Adrenomedullin inhibits angiotensin II-induced contraction in human aortic smooth muscle cells

The vasodilating peptide adrenomedullin (AM) has been reported to regulate vascular tone as well as proliferation and differentiation of various cell types in an autocrine/paracrine manner. Our study was designed to investigate the effect of AM on Ang II-induced contraction on human aortic smooth muscle cells (HASMC) in vitro, evaluating the signal pathways involved. Our findings indicate that AM was able to inhibit HASMC Ang II-induced contraction (IC50 19 nM). AM stimulated cAMP production in a dose-dependent fashion as well. SQ 22.536, an adenylate cyclase inhibitor, and KT5720, a PKA inhibitor, blunted the AM effect, suggesting that it was mediated by the activation of the cAMP transduction pathway. Our results suggest that AM plays a role in the regulation of HASMC contraction by antagonizing the Ang II effects and may be involved in conditions of altered regulation of the blood vessels.     

Nox5 mediates PDGF-induced proliferation in human aortic smooth muscle cells

The proliferation of vascular smooth muscle cells is important in the pathogenesis of many vascular diseases. Reactive oxygen species (ROS) produced by NADPH oxidases in smooth muscle cells have been shown to participate in signaling cascades regulating proliferation induced by platelet-derived growth factor (PDGF), a powerful smooth muscle mitogen. We sought to determine the role of Nox5 in the regulation of PDGF-stimulated human aortic smooth muscle cell (HASMC) proliferation. Cultured HASMC were found to express four isoforms of Nox5. When HASMC stimulated with PDGF were pretreated with N-acetyl cysteine (NAC), proliferation was significantly reduced. Proliferation induced by PDGF was also heavily dependent on JAK/STAT activation, as the JAK inhibitor, AG490, was able to completely abolish PDGF-stimulated HASMC growth. Specific knockdown of Nox5 with a siRNA strategy reduced PDGF-induced HASMC ROS production and proliferation. Additionally, siRNA to Nox5 inhibited PDGF-stimulated JAK2 and STAT3 phosphorylation. ROS produced by Nox5 play an important role in PDGF-induced JAK/STAT activation and HASMC proliferation.     

Phenotypic changes of adrenomedullin receptor components, RAMP2, and CRLR mRNA expression in cultured rat vascular smooth muscle cells.

Adrenomedullin is known to inhibit cell proliferation in cultured rat vascular smooth muscle cells, through a cAMP-dependent process. The calcitonin receptor-like receptor could function as an adrenomedullin receptor when co-expressed with receptor activity-modifying protein 2. To determine whether vascular adrenomedullin receptor components, the calcitonin receptor-like receptor and the receptor activity-modifying protein 2, phenotypically change during in vitro culture conditions, we examined the expression of adrenomedullin receptor components, adrenomedullin-induced cAMP production, and the inhibition of cell proliferation in culture rat vascular smooth muscle cells during serial passages. The results demonstrated that the receptor activity-modifying protein 2 and calcitonin receptor-like receptor mRNAs increased in a passage-dependent manner in rat vascular smooth muscle cells. Furthermore, the responses of both the elevation of cAMP and the inhibition of cell proliferation became larger in vascular smooth muscle cells with an increasing number of passages. The results suggest that the increase in functional AM receptor during phenotypic change may in part contribute to the development of vascular lesions, such as in atherosclerosis.     

Increased expression of H11 kinase stimulates glycogen synthesis in the heart

Mechanical forces related to pressure and flow are important for cell hypertrophy and proliferation. There are still a few studies that examine responses of human vascular smooth muscle cells to pure pressure stress (transmural pressure). It is unclear as to which mechanisms are involved in cellular responses to pressure elevation. On the other hand, although the involvement of the local renin-angiotensin system (RAS) in pressure-induced responses was reported, the results were contradictory. It still remains to be determined whether RAS in human vascular smooth muscle cells is activated by pure pressure stress. We studied the upstream signal transduction events of extracellular signal kinase (ERK) in response to atmospheric pressure stress and involvement of angiotensin II in pressure-induced cell proliferation in human aortic smooth muscle cells (HASMC). A pressure-loading apparatus was set up to examine the effects of atmospheric pressure on human aortic smooth muscle cells. Pressure application of 160 mmHg for 3 h produced cell proliferation and activated ERK and c-JUN N-terminal kinase (JNK). ACE inhibitor suppressed all of them. ERK kinase (MEK) inhibitor also suppressed cell proliferation stimulated by pure pressure. The phosphorylated c-Src was increased by pure pressure stress. The treatment with c-Src kinase inhibitor suppressed pressure-induced proliferative response. In summary, our study found that ERK activation mediated pure pressure-induced proliferative response of HASMC. This activation was partly mediated by c-Src.     

Screening and functional prediction of differentially expressed circRNAs in proliferative human aortic smooth muscle cells

Vascular smooth muscle cell (VSMC) proliferation is the pathological base of vascular remodelling diseases. Circular RNAs (circRNAs) are important regulators involved in various biological processes. However, the function of circRNAs in VSMC proliferation regulation remains largely unknown. This study was conducted to identify the key differentially expressed circRNAs (DEcircRNAs) and predict their functions in human aortic smooth muscle cell (HASMC) proliferation. To achieve this, DEcircRNAs between proliferative and quiescent HASMCs were detected using a microarray, followed by quantitative real‐time RT‐PCR validation. A DEcircRNA‐miRNA‐DEmRNA network was constructed, and functional annotation was performed using Gene Ontology (GO) and KEGG pathway analysis. The function of hsa_circ_0002579 in HASMC proliferation was analysed by Western blot. The functional annotation of the DEcircRNA‐miRNA‐DEmRNA network indicated that the four DEcircRNAs might play roles in the TGF‐β receptor signalling pathway, Ras signalling pathway, AMPK signalling pathway and Wnt signalling pathway. Twenty‐seven DEcircRNAs with coding potential were screened. Hsa_circ_0002579 might be a pro‐proliferation factor of HASMC. Overall, our study identified the key DEcircRNAs between proliferative and quiescent HASMCs, which might provide new important clues for exploring the functions of circRNAs in vascular remodelling diseases.     

Cyclin D1 Is a Bona Fide Target Gene of NFATc1 and Is Sufficient in the Mediation of Injury-induced Vascular Wall Remodeling

Platelet-derived growth factor BB induced cyclin D1 expression in a time- and nuclear factor of activated T cells (NFAT)-dependent manner in human aortic smooth muscle cells (HASMCs), and blockade of NFATs prevented HASMC DNA synthesis and their cell cycle progression from G₁ to S phase. Selective inhibition of NFATc1 by its small interfering RNA also blocked HASMC proliferation and migration. Characterization of the cyclin D1 promoter revealed the presence of several NFAT binding sites, and the site at nucleotide −1333 was found to be sufficient in mediating platelet-derived growth factor BB-induced cyclin D1 promoter-luciferase reporter gene activity. In addition to its role in cell cycle progression, cyclin D1 mediated HASMC migration in an NFATc1-dependent manner. Balloon injury-induced cyclin D1-CDK4 activity requires NFAT activation, and adenovirus-mediated transduction of cyclin D1 was found to be sufficient to overcome the blockade effect of NFATs by VIVIT on balloon injury-induced vascular wall remodeling events, including smooth muscle cell migration from the medial to luminal region, their proliferation in the intimal region, and neointima formation. Together, these results provide more mechanistic evidence for the role of NFATs, particularly NFATc1, in the regulation of HASMC proliferation and migration as well as vascular wall remodeling. NFATc1 could be a potential therapeutic target against the renarrowing of artery after angioplasty.     

Adrenomedullin is an autocrine/paracrine growth factor for rat vascular smooth muscle cells

Adrenomedullin is a potent vasodilator peptide secreted by vascular endothelial and smooth muscle cells. Adrenomedullin stimulates the proliferation of quiescent rat vascular smooth muscle cells (VSMCs) via p42/p44 ERK/MAP kinase activation. Recently, receptor-activity-modifying proteins (RAMPs) have been shown to transport calcitonin-receptor-like-receptor (CRLR) to the cell surface to present either as CGRP receptor or adrenomedullin receptor. We investigated whether adrenomedullin acts as an autocrine/paracrine growth factor for cultured rat VSMCs and whether coexpressions of RAMP isoform and CRLR may mediate p42/p44 ERK/MAP kinase activation by adrenomedullin. Adrenomedullin dose-dependently stimulated the proliferation of quiescent rat VSMCs, and this effect was inhibited by an adrenomedullin receptor antagonist, a MAP kinase kinase inhibitor and phosphatidylinositol 3-kinase inhibitors. Addition of either CGRP(8-37) or anti-adrenomedullin antibody to exponentially growing rat VSMCs inhibited the serum-induced cell proliferation, suggesting its role as an autocrine/paracrine growth factor. Cotransfection of RAMP2 or RAMP3 with CRLR into rat VSMCs potentiated activation of cAMP activity, but not of p42/p44 ERK/MAP kinase activity in response to adrenomedullin. Our results suggest that adrenomedullin is an autocrine/paracrine growth factor for rat VSMCs via p42/p44 ERK/MAP kinase and phosphatidylinositol 3-kinase pathways and that it is not mediated by human RAMP-CRLR receptors.     

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.     

Pure atmospheric pressure promotes an expression of osteopontin in human aortic smooth muscle cells

Although the types of pathophysiological stimulation that initiate an overexpression of OPN have yet to be determined, we hypothesized that mechanical stress is one of the candidates which initiates OPN expression in vascular smooth muscle cells. Cell proliferation assay indicated that a pure atmospheric pressure of 160 mmHg activated cell proliferation by 11% in human aortic smooth muscle cells (HASMC) compared to nonpressurized controls. Immunoblot analysis probed with an anti-OPN antibody demonstrated a 50% increase in OPN. Dual-luciferase reporter assay demonstrated that OPN promoter, corresponding to the -771 through -1 region of OPN gene, was highly responsive to pure atmospheric pressure by ten times that of the control. From these observations, we concluded that pure atmospheric pressure directly promotes an expression of OPN in HASMC, with these results also suggesting that high blood pressure-mediated mechanical compression is involved in the process of atherosclerosis and remodeling via OPN expression in HASMC.     

PPARgamma inhibits the expression of c-MET in human gastric cancer cells through the suppression of Ets

We have reported that plasma adrenomedullin (AM) in hyperglycemic patients was significantly increased compared with normal volunteers. In this report we examined the effects of hyperglycemia on AM expression in the vasculature, the main site of AM production. AM mRNA level in the aorta was higher in the diabetic rats than in the control rats. AM mRNA level and protein kinase C (PKC) activity in cultured vascular smooth muscle cells (VSMC) increased as the glucose concentration in the medium changed from 100mg/dl to 450mg/dl. PKC inhibitors blocked this increase of AM mRNA. Similar osmotic change with mannitol had no effect on AM expression. We conclude that (1) hyperglycemia increases vascular AM expression through PKC-dependent pathway, and (2) the elevated plasma AM in hyperglycemic patients originates from the glucose induced vascular AM expression. We propose the possible role of AM in the pathogenesis of diabetic vascular complications.     

Berberine suppresses in vitro migration of human aortic smooth muscle cells through the inhibitions of MMP-2/9, u-PA,AP-1, and NF-κB

Berberine, a type of isoquinoline alkaloid isolated from Chinese medicinal herbs, has been reported to have various pharmacological activities. Studies have demonstrated that berberine has beneficial effects on vascular remodeling and alleviates restenosis after vascular injury. However, its mechanism of action on vascular smooth muscle cell migration is not fully understood. We therefore investigated the effect of berberine on human aortic smooth muscle cell (HASMC) migration. Boyden chamber assay was performed to show that berberine inhibited HASMC migration dosedependently. Real-time PCR and Western blotting analyses showed that levels of matrix metalloproteinase (MMP)-2, MMP-9, and urokinase-type plasminogen activator (u-PA) were reduced by berberine at both the mRNA and protein levels. Western blotting assay further confirmed that activities of c-Fos, c-Jun, and NF-κB were significantly attenuated. These results suggest that berberine effectively inhibited HASMC migration, possibly by down-regulating MMP-2, MMP-9, and u-PA; and interrupting AP-1 and NF-κB mediated signaling pathways. [BMB Reports 2014; 47(7): 388-392]     

Comparison of doxycycline and minocycline in the inhibition of VEGF-induced smooth muscle cell migration

Smooth muscle cell migration plays an important role during angiogenesis and vascular remodeling. In this study, we examined the effects of doxycycline and minocycline on vascular endothelial growth factor (VEGF)-induced human aortic smooth muscle cell (HASMCs) migration, and explored the mechanisms in which doxycycline or minocycline inhibit HASMC migration. We demonstrated that both doxycycline and minocycline attain consistent anti-angiogenic effects in the inhibition of HASMC migration via a different signal pathway (p<0.05). This effect is through attenuating VEGF-induced matrix metalloproteinase-9 (MMP-9) activity (p<0.05). Doxycycline could increase tissue inhibitors of metalloproteinases-1 (TIMP-1) expression while minocycline down-regulated PI3K/Akt phosphorylation in HASMC. Our study suggests that doxycycline has a stronger ability to inhibit MMP secretion in HASMC by up-regulating endogenous MMPs inhibitor TIMP-1, while minocycline implements anti-angiogenic effect through inhibiting HASMC migration by down-regulating PI3K/Akt pathway.     

Acetylshikonin attenuates angiotensin II-induced proliferation and motility of human brain smooth muscle cells by inhibiting Wnt/β-catenin signaling

Cerebrovascular smooth muscle cell proliferation and migration contribute to hyperplasia in case of cerebrovascular remodeling and stroke. In the present study, we investigated the effects of acetylshikonin, the main ingredient of a Chinese traditional medicine Zicao, on human brain vascular smooth muscle cell (HBVSMCs) proliferation and migration induced by angiotensin II (AngII), and the underlying mechanisms. We found that acetylshikonin treatment significantly inhibited AngII-induced HBVSMCs proliferation and cell cycle transition from G1 to S phase. Wound-healing assay and Transwell assay showed that AngII-induced cell migration and invasion were markedly attenuated by acetylshikonin. In addition, AngII challenge significantly induced Wnt/β-catenin signaling activation, as evidenced by increased β-catenin phosphorylation and nuclear translocation and GSK-3β phosphorylation. However, acetylshikonin treatment inhibited the activation of Wnt/β-catenin signaling. Consequently, western blotting analysis revealed that acetylshikonin effectively reduced the expression of downstream target genes in AngII-treated cells, including c-myc, survivin and cyclin D1, which contributed to the inhibitory effect of acetylshikonin on HBVSMCs proliferation. Further, stimulation with recombinant Wnt3a dramatically reversed acetylshikonin-mediated inhibition of proliferation and cell cycle transition in HBVSMCs. Our study demonstrates that acetylshikonin prevents AngII-induced cerebrovascular smooth muscle cells proliferation and migration through inhibition of Wnt/β-catenin pathway, indicating that acetylshikonin may present a potential option for the treatment of cerebrovascular remodeling.