Tumor necrosis factor-alpha regulates insulin-like growth factor-1 and insulin-like growth factor binding protein-3 expression in vascular smooth muscle.

BACKGROUND: Inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), IL-6, and interferon gamma (IFN-gamma) may change coronary plaque integrity by altering vascular smooth muscle cell (VSMC) survival and modifying the extracellular matrix. Insulin-like growth factor-1 (IGF-1) prevents apoptosis, promotes matrix formation, and can decrease TNF-alpha or IL-1beta--induced proteoglycan degradation. METHODS AND RESULTS: To determine the effects of cytokines on the IGF-1 system, rat aortic VSMCs were exposed to TNF-alpha (10 to 500 ng/mL), IL-1beta (20 pg to 10 ng/mL), IL-6 (100 pg to 15 ng/mL), or IFN-gamma (10 to 600 U/mL). IL-1beta, IL-6, and IFN-gamma did not regulate IGF-1, IGF-1 receptor (R), or IGF binding proteins (IGFBPs). However, TNF-alpha markedly decreased IGF-1 mRNA (85% reduction at 24 hours) and increased IGFBP-3 mRNA and protein (300% increase at 24 hours). These changes were blocked by actinomycin D, consistent with a transcriptional mechanism. Experiments using TNF binding protein-1 indicated that these effects were not attributable to secretion of an autocrine factor. Anti--IGFBP-3 antibodies increased VSMC DNA synthesis 3-fold. In addition, apoptosis induced by TNF-alpha, IFN-gamma, and Fas ligand was markedly reduced by desamino-(1-3)-IGF-1. CONCLUSIONS: TNF-alpha, a cytokine that is upregulated in atherosclerotic plaques, reduces IGF-1 and increases IGFBP-3 in VSMCs, likely leading to a reduction in bioactive IGF-1. Because IGF-1 is important for growth and survival of VSMCs, its downregulation by TNF-alpha possibly plays a crucial role in acute and chronic coronary syndromes by decreasing VSMC viability and promoting plaque instability.

Oxidized low-density lipoprotein is associated with apoptosis of vascular smooth muscle cells in human atherosclerotic plaques.

BACKGROUND: Cytotoxic oxidized LDL (oxLDL) has been shown to promote apoptosis in cultured vascular smooth muscle cells (VSMCs). We investigated the localization of oxLDL and its association with apoptosis and the expression of apoptosis-related proteins in early and advanced atherosclerotic lesions. METHODS AND RESULTS: Atherosclerotic plaques (n=23) from patients undergoing aortic, carotid, or femoral arterial surgery were studied. In early lesions, oxLDL was located predominantly in the superficial intima and in the media just beneath the internal elastic lamina. Medial VSMCs staining positive for oxLDL showed expression of BAX, a proapoptotic protein of the BCL-2 family. Apoptosis, as detected by DNA in situ terminal deoxynucleotidyl transferase end-labeling (TUNEL), was not present in these early lesions. In advanced plaques, areas of the intima positive for oxLDL showed lower alpha-smooth muscle actin immunoreactivity (P<0.01) and higher BAX immunoreactivity (P<0.05). Furthermore, these areas showed an increased number of apoptotic VSMCs (P<0.01). Western blot analysis revealed that oxLDL increases BAX expression in cultured human coronary VSMCs. CONCLUSIONS: We conclude that in early atherosclerotic lesions, oxLDL-positive VSMCs express BAX, which increases the susceptibility of these cells to undergo apoptosis. This could be important in our understanding of the transition of early lesions into advanced atherosclerotic plaques, which are characterized by regions of cell death. In advanced plaques, oxLDL-positive areas of the intima show higher BAX immunoreactivity and TUNEL-positive VSMCs, and this may contribute to plaque instability and rupture.

Estradiol decreases IGF-1 and IGF-1 receptor expression in rat aortic smooth muscle cells. Mechanisms for its atheroprotective effects.

Insulin-like growth factor (IGF-1) is a potent mitogen for vascular smooth muscle cells. Both IGF-1 and its receptor have been shown to be highly expressed in atherosclerotic lesions. Here we investigated whether part of the vasculoprotective properties of E(2) may be mediated by its negative regulation of the IGF-1 system. HeLa cells, which do not contain endogenous estrogen receptors (ER), were transiently transfected with IGF-1R promoter constructs with or without a plasmid encoding human ERalpha or ERbeta and treated with 100 nm 17beta-estradiol (E(2)) for 24 h. E(2) treatment decreased basal luciferase activity by 51%, and this effect was dependent on co-expression of ERalpha, whereas no repression was observed with ERbeta. A mutation within the DNA binding domain of the ERalpha abolished the repressor function of the ER receptor. Similarly, E(2) decreased IGF-1R transcription by 21% in rat aortic smooth muscle cells (RASMC), which express endogenous ER. This effect was specific for E(2), because it was inhibited by an antiestrogen and because progesterone did not have any effect on IGF-1R expression in HeLa or RASMC transfected with progesterone receptor. Accordingly, E(2) decreased IGF-1R and IGF-1 mRNA in RASMC by 47% and 33%. Western blot analysis and radioligand binding studies showed that E(2) also dose-dependently decreased IGF-1R protein expression in RASMC by 40% and 30%, respectively, and that IGF-1 protein was reduced by 43%. Repression of IGF-1R promoter activity by a combination of ERalpha and E(2) did not appear to be mediated via direct binding of ER to the IGF-1R promoter but rather by inhibition of SP1 binding to the IGF-1R promoter. Thus, E(2) down-regulates IGF-1R and IGF-1 expression in vascular smooth muscle cells. This may have important implications for the understanding of the beneficial effects of estrogen in the cardiovascular system.

Differential effects of low density lipoproteins on insulin-like growth factor-1 (IGF-1) and IGF-1 receptor expression in vascular smooth muscle cells.

Low density lipoproteins (LDLs) play an important role in the pathogenesis of atherosclerosis. LDL has been shown to be mitogenic and proapoptotic for vascular smooth muscle cells. However, the mechanisms are poorly understood and may result from an alteration in intracellular mitogenic signaling either directly by LDL or indirectly through an autocrine effect involving growth factor secretion and/or growth factor receptor expression. Insulin-like growth factor-1 (IGF-1) is an autocrine/paracrine factor for vascular smooth muscle cells and has potent anti-apoptotic effects. Thus, we hypothesized that part of the proliferative responses to LDLs may be explained by its modulation of IGF-1 or IGF-1 receptor (IGF-1R) expression. Treatment of rat vascular smooth muscle cells with increasing doses of native LDL dose-dependently increased IGF-1 mRNA by up to 2.6-fold; however, native LDL had no effect on IGF-1R mRNA expression. In contrast, the same doses of oxidized LDL significantly reduced IGF-1 and IGF-1R mRNA by 80 and 61%, respectively, and reduced IGF-1 and IGF-1R protein expression by 63 and 46%. In addition, native and oxidized LDL significantly increased IGF-1-binding protein-2 and IGF-1-binding protein-4 expression as measured by Western ligand blot. Most interestingly, anti-IGF-1 antiserum completely inhibited LDL-induced but not serum-induced increase in (3)H-thymidine incorporation, indicating a requirement for IGF-1 in the LDL-stimulated mitogenic signaling pathway. In summary, these results suggest that native and oxidized LDLs have differential effects on IGF-1 and IGF-1R expression. Because IGF-1 is a potent survival factor for vascular smooth muscle cells, our findings suggest that moderately oxidized LDL may favor proliferation of smooth muscle cells, whereas oxidized LDL may contribute to plaque apoptosis by local depletion of IGF-1 and IGF-1R.

Angiotensin II activation of insulin-like growth factor 1 receptor transcription is mediated by a tyrosine kinase-dependent redox-sensitive mechanism.

We have recently shown that angiotensin II activation of insulin-like growth factor 1 receptor (IGF-1R) transcription is a critical requirement for angiotensin-stimulated vascular smooth muscle cell growth; therefore, we examined the signaling pathway involved. In rat aortic smooth muscle cells, the antioxidants N-acetyl-L-cysteine (5 mmol/L) and pyrrolidine dithiocarbamate (100 micromol/L) completely inhibited angiotensin II-stimulated increases in IGF-1R mRNA and protein levels, suggesting the involvement of reactive oxygen species. Indeed, catalase abolished the Ang II-stimulated increase of IGF-1R protein expression, and accordingly, H(2)O(2) (0.2 mmol/L) or the oxidized products of linoleic acid, hydroperoxyoctadecadienoic acids (10 micromol/L), increased IGF-1R mRNA levels at 3 hours by 74+/-20% and 107+/-22% and increased receptor number at 24 hours by 51+/-6.7% and 55+/-7.4%, respectively. The protein tyrosine kinase inhibitors genistein and tyrphostin A25 also blocked angiotensin II increases in IGF-1R mRNA and protein levels and blocked the ability of hydroperoxyoctadecadienoic acids and H(2)O(2) to increase IGF-1R expression, suggesting that oxidative stress may be an early event in the angiotensin II signaling cascade. Furthermore, calcium chelation inhibited the angiotensin II effect. Transient transfection assays revealed that a (-2350)+640 IGF-1R promoter/luciferase construct was fully responsive to angiotensin II stimulation (127+/-20% increase). Ten millimoles per liter hydroperoxyoctadecadienoic acids and 0.2 mmol/L H(2)O(2) increased luciferase activity by 79+/-8.5% and 63+/-12%, respectively, and 5 mmol/L N-acetyl-L-cysteine blocked the angiotensin II-induced upregulation of luciferase activity by 70%. These data suggest that angiotensin II stimulates IGF-1R gene transcription via calcium-dependent activation of protein tyrosine kinase activity that lies downstream from an oxidant stimulus. These findings provide key insights into the signaling mechanisms whereby angiotensin II exerts its growth-promoting effects on the vasculature.

Distinct and common pathways in the regulation of insulin-like growth factor-1 receptor gene expression by angiotensin II and basic fibroblast growth factor.

Angiotensin II (Ang II) and basic fibroblast growth factor (bFGF) are important modulators of cell growth under physiological and pathophysiological conditions. We and others have previously shown that these growth factors increase insulin-like growth factor-1 receptor (IGF-1R) number and mRNA in vascular smooth muscle cells and that this effect is transcriptionally regulated. To study the mechanisms and the signaling pathways involved, IGF-1R promoter reporter constructs were transiently transfected in CHO-AT1 cells that overexpress angiotensin AT1 receptors. Our findings indicate that Ang II and bFGF significantly increased IGF-1R promoter activity up to 7- and 3-fold, respectively. The effect induced by Ang II was mediated via a tyrosine kinase-dependent mechanism, since tyrphostin A25 largely inhibited the Ang II-induced increase in promoter activity. In addition, co-transfection of dominant negative Ras, Raf, and MEK1 or pretreatment with the MEK inhibitor PD 98059 dose-dependently decreased both the Ang II- and bFGF-induced increase in IGF-1R transcription and protein expression, suggesting that the Ras-Raf-mitogen-activated protein kinase kinase pathway is required for both growth factors. Reactive oxygen species have been shown to act as second messengers in Ang II-induced signaling, and activation of the transcription factor NF-kappaB is redox-sensitive. While co-transfection of dominant negative IkappaBalpha mutant completely inhibited the Ang II-induced increase in transcription, it had no effect on the bFGF signaling. In contrast, co-transfection studies indicated that the transcription factors STAT1, STAT3, and c-Jun and the Janus kinase 2 kinase are required in the signaling pathway of bFGF, whereas only dominant c-Jun inhibited the Ang II-induced effect. In summary, these data demonstrate that Ang II and bFGF increase IGF-1R gene transcription via distinct as well as shared pathways and have important implications for understanding growth-stimulatory effects of these growth factors on vascular cells.

Angiotensin II increases macrophage-mediated modification of low density lipoprotein via a lipoxygenase-dependent pathway.

The molecular and cellular mechanisms by which hypertension enhances atherosclerosis are poorly understood. Angiotensin II (Ang II) has been implicated in the regulation of cellular lipoxygenases (LO), which are thought to play a role in atherogenesis by inducing oxidative modification of low density lipoprotein (LDL). We sought to test the hypothesis that Ang II would stimulate murine macrophage LO activity (which has both 12- and 15-LO activity). Competitive binding studies revealed the presence of Ang II AT1 receptors on mouse peritoneal macrophages (MPM) and J-774 cells, but not on the RAW cell line. Valsartan, a specific AT1 receptor antagonist inhibited Ang II binding, whereas PD 123319, an AT2 receptor antagonist did not. Incubation of MPM or J-774 cells with Ang II (10 pM to 1 microM) for 24 h led to a 2.5-3.5-fold increase in LO activity, measured as generated 13-HODE or 12(S)-HETE. This stimulation was inhibited by valsartan, but not by PD 123319. In contrast, Ang II did not stimulate LO activity in RAW macrophages. Semiquantitative reverse transcriptase-polymerase chain reaction showed a 2-3-fold increase in LO mRNA in MPM, but not in RAW cells after treatment with Ang II. Ang II also induced an increase in 12-LO protein. In addition, pretreatment of J-774 cells with Ang II increased in a dose-dependent manner the ability of the cells to modify LDL, resulting in greater chemotactic activity for monocytes, typical of minimally modified LDL. This stimulation was inhibited by AT1 receptor blockade. In summary, these data suggest that Ang II increases macrophage LO activity via AT1 receptor-mediated mechanisms and this further increases the ability of the cells to generate minimally oxidized LDL. These studies provide a link between hypertension and the associated increased atherosclerosis observed in hypertensive patients.

Local application of angiotensin II to the rat carotid artery induces adventitial thickening.

The aim of this study was to determine the effects of angiotensin II (Ang II) on the vascular wall, that are independent of its effects on systemic blood pressure. Ang II has been shown to have different effects on vascular growth in vitro and in vivo. The generally weak effects of Ang II in cell culture may be due to the absence of blood-borne factors and interactions between different cell types, dedifferentiation of cells and receptor loss. On the other hand, studies with administration of Ang II in vivo are complicated by effects on blood pressure and it is not clear whether the effects of Ang II are direct or secondary. In order to overcome some of these limitations, we delivered Ang II locally to the left carotid artery of normotensive rats with a perivascular drug delivery system, in doses that did not affect systemic blood pressure. Ang II was applied perivascularly to the artery in doses of 0.05, 0.5, 5 and 50 ng/h for 14 days. A vehicle-treated group and the right carotid artery served as controls. Other groups received noradrenaline as a control for the vasoconstrictive actions of Ang II. Ang II, but not noradrenaline, induced a dose-dependent thickening of the adventitia with increased cellularity which was characterized by DNA synthesis, neovascularization, and collagen deposition. Administration of specific ligands for the AT1 and AT2 receptors in the presence or absence of Ang II indicates that the increased cellularity and collagen deposition are mediated via the AT1 receptor, are inhibited via stimulation of the AT2 receptor, and that the angiogenesis may be mediated via the AT2 receptor. In conclusion, Ang II, independent of its effect on systemic blood pressure, induces a specific and receptor-dependent adventitial growth response when delivered perivascularly to the carotid artery.

Structural adaptation to ischemia in skeletal muscle: effects of blockers of the renin-angiotensin system.

OBJECTIVE: To investigate the effects of long-term treatment with blockers of the renin-angiotensin system on capillarization and growth of fibers in ischemic hind-limb muscles and in muscles under normal growth conditions. METHODS: Ischemia was induced by partial ligation of the left common iliac artery. RESULTS: Ischemia resulted in a significant increase in capillary and fiber density in the soleus muscle, a significant decrease in mean fiber size and a decrease in muscle cross-sectional area after 4 weeks compared with the contralateral nonischemic muscle. Ischemia also significantly decreased the muscle: body weight ratio of the left soleus muscle. We observed no significant effect on total number of capillaries and capillary: fiber ratio, suggesting that ischemia did not result in an increase in capillarization in this muscle. Treatments with subhypotensive and with hypotensive doses of the angiotensin converting enzyme (ACE) inhibitor benazeprilat, the angiotensin (Ang) II AT1 antagonist valsartan, or the Ang II AT2 antagonist PD 123 319 for 4 weeks did not influence any of the above-described changes in the normal and ischemic muscles and treatment effects were also independent of the degree of reduction of blood pressure. CONCLUSION: Treatments with an ACE inhibitor and with Ang II receptor antagonists in dose ranges that moderately lower blood pressure do not influence vessel density and any of the other structural adaptations after hind-limb ischemia. Administrations of ACE inhibitors and Ang II AT1 antagonists may therefore be adequate and beneficial therapies under ischemic conditions, such as in the treatment of hypertension complicated by intermittent claudication, for which treatment must not increase ischemia.

Effects of prolonged blockade of the renin angiotensin system on striated muscle microcirculation of spontaneously hypertensive rats.

Changes in microcirculation play an important role in the pathogenesis and maintenance of hypertension. The changes can be due to an alteration in vessel diameter or in the number of small blood vessels. In this study, the effects of prolonged administration of different blockers of the renin angiotensin system on the microcirculation of the cutaneous maximus muscle of young spontaneously hypertensive rats were determined by using the dorsal microcirculatory chamber model. Animals were treated with the angiotensin-converting enzyme inhibitor (ACE inhibitor) benazeprilat (3 mg/kg/d) or the specific angiotensin II AT1 receptor antagonist valsartan (3 mg/kg/d) for 4 weeks. Blood pressure was significantly lowered by 22 to 33% and to a similar extent in both treatment groups, whereas blood pressure in the control group continued to rise. Microvascular diameters and density were measured before and during the drug treatment and compared with those in the control group. There was no significant effect of either of the drug treatments on vascular diameters when compared with the control group for any vessel type (arterioles or venules). In contrast, there was a significant decrease in small arteriolar and venular density and in large venular density after treatment with the ACE inhibitor, whereas the angiotensin II AT1 receptor antagonist had no significant effect. The data do not suggest a role for angiotensin II in the long-term control of striated muscle microvascular tone. However, angiotensin II may be involved in microvascular growth via a non-AT1 receptor-mediated mechanism, or other vasoactive peptides degraded by ACE may contribute to the effects of the ACE inhibitor.