migration and growth are under the control of cross-talk between aldosterone and Ang II. A synergistic mechanism leads to potentiation of signaling pathways activated by each agent. The genomic and non-genomic mechanisms activated by aldosterone cooperate with Ang II to regulate vascular tone and gene expression of pro-inflammatory and pro-fibrotic molecules. This cross-talk is dependent on the non-receptor tyrosine kinase c-Src, and on receptor tyrosine kinases, EGFR and PDGFR, and leads to activation of MAP kinases and growth, migration and inflammatory effects. These new findings will contribute to development of better treatments for conditions in which the RAAS is excessively activated.     

Physiological effects of androgens on human vascular endothelial and smooth muscle cells in culture

Androgenic hormones are associated with atherosclerotic cardiovascular disease, although the underlying cellular and molecular mechanisms remain unclear. This study examines the impact of androgens on the physiology of human vascular endothelial cells (EC) and smooth muscle cells (SMC) in culture. Cells were incubated with testosterone, dihydrotestosterone (DHT) or dehydroepiandrosterone (DHEA) at various physiological concentrations (5-50 nM) in the present or absence of an androgen receptor (AR) blocker flutamide (100 nM). Cell growth and death, DNA and collagen synthesis, and gene protein expression were assessed. It was shown that: (1) DHEA protected EC from superoxide injury via AR-independent mechanisms; (2) testosterone induced DNA synthesis and growth in EC via an AR-independent manner with activation of ERK1/2 activity; (3) DHT inhibited DNA synthesis and growth in EC in an AR-dependent manner; (4) testosterone and DHT enhanced ERK1/2 activation and proliferation in SMC via AR-independent and -dependent pathways, respectively; and (5) these androgens did not significantly affect collagen synthesis in SMC. We conclude that androgens possess multiple effects on vascular cells via either AR-dependent or -independent mechanisms. Testosterone and DHEA may be “beneficial” in preventing atherosclerosis by improving EC growth and survival; in contrast, stimulation of VSMC proliferation by testosterone and DHT is potentially “harmful”. The relationship of these in vitro effects by androgens to in vivo vascular function and atherogenesis needs to be further clarified.     

Differential effects of PDGF and PDGF-BB on vascular smooth muscle cells

Several biological effects of recombinant PDGF-BB and PDGF obtained from human platelets were examined with vascular smooth muscle cells. Although PDGF and PDGF-BB were equally potent mitogens for these cells, 5 fold higher levels of PDGF were required to displace 125I-PDGF-BB binding than PDGF-BB itself. Higher concentrations of PDGF relative to PDGF-BB were also required to stimulate the phosphorylation of a 163K protein in membrane preparations. PDGF-BB, but not PDGF, treatment of intact cells resulted in the phosphorylation on tyrosine residues of 168, 53, 48, and 45K proteins. The data suggest that PDGF and PDGF-BB stimulate smooth muscle cell mitogenesis by different mechanisms.     

Heparin interactions with cultured human vascular endothelial and smooth muscle cells: incidence on vascular smooth muscle cell proliferation

The binding, internalization, and metabolism of [3H]-heparin by human umbilical vein endothelial cells (HUVEC) and human umbilical arterial smooth muscle cells (HUASMC) have been characterized using size-exclusion HPLC. Incubation of HUVEC with [3H]-heparin demonstrated selective binding of high-molecular-weight (MW) components (MW = 21 kd), which was followed by rapid, temperature-dependent internalization. Over the next 3 hours, this internalized [3H]-heparin was degraded to low-MW fragments (MW = 0.9 kd). Primary cultures of HUASMC selectively bound extremely high-MW components (MW = 40 kd) and also smaller components whose MW (0.9 kd) corresponded to that of the heparin metabolite(s) formed by HUVEC. Subcultured HUASMC bound only the 40-kd components. Internalization of heparin by smooth muscle cells (SMC) was significantly slower than that determined for HUVEC, and even after 4 hours there was no evidence of the heparin being metabolized. However, when incubating primary rabbit aortic SMC with purified low-MW heparin fragment(s) produced in culture by HUVEC, a significantly lower proliferative response of these cells (IC50 = 18.4 micrograms/ml) was obtained. Virtually no effect was observed with subcultured SMC in the range of the tested concentrations (0-20 micrograms/ml). These fragments were 10- to 15-fold more effective in inhibiting primary SMC growth than was standard heparin. Furthermore, heparin fractions in the same range of molecular weights, purified either after nitrous acid or heparinase depolymerization of standard heparin, showed no activity on primary SMC growth, thus indicating a high degree of selectivity of the heparin metabolite(s) produced by HUVEC in culture.     

Intercellular communication in cultured human vascular smooth muscle cells

Intercellular communication through gap junction channelsplays a fundamental role in regulating vascular myocyte tone. We investigated gap junction channel expression and activity in myocytes from the physiologically distinct vasculature of the human internal mammary artery (IMA, conduit vessel) and saphenous vein (SV,capacitance vessel). Northern and Western blots documented the presenceof connexin43 (Cx43) in frozen tissues and cultured cells from both vessels. Northern blots also confirmed the presence of Cx40 mRNA incultured IMA and SV myocytes. Dual whole cell patch-clamp experiments revealed that macroscopic junctional conductance was voltage dependent and characteristic of that observed for Cx43. In the majority ofrecords, in both vessels, single-channel activity was dominated by amain-state conductance of 120 pS, with subconducting events comprisingless than 10% of the amplitude histograms. However, some recordsshowed "atypical" unitary events that had a conductance similar toCx40 (~140-160 pS), but gating behavior like that of Cx43. Assuch, it is conceivable that the presence and coexpression of Cx40 andCx43 in IMA and SV myocytes may result in heteromeric channelformation. Nonetheless, in terms of gating, Cx43-like behavior clearly dominates.

     

Autophagy-lysosomal defect in human CADASIL vascular smooth muscle cells

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a familial progressive degenerative disorder and is caused by mutations in NOTCH3 gene. Previous study reported that mutant NOTCH3 is more prone to form aggregates than wild-type NOTCH3 and the mutant aggregates are resistant to degradation. We hypothesized that aggregation or accumulation of NOTCH3 could be due to impaired lysosomal-autophagy machinery in VSMC.Here, we investigated the possible cause of accumulation/aggregation of NOTCH3 in CADASIL using cerebral VSMCs derived from control and CADASIL patients carrying NOTCH3^R133C mutation. Thioflavin-S-staining confirmed the increased accumulation of aggregated NOTCH3 in VSMC^R133C compared to VSMC^WT. Increased levels of the lysosomal marker, Lamp2, were detected in VSMC^R133C, which also showed co-localization with NOTCH3 using double-immunohistochemistry. Increased level of LC3-II/LC3-I ratio was observed in VSMC^R133C suggesting an accumulation of autophagosomes. This was coupled with the decreased co-localization of NOTCH3 with LC3, and Lamp2 and, further, increase of p62/SQSTM1 levels in VSMC^R133C compared to the VSMC^WT. In addition, Western blot analysis indicated phosphorylation of p-ERK, p-S6RP, and p-P70 S6K. Altogether, these results suggested a dysfunction in the autophagy-lysosomal pathway in VSMC^R133C.The present study provides an interesting avenue of the research investigating the molecular mechanism of CADASIL.     

Divergent effects of estrogen and nicotine on Rho-kinase expression in human coronary vascular smooth muscle cells

Recent studies have demonstrated that up-regulated Rho-kinase plays an important role in the pathogenesis of coronary arteriosclerosis and vasospasm. We have shown that inflammatory stimuli, such as angiotensin II and interleukin-1beta, up-regulate Rho-kinase expression and activity in human coronary vascular smooth muscle cells, for which intracellular signal transduction mediated by protein kinase C and NF-kappaB is involved. Here, we show that estrogen down-regulates while nicotine up-regulates Rho-kinase and that nicotine counteracts the inhibitory effect of estrogen on angiotensin II-induced Rho-kinase expression. Furthermore, we demonstrated that the intracellular signal transduction of the inhibitory effect of estrogen is mediated by an estrogen receptor. These results demonstrate that inflammatory stimuli up-regulate Rho-kinase, for which estrogen (mediated by an estrogen receptor) and nicotine exert divergent inhibitory and stimulatory effects on the Rho-kinase expression, respectively, and may explain in part why the incidence of arteriosclerotic and vasospastic disorders is increased in postmenopausal women and smokers.     

Divergent effects of estrogen and nicotine on Rho-kinase expression in human coronary vascular smooth muscle cells

Recent studies have demonstrated that up-regulated Rho-kinase plays an important role in the pathogenesis of coronary arteriosclerosis and vasospasm. We have shown that inflammatory stimuli, such as angiotensin II and interleukin-1β, up-regulate Rho-kinase expression and activity in human coronary vascular smooth muscle cells, for which intracellular signal transduction mediated by protein kinase C and NF-κB is involved. Here, we show that estrogen down-regulates while nicotine up-regulates Rho-kinase and that nicotine counteracts the inhibitory effect of estrogen on angiotensin II-induced Rho-kinase expression. Furthermore, we demonstrated that the intracellular signal transduction of the inhibitory effect of estrogen is mediated by an estrogen receptor. These results demonstrate that inflammatory stimuli up-regulate Rho-kinase, for which estrogen (mediated by an estrogen receptor) and nicotine exert divergent inhibitory and stimulatory effects on the Rho-kinase expression, respectively, and may explain in part why the incidence of arteriosclerotic and vasospastic disorders is increased in postmenopausal women and smokers.     

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.     

Interaction between aldosterone and vasopressin on vascular smooth muscle permeability to sodium

The s.c. injection of aldosterone (10 micrograms/kg) induces a release of vasopressin. The peak of plasma vasopressin level occurs at the same time as the late in vivo effect of aldosterone on passive 22Na efflux from arterial smooth muscle. These results indicate that vasopressin mediates the delayed in vivo effects of aldosterone on ouabain-insensitive 22Na efflux, since on the other hand, it has been possible to show that the action of the peptide is accelerated by a previous exposure to the mineralocorticoid. Indeed, after a 120-min pretreatment with 10(-8) M aldosterone, vasopressin induces an effect on 22Na efflux in 30 min, as opposed to the 120 min needed in the absence of the steroid.     

Energetic effects of adenosine on vascular smooth muscle

Adenosine (Ado) is a naturally occurring compound that has several important cardiovascular actions, including activation of ATP-sensitive K(+) channels in vascular smooth muscle, vasorelaxation, and an effect to alter glucose metabolism of cardiac muscle. The metabolic effects of Ado on vascular smooth muscle have not been defined and were examined in this study. Porcine carotid artery strips were incubated in the presence and absence of 0.5 mM Ado. Compared with the control, Ado had no effect on glucose uptake, glucose oxidation, or fatty acid (octanoate) oxidation. Ado suppressed glycolysis but enhanced glycogen synthesis. Relative to the rate of glycolysis, Ado increased lactate production. Ado stimulated O(2) consumption by 52 +/- 10%, altered the activities of the tricarboxylic acid cycle and malate-aspartate shuttle, and increased the content of ATP, ADP, AMP, and phosphocreatine. Alteration in the metabolic variables by Ado could not be attributed to diminished energy requirements of reduced resting muscle tone of the arterial strips. Relaxation of the arterial strips in response to Ado were abolished in arteries incubated under hypoxic conditions (95% N(2)-5% CO(2)). Hypoxia was associated with increased ADP content. It is concluded that Ado affected glucose metabolism indirectly. The metabolic and energetic effects of 0.5 mM Ado are mediated by alterations in the concentrations of AMP, ATP, and phosphorylation potential (ATP/ADP).     

Use of human vessels and human vascular smooth muscle cells in pharmacology

Relatively limited information is available regarding the mechanisms controlling vasomotricity in human vessels. Isolated vessels obtained from patients undergoing surgery were used to characterize the role of endothelial factors and to study coupling mechanisms between receptors, intracellular calcium, and contraction. However, these investigations are limited by the availability of tissues and many uncontrolled factors. Cultured human vascular cells were also used, were these cells rapidly lose at least some of their differentiated characters. Recently, a human blood vessel equivalent was constructed in vitro from cultured cells, using tissue engineering. This technique allowed us to obtain vessel equivalents containing intima, media, and adventitia layers or tubular media layer only. Contraction and rises in intracellular calcium produced by agonists were studied, indicating that such human vessel equivalents may provide valuable models for pharmacological studies.     

Molecular basis of the effects of mechanical stretch on vascular smooth muscle cells

The pulsatile nature of blood pressure and flow creates hemodynamic stimuli in the forms of cyclic stretch and shear stress, which exert continuous influences on the constituents of the blood vessel wall. Vascular smooth muscle cells (VSMCs) use multiple sensing mechanisms to detect the mechanical stimulus resulting from pulsatile stretch and transduce it into intracellular signals that lead to modulations of gene expression and cellular functions, e.g., proliferation, apoptosis, migration, and remodeling. The cytoskeleton provides a structural framework for the VSMC to transmit mechanical forces between its luminal, abluminal, and junctional surfaces, as well as its interior, including the focal adhesion sites, the cytoplasm, and the nucleus. VSMCs also respond differently to the surrounding structural environment, e.g., two-dimensional versus three-dimensional matrix. In vitro studies have been conducted on cultured VSMCs on deformable substrates to elucidate the molecular mechanisms by which the cells convert mechanical inputs into biochemical events, eventually leading to functional responses. The knowledge gained from research on mechanotransduction in vitro, in conjunction with verifications under in vivo conditions, will advance our understanding of the physiological and pathological processes involved in vascular remodeling and adaptation in health and disease.