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Background: Transforming growth factor beta (TGF-β1) is a pleiotropic cytokine, which is deregulated in atherosclerosis; however the role of age in this process is unknown. We aimed to assess whether TGF-β1 signaling is affected by age., Methods: Vascular smooth muscle cells (VSMC) were obtained from patients undergoing abdominal surgery. Levels of TGF-β1 were measured by ELISA in sera from 169 patients undergoing coronary artery bypass grafting (CABG). The p27 expression was determined by Western blot from internal mammary arteries (IMA) obtained from CABG patients (n=13). In VSMC from these patients undergoing abdominal surgery, secretion of TGF-β1 was determined by ELISA of cell-conditioned media., Results: In VSMC from aged patients we observed a lower TGF-β1 secretion, measured as TGF-β1 concentration in cell conditioned medium (p<0.001). This effect was correlated to an age-dependent decrease of p27 expression in IMA from aged CABG patients. In a similar manner, there was an age-dependent decrease of serum TGF-β1 levels in CABG patients (p=0.0195)., Conclusions: VSMC from aged patients showed a higher degree of cellular senescence and it was associated to a lower TGF-β1 secretion and signaling.

Mercury exposure is known to increase cardiovascular risk but the underlying cellular mechanisms remain undetermined. We analyzed whether chronic exposure to HgCl2 affects vascular structure and the functional properties of vascular smooth muscle cells (VSMC) through oxidative stress/cyclooxygenase-2 dependent pathways. Mesenteric resistance arteries and aortas from Wistar rats treated with HgCl2 (first dose 4.6mgkg(-1), subsequent doses 0.07mgkg(-1)day(-1), 30days) and cultured aortic VSMC stimulated with HgCl2 (0.05-5μg/ml) were used. Treatment of rats with HgCl2 decreased wall thickness of the resistance and conductance vasculature, increased the number of SMC within the media and decreased SMC nucleus size. In VSMCs, exposure to HgCl2: 1) induced a proliferative response and a reduction in cell size; 2) increased superoxide anion production, NADPH oxidase activity, gene and/or protein levels of the NADPH oxidase subunit NOX-1, the EC- and Mn-superoxide dismutases and cyclooxygenase-2 (COX-2); 3) induced activation of ERK1/2 and p38 MAPK. Both antioxidants and COX-2 inhibitors normalized the proliferative response and the altered cell size induced by HgCl2. Blockade of ERK1/2 and p38 signaling pathways abolished the HgCl2-induced Nox1 and COX-2 expression and normalized the alterations induced by mercury in cell proliferation and size. In conclusion, long exposure of VSMC to low doses of mercury activates MAPK signaling pathways that result in activation of inflammatory proteins such as NADPH oxidase and COX-2 that in turn induce proliferation of VSMC and changes in cell size. These findings offer further evidence that mercury might be considered an environmental risk factor for cardiovascular disease., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aims/hypothesis: Inflammation is a common feature in cardiovascular diseases, including diabetes mellitus. In addition to the well-known inflammatory role of cyclo-oxygenase-2 (COX-2), this protein has also been implicated in apoptosis resistance in tumour cells. Vascular smooth muscle cells (VSMC) from diabetic patients are also resistant to apoptosis because of an increased abundance of B cell lymphoma 2 protein (BCL2). In this work, we investigated whether overproduction of COX-2 was involved in the resistance to apoptosis in VSMC from diabetic patients., Methods: VSMC were obtained from internal mammary arteries from patients who had undergone coronary artery bypass graft surgery. Apoptosis was measured by DNA fragmentation, BCL2 degradation and cytochrome c release., Results: Apoptosis induced by C-reactive protein in cells from non-diabetic patients was mediated by COX-2. VSMC from diabetic patients showed higher basal levels of COX-2 compared with those from non-diabetic patients. Transfection of VSMC from non-diabetic patients with a plasmid containing COX-2 (also known as PTGS2) increased basal production of COX-2 and BCL2 and mimicked the resistance to apoptosis that occurs in diabetic patients. We also found a significant correlation (R = 0.846, p = 0.016) between COX-2 and BCL2 production in arterial rings from diabetic patients measured by confocal microscopy. However, inhibition of COX-2 production by small interfering RNA proved unable to reverse BCL2 production in diabetic VSMC., Conclusions/interpretation: These results suggest a link between inflammation (COX-2) and apoptosis resistance (BCL2) in the arteries of diabetic patients. This relationship is not causative and the common production of these two proteins may be co-regulated by shared regulatory elements in diabetes.

Background: We aimed to test the antiproliferative effect of acetylsalicylic acid (ASA) on vascular smooth muscle cells (VSMC) from bypass surgery patients and the role of transforming growth factor beta 1 (TGF-beta1)., Methodology/principal Findings: VSMC were isolated from remaining internal mammary artery from patients who underwent bypass surgery. Cell proliferation and DNA fragmentation were assessed by ELISA. Protein expression was assessed by Western blot. ASA inhibited BrdU incorporation at 2 mM. Anti-TGF-beta1 was able to reverse this effect. ASA (2 mM) induced TGF-beta1 secretion; however it was unable to induce Smad activation. ASA increased p38(MAPK) phosphorylation in a TGF-beta1-independent manner. Anti-CD105 (endoglin) was unable to reverse the antiproliferative effect of ASA. Pre-surgical serum levels of TGF-beta1 in patients who took at antiplatelet doses ASA were assessed by ELISA and remained unchanged., Conclusions/significance: In vitro antiproliferative effects of aspirin (at antiinflammatory concentration) on human VSMC obtained from bypass patients are mediated by TGF-beta1 and p38(MAPK). Pre-surgical serum levels of TGF- beta1 from bypass patients who took aspirin at antiplatelet doses did not change.

In many vascular smooth muscle cells, physiological and pharmacological agonists initiate oscillatory fluctuations in intracellular Ca(2+) to initiate and maintain vasoconstriction. These oscillations are supported by the underlying cellular ultrastructure, particularly the close apposition between the plasma membrane (PM) and superficial sarcoplasmic reticulum (SR), the so-called PM-SR junctions, which are important for SR Ca(2+) refilling. We hypothesize that the disappearance of PM-SR junctions during aging and/or disease is directly related to the disappearance of agonist-induced Ca(2+) oscillations. We compared phenylephrine-mediated Ca(2+) signals and contraction in human and murine smooth muscle cells in small mesenteric arteries and also employed electron microscopy to examine the cytoplasmic distribution of the SR. Phenylephrine elicited tonic contractions in both types of vessels, asynchronous Ca(2+) oscillations in the mouse mesenteric smooth muscle cells, but only single transient Ca(2+) signals in the human mesenteric smooth muscle cells. While nifedipine inhibited 90% of the phenylephrine-induced tonic contraction in mouse mesenteric arteries, it only slightly attenuated tonic contraction in human mesenteric arteries, although the nifedipine-resistant component was abolished by the Rho-kinase blocker 1-(5-Isoquinolinylsulfonyl)homopiperazine dihydrochloride (HA-1077). Furthermore, superficial SR was found to be abundant in the mouse vessels and many PM-SR junctions were observed, but the smooth muscle of human mesenteric arteries had far less peripheral SR and was almost devoid of PM-SR junctions. As PM-SR junctions are essential for the maintenance of Ca(2+) oscillations, the change in Ca(2+) signalling pattern in the relatively old human patients was due to impaired SR refilling., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

The intracellular uptake of Angiotensin II has been described, although its physiological role is not yet understood. We aimed to study the role of Angiotensin II internalization in Angiotensin II-induced apoptosis. Vascular smooth muscle cells were cultured from male Wistar-Kyoto rats and treated with Angiotensin II (1 microM, 48 h). Apoptosis was assessed by DNA fragmentation, cell cytometry and caspase-3 activity. The Angiotensin AT(1) receptor antagonist irbesartan (0.1-10 microM) and the inhibitors of Angiotensin II internalization phenylarsine oxide (PAO, 20 microM), but not the AT(2) receptor antagonist PD123319 (S-(+)-1-[(4-(Dimethylamino)-3-methylphenyl)methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid di(trifluoroacetate) salt), decreased Angiotensin II-mediated apoptosis. Pre-treatment with irbesartan, but not with PD123319, blocked Angiotensin II internalization. We found a strong correlation between intracellular Angiotensin II staining and Angiotensin II-induced apoptosis for all compared groups. We therefore conclude that internalization of Angiotensin II is involved in apoptosis of vascular smooth muscle cells induced by this peptide.

Alterations in vascular wall remodeling are a typical complication in type 2 diabetes mellitus due to an imbalance between cell proliferation and apoptosis. In this context, we have previously shown that vascular smooth muscle cells (VSMC) from diabetic patients were resistant to induced apoptosis. Thiazolidinediones, such as pioglitazone, seem to exert direct antiatherosclerotic effects on type 2 diabetes. Here, we aimed to study whether pioglitazone was able to induce apoptosis in VSMC from diabetic patients (DP) and, if so, whether the transforming growth factor (TGF)-beta1/Smad-2 pathway was involved. We isolated human internal mammary artery VSMC from patients who had undergone coronary-artery bypass graft. Pioglitazone (100 microM) induced apoptosis in human VSMC from diabetic and nondiabetic patients (NDP), analyzed by DNA fragmentation and by degradation of Bcl-2, in high-glucose-containing medium (15 and 25 mM). This apoptotic effect was inhibited by the activin receptor-like kinase-4/5/7/Smad2 inhibitor 4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide (SB-431542), denoting that the TGF-beta1/Smad-2 pathway was involved. Pioglitazone rapidly increased the extracellular TGF-beta1 levels and concomitantly induced phosphorylation of Smad2 in VSMC from DP and NDP. Thus, we demonstrated that pioglitazone induced apoptosis in human VSMC from DP, which are strongly resistant to the induced apoptosis. This effect of pioglitazone might contribute in the treatment of alterations of vascular remodeling in type 2 diabetes mellitus.

Both Angiotensin II and transforming growth factor beta-1 (TGF-beta1) are important mediators of vascular smooth muscle cell function and have been reported to mediate the balance between proliferation and apoptosis. Some crosstalk between Angiotensin II and TGF-beta1 in end-organ hypertension has been established. However, whether TGF-beta1 is able to mediate Angiotensin II-induced vascular cell damage remains unknown. Vascular smooth muscle cells were obtained from rat thoracic aorta and cultured in 10% foetal calf serum. In all experiments, medium was changed to a low-serum (0.4% foetal calf serum) or serum-free one with or without Angiotensin II. Apoptosis was assessed by DNA fragmentation, DNA synthesis was measured as bromo-deoxyuridine uptake. TGF-beta1 production was determined by Enzyme-linked Immunosorbent Assay (ELISA) from cell conditioned media, RT-PCR from cell lysates and confocal immunostaining of fixed cells. Angiotensin II induced apoptosis in the absence of DNA synthesis when coincubated at 1 microM. Neither the specific anti-TGF-beta1 monoclonal antibody (50 microg/ml) nor the novel activin-like kinase (ALK)-4/5/7 synthetic inhibitor SB-431542 (4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide) at 10 microM were able to inhibit this effect. Angiotensin II induced expression of TGF-beta1 without further secretion of this cytokine. This effect was not affected by incubation with the AT1 inhibitor irbesartan (10 microM). A pharmacological approach to TGF-beta1 inhibition would be unable to reverse the apoptotic effect of Angiotensin II on vascular smooth muscle cells.

An emerging body of evidence suggests that vascular remodeling in diabetic patients involves a perturbation of the balance between cell proliferation and cell death. Our aim was to study whether arteries and vascular smooth muscle cells (VSMCs) isolated from diabetic patients exhibit resistance to apoptosis induced by several stimuli. Internal mammary arteries (IMAs) were obtained from patients who had undergone coronary artery bypass graft surgery. Arteries from diabetic patients showed increasing levels of Bcl-2 expression in the media layer, measured by immunofluorescence and by Western blotting. Human IMA VSMCs from diabetic patients showed resistance to apoptosis, measured as DNA fragmentation and caspase-3 activation, induced by C-reactive protein (CRP) and other stimuli, such as hydrogen peroxide and 7beta-hydroxycholesterol. The diabetic cells also exhibited overexpression of Bcl-2. Knockdown of Bcl-2 expression with Bcl-2 siRNA in cells from diabetic patients reversed the resistance to induced apoptosis. Consistent with the above, we found that pretreatment of nondiabetic VSMCs with high glucose abolished the degradation of Bcl-2 induced by CRP. Moreover, cell proliferation was increased in diabetic compared with nondiabetic cells. This differential effect was potentiated by glucose. We conclude that the data provide strong evidence that arterial remodeling in diabetic patients results from a combination of decreased apoptosis and increased proliferation.

Antioxidants such as flavonoids afford protection against oxysterols-induced toxicity. We have investigated the effect of kaempferol and rutin, active components of red wine, in the apoptosis induced by 7beta-hydroxycholesterol in rat vascular smooth muscle cells. 7beta-Hydroxycholesterol induced apoptosis in vascular smooth muscle which include BcL-x(L) degradation, caspase-3 activation and DNA fragmentation. The apoptosis induced by 7beta-hydroxycholesterol was prevented by pretreatment with kaempferol (10-30 microM), but not with rutin. Interestingly preincubation with the estrogen receptor alpha antagonist ICI 182,780 (1 microM) prior to kaempferol partially reverted the antiapoptotic effect of this flavonoid on caspase-3 activation and DNA fragmentation induced by 7beta-hydroxycholesterol. In conclusion, the flavonoid kaempferol, unlike rutin, diminished the apoptosis induced by a component of oxidized low-density lipproteins (oxLDL). This effect was partially mediated by the estrogen receptor alpha.

Thiazolidinediones, such as pioglitazone, seem to exert direct antiatherosclerotic and antirestenotic effects on type 2 diabetes, in part due to an induction of vascular smooth muscle cell (VSMC) apoptosis. We aimed to study the role of transforming growth factor (TGF)-beta in rat aortic VSMC. Pioglitazone at 100 micromol/l increased apoptosis without affecting DNA synthesis, and this effect was reversed by an anti-TGF-beta1 antibody. Extracellular TGF-beta1 levels were rapidly increased after treatment with pioglitazone in a peroxisome proliferator-activated receptor (PPAR)-gamma-dependent mechanism because this secretion was blocked by the PPAR-gamma inhibitor GW9662. Pioglitazone subsequently increased the nuclear recruitment of phospho-Smad2, without any effect on protein expression. According to our results, we propose that the apoptotic effect of pioglitazone on VSMC depends on the following sequence: PPAR-gamma activation, TGF-beta1 release, and selective phospho-Smad2 nuclear recruitment. Management of Smad signaling on VSMC might provide future clinical benefits in vascular diseases.

Background: Acetylsalicylic acid (ASA) inhibits cell proliferation. This may be mediated by transforming growth factor-beta (TGF-beta). TGF-beta directly stops cell proliferation, restrains cells in G(0), and inhibits the uptake of platelet-derived growth factor and insulin-like growth factor. These effects are identical to those observed with ASA treatment., Methods and Results: We cultured rat thoracic aorta vascular smooth muscle cells and measured cytotoxicity, cell proliferation, cell cycle, transcription of TGF-beta1, and concentration of TGF-beta1 in supernatant medium. ASA dose-dependently restrained cells in G(0) phase with no cytotoxic effect and inhibited cell proliferation by 30.86%. Anti-TGF-beta1 reversed this inhibition by 30.21%. However, ASA treatment decreased TGF-beta1 transcription and had no significant effect on TGF-beta1 concentration., Conclusions: TGF-beta seems to play an important role in ASA-mediated inhibition of cell proliferation. Therefore, treatment with ASA prevents coronary disease not only by means of its antiplatelet properties but also by an important inhibition of plaque growth. This relationship between ASA and TGF-beta explains many other effects, such as cancer chemoprevention, immunomodulation, and wound healing. The aim of this study was to demonstrate this link.