Deficiency of haematopoietic-cell-derived IL-10 does not exacerbate high-fat-diet-induced inflammation or insulin resistance in mice.

AIMS/HYPOTHESIS: Recent work has identified the important roles of M1 pro-inflammatory and M2 anti-inflammatory macrophages in the regulation of insulin sensitivity. Specifically, increased numbers of M2 macrophages and a decrease in M1 macrophages within the adipose tissue are associated with a state of enhanced insulin sensitivity. IL-10 is an anti-inflammatory cytokine and is a critical effector molecule of M2 macrophages. METHODS: In the present study, we examined the contribution of haematopoietic-cell-derived IL-10 to the development of obesity-induced inflammation and insulin resistance. We hypothesised that haematopoietic-cell-restricted deletion of IL-10 would exacerbate obesity-induced inflammation and insulin resistance. Lethally irradiated wild-type recipient mice receiving bone marrow from either wild-type or Il10-knockout mice were placed on either a chow or a high-fat diet for a period of 12 weeks and assessed for alterations in body composition, tissue inflammation and glucose and insulin tolerance. RESULTS: Contrary to our hypothesis, neither inflammation, as measured by the activation of pro-inflammatory stress kinases and gene expression of several pro-inflammatory cytokines in the adipose tissue and liver, nor diet-induced obesity and insulin resistance were exacerbated by the deletion of haematopoietic-cell-derived IL-10. Interestingly, however, Il10 mRNA expression and IL-10 protein production in liver and/or adipose tissue were markedly elevated in Il10-knockout bone-marrow-transplanted mice relative to wild-type bone marrow-transplanted mice. CONCLUSIONS/INTERPRETATION: These data show that deletion of IL-10 from the haematopoietic system does not potentiate high-fat diet-induced inflammation or insulin resistance.

Increased expression of the DNA-binding cytokine HMGB1 in human atherosclerotic lesions: role of activated macrophages and cytokines.

OBJECTIVE: Atherosclerosis is a chronic inflammatory response of the arterial wall to injury. High-mobility group box 1 (HMGB1) is a DNA-binding protein, which on release from cells exhibits potent inflammatory actions. We examined its expression in atherosclerotic lesions and regulation by cytokines. METHODS AND RESULTS: In atherosclerotic lesions, HMGB1 protein is expressed by endothelial cells, some intimal smooth muscle cells, and macrophages. As atherosclerosis develops and progresses from fatty streaks to fibrofatty lesion, the number of HMGB1-producing macrophages increases markedly. Studies using the THP-1 cell line indicated that HMGB1 mRNA expression could be markedly upregulated by inflammatory cytokines, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and also transforming growth factor (TGF)-beta. IFN-gamma, TNF-alpha, TWEAK, and TGF-beta induced an intracellular redistribution of HMGB1 and stimulated secretion by THP-1 cells and human blood monocytes. Inhibitors of MEK1/MEK2, protein kinase C, and PI-3/Akt, which inhibit lysosomal degranulation and mRNA translation, attenuated cytokine-induced HMGB1 secretion. CONCLUSIONS: Macrophage is the major cell type responsible for HMGB1 production in human atherosclerotic lesions. Inflammatory cytokines and TGF-beta increase HMGB1 expression and secretion by monocyte/macrophages. HMGB1 appears to be a common mediator of inflammation induced by inflammatory cytokines and is likely to contribute to lesion progression and chronic inflammation.

Eplerenone suppresses constrictive remodeling and collagen accumulation after angioplasty in porcine coronary arteries.

BACKGROUND: Coronary artery angioplasty triggers healing that causes constrictive remodeling. Because collagen accumulation correlates with constrictive remodeling and aldosterone has been implicated in collagen accumulation, we examined how aldosterone and the mineralocorticoid receptor antagonists spironolactone and eplerenone affect remodeling and collagen in porcine coronary and iliac arteries after angioplasty. METHODS AND RESULTS: Twenty-four pigs were allocated into 4 treatment groups: oral eplerenone (100 mg/d), oral spironolactone (200 mg/d), subcutaneous aldosterone (400 microgram/d), or no treatment. Twenty-eight days after angioplasty of the coronary arteries, eplerenone increased total vessel area by 30% (P<0.05) and luminal area by nearly 60% (P<0.05) compared with the no-treatment group, without affecting neointima size. These effects were accompanied by a 65% reduction in neointimal and medial collagen density (both P<0.05). Spironolactone was less effective, and aldosterone tended to exert opposite effects on coronary artery structure after angioplasty. These effects were not observed in angioplastied iliac arteries. CONCLUSIONS: Eplerenone attenuates constrictive remodeling after coronary artery angioplasty by mechanisms involving reduction in collagen accumulation, which thus appears to be an important contributor to constrictive remodeling of angioplastied coronary arteries.

Response to balloon injury is vascular bed specific: a consequence of de novo vessel structure?

Relative contributions of remodelling and neointimal hyperplasia to restenosis after coronary angioplasty have been inferred from studies using iliofemoral arteries, despite differences in structure/function and smooth muscle cell lineage. We compared the response to balloon overstretch injury of coronary arteries (C, n = 16) and similar sized branches of the iliac arteries (I, n = 18) using preinjury vessel diameter (P). inflated balloon size in vivo (B) and the manufacturer predicted inflated size (M) to examine arterial compliance, as well as resulting injury and morphology in perfusion fixed vessels. Despite similar degrees of oversizing (M/P) in the coronary and iliac arteries (C, 1.44 +/- 0.04; I, 1.51 +/- 0.02), the compliance to overstretch (B-P/M-P) was significantly greater in the coronary than the iliac arteries (C, 0.71 +/- 0.05; I, 0.51 +/- 0.03) (P <0.05) and was associated with a higher injury score (C, 1.64 +/- 0.31; I, 0.39 +/- 0.18 P < 0.05)--only 5/18 iliac vessels had rupture of the IEL compared with 13/16 in the coronary bed. In a subgroup of animals whose vessels (C:n = 7; I:n = 8) were perfusion fixed 28 days after injury, coronary arteries had greater intimal area (C:1.03 +/- 0.42; I:0.10 +/- 0.03 mm2, P < 0.05) but larger luminal area (C:1.61 +/- 0.71; 1:0.76 +/- 0.51, P < 0.05) due to greater area within EEL (C:3.38 +/- 0.49;1:] .49 +/- 0.54, P < 0.05) or less inward remodelling. The injuries resulting from similar strategies of balloon overstretch in the coronary and the iliac arteries are different and affect healing responses--iliac arteries remodel more while coronary arteries develop more intimal hyperplasia. These results indicate that caution is warranted when extrapolating results from the iliac to the coronary artery when investigating restenosis after angioplasty.

Blood flow regulates the development of vascular hypertrophy, smooth muscle cell proliferation, and endothelial cell nitric oxide synthase in hypertension.

Blood flow participates in vascular remodeling during development and growth by regulating cell apoptosis and proliferation. However, its significance in the development of vascular hypertrophy and vascular remodeling in hypertensive patients is not known. We investigated how changing blood flow through the common carotid artery (CA) of young adult rats rendered hypertensive via aortic coarctation affects CA hypertrophy and/or remodeling responses to hypertension. Blood flow was reduced by approximately 50% as a result of ligation of the external CA immediately after hypertension was induced, and the effects of that procedure were compared with those in similarly treated normotensive rats. Reducing blood flow in the hypertensive animals markedly augmented the development of CA hypertrophy over the ensuing 14 days by increasing the vessel wall cross-sectional area. In those animals, CA lumen size was unaltered by reducing blood flow, as was CA structure in normotensive animals. The greater hypertrophy in the hypertensive animals with reduced blood flow was associated with enhanced smooth muscle cell (SMC) proliferation 3 days after the hemodynamic changes were induced. There also appeared to be more extensive remodeling of the endothelium in the hypertensive animals with normal flow; this was indicated by the greater frequency of apoptotic endothelial cells at that time. This reduction in blood flow also attenuated endothelial cell nitric oxide synthase expression in hypertensive animals but not in normotensive animals. Severe reductions in blood flow ( approximately 90%) were required to reduce endothelial cell nitric oxide synthase in the normotensive animals. Increasing CA nitric oxide levels by perivascular application of S-nitroso-N-acetylpenicillamine (SNAP) to the CAs of hypertensive animals with reduced endothelial cell nitric oxide synthase attenuated the greater SMC proliferation. Thus, reduced blood flow in hypertensive animals promotes hypertrophy by enhancing SMC proliferation via mechanisms that reduce the inhibitory effects of nitric oxide on SMC proliferation.

Distinct patterns of transforming growth factor-beta isoform and receptor expression in human atherosclerotic lesions. Colocalization implicates TGF-beta in fibrofatty lesion development.

BACKGROUND: Some animal studies suggest that transforming growth factor-beta (TGF-beta) protects vessels from atherosclerosis by preventing intima formation, but others indicate a role in vessel proteoglycan accumulation and lipoprotein retention. To distinguish between these possibilities in humans, immunohistochemical studies were performed examining the coexpression of TGF-beta isoforms and the TGF-beta receptors ALK-5 and TbetaR-II in aorta during the various stages of atherosclerotic lesion development. METHODS AND RESULTS: The spatial relationships between TGF-beta1, TGF-beta3, ALK-5, and TbetaR-II expression were compared in aortic segments from 21 subjects. Nonatherosclerotic intima contained predominantly TGF-beta1, low concentrations of TbetaR-II, and barely detectable amounts of ALK-5. In contrast, fatty streaks/fibrofatty lesions contained high concentrations of both TGF-beta isoforms. Smooth muscle cells (SMCs), macrophages, and foam cells of macrophage and SMC origin contributed to these high levels. These lesions also contained high, colocalized concentrations of ALK-5 and TbetaR-II. Despite fibrous plaques containing TGF-beta1, its receptors were at detection limits. We found no evidence for truncated TbetaR-II expression in either normal intima or the various atherosclerotic lesions. CONCLUSIONS: TGF-beta appears to be most active in lipid-rich aortic intimal lesions. The findings support the hypothesis that TGF-beta contributes primarily to the pathogenesis of lipid-rich atherosclerotic lesions by stimulating the production of lipoprotein-trapping proteoglycans, inhibiting smooth muscle proliferation, and activating proteolytic mechanisms in macrophages.

Sympatho-adrenal mechanisms regulating cardiovascular hypertrophy in primary hypertension: a role for rilmenidine?

OBJECTIVE: Overactivity of the sympatho-adrenal system has long been considered a major factor contributing to blood pressure elevation in primary hypertension in humans and experimental animals. Our aim has been to elucidate its role in the development of cardiovascular hypertrophy in hypertensives. METHODS: Two studies have been performed in spontaneously hypertensive rats (SHR). One involved irreversible inhibition of the sympatho-adrenal system in newborn SHR using a sympathectomy procedure combined with prolonged alpha1-adrenoceptor blockade. The other involved reversible, long-term inhibition of the sympatho-adrenal system in young but mature SHR, by treatment with rilmenidine, a centrally active antihypertensive agent interacting with imidazoline receptors. Their effects on cardiovascular structure were examined. RESULTS: Sympathectomy plus alpha1-adrenoceptor blockade prevented the development of cardiac and vascular hypertrophy in adolescent SHR and these effects were maintained later in life. Rilmenidine administered to older (9-week) SHR also attenuated cardiac hypertrophy, abolished perivascular fibrosis associated with the intramyocardial vessels, and normalized vessel structure in the richly sympathetically innervated mesenteric vasculature. These effects were only partially related to the level of blood pressure reduction. CONCLUSIONS: Inhibition of the sympatho-adrenal system not only reduces blood pressure to normotensive levels in SHR but also has beneficial effects on cardiovascular structure, potentially reducing risk factors for cardiac and renal abnormalities frequently seen in long-term hypertensives. Therapeutically, these effects are likely to be achieved with rilmenidine.

Inhibition of protein tyrosine kinases attenuates increases in expression of transforming growth factor-beta isoforms and their receptors following arterial injury.

Transforming growth factor-beta 1 (TGF-beta 1) has been implicated in neointima formation in mechanically injured vessels and in restenosis after angioplasty. To further understand the significance of TGF-beta s in neointima formation, we examined the temporal expression of three TGF-beta isoforms (-beta 1, -beta 2, and -beta 3), their receptors (ALK-2, ALK-5, and T beta RII), and two putative TGF-beta responses (elevations in alpha v and beta 3 integrin mRNAs) in balloon catheter-injured rat carotid arteries and their dependency on tyrosine kinase activity. Using a standardized reverse transcriptase-polymerase chain reaction assay optimized to estimate mRNA levels, we observed distinct patterns of mRNA regulation for TGF-beta 1, -beta 2, and -beta 3 during the 48 hours immediately after injury, which were localized to the vessel's media. TGF-beta 1 mRNA increased 10-fold during this time while TGF-beta 3 mRNA also increased almost 2-fold. There were also increases in mRNAs encoding the TGF-beta type I receptors ALK-5 and ALK-2, as well as the type II receptor (T beta RII). Eight hours after the injury, mRNA levels for ALK-2 and ALK-5 were on average 2-fold higher; mRNA encoding the type II receptor increased approximately 3-fold by 24 hours. There were also associated increases in TGF-beta 1, TGF-beta 3, ALK-5, and T beta RII immunoreactive peptide levels. Peak increases in mRNAs for integrins alpha v and beta 3 averaged approximately 2-fold and 2.5-fold, respectively. Perivascular administration of the tyrosine kinase inhibitor genistein at the time of vessel injury markedly (> 85%) inhibited elevations in mRNAs encoding TGF-beta 1, TGF-beta 3, T beta RII, and the two integrins alpha v and beta 3, while application of its inactive chemically similar homologue daidzein did not prevent the injury-induced elevations in mRNA levels. Since the increases in integrins alpha v and beta 3 mRNA could be theoretically attributed to TGF-beta actions despite being dependent on tyrosine kinase activity, we examined whether the observed elevations in integrins alpha v and beta 3 were due to TGF-beta 1 secretion, using cultured rat carotid artery smooth muscle cells. TGF-beta 1 neutralizing antibodies specifically inhibited elevations in integrins alpha v and beta 3 mRNAs due to platelet-derived growth factor-BB and fibroblast growth factor-2. We conclude that multiple components of the TGF-beta system in vessels are activated following injury and influence expression of integrin receptors important for smooth muscle cell migration. Activation of the TGF-beta system appears to be highly dependent on tyrosine kinases.

Role of angiotensin II in early cardiovascular growth and vascular amplifier development in spontaneously hypertensive rats.

OBJECTIVE: To examine the role played by angiotensin II (AII) in the development of prehypertensive vascular hypertrophy in the spontaneously hypertensive rat (SHR) and to determine whether normalization of prehypertensive vascular hypertrophy attenuates the development of hypertension. DESIGN: Male SHR and Wistar-Kyoto (WKY) rats were treated from age 10 days until age 6 weeks with perindopril, an angiotensin converting enzyme (ACE) inhibitor, or with losartan, a type 1 AII receptor antagonist. METHODS: At termination of treatment, or 8 weeks after cessation of treatment, vascular growth was assessed by measurement of hindquarter resistance properties and of the medial cross-sectional area of first-order mesenteric arteries. The growth of the heart was assessed by measurement of the left ventricle:body weight ratio. RESULTS: Perindopril and losartan treatment of SHR and WKY rats led to a heterogeneous response in the vasculature, resulting in a reduction in perfusion pressures at maximum dilatation and constriction in the hindquarter vasculature but no significant change in medial cross-sectional area of small mesenteric arteries. Neither perindopril nor losartan treatment affected the growth of the left ventricle in the SHR. After the cessation of treatment the development of hypertension in the losartan- and perindopril-treated SHR did not differ from that in controls. CONCLUSION: These results suggest that AII, acting via angiotensin type 1 receptors, plays an important role in determining the early post-natal reactivity of the hindquarter vasculature but not the medial cross-sectional area of the mesenteric vasculature, which implies that different growth regulatory mechanisms are operating in the two vascular beds. The lack of effect in some vascular beds, together with the lack of effect on the heart, may account for the absence of a persistent effect on the blood pressure.

Vascular hypertrophy in renal hypertensive spontaneously hypertensive rats.

Vascular smooth muscle cells isolated from spontaneously hypertensive rats (SHR) replicate faster in vitro than do cells from Wistar-Kyoto (WKY) rats, suggesting that the vascular hypertrophy seen early in the life of SHR might be at least partially caused by abnormal cellular growth properties in vivo. To test whether specific growth stimuli produce more extensive hypertrophy in SHR than WKY rats, we compared their cardiovascular growth responses to two-kidney, one clip renal hypertension. Six-week-old animals were subjected to either renal artery clipping or sham operation. Four weeks after renal artery clipping, there was a proportionately smaller rise in systolic blood pressure in SHR than WKY rats (21% and 44%, respectively); however, the overall level of systolic blood pressure achieved in the two rat strains differed by less than 10 mm Hg (4%). Limitations in the blood pressure responses of SHR to renal artery clipping were not due to inadequate development of left ventricular hypertrophy, as this was greater in SHR than WKY rats; however, aortic hypertrophy was similar in both strains. Aortic DNA content changes in SHR were consistent with a significant hyperplasia of medial smooth muscle cells, whereas in WKY rats, there was cellular hypertrophy. Small and medium-sized arteries of the mesenteric vasculature were also hypertrophied in SHR, and the medial cross-sectional area increased by 63% and 114%, respectively, compared with increases of only 15% and 23% in WKY rats. Strain differences between the sham-operated rat groups were small.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmentally regulated transforming growth factor-beta 1 action on vascular smooth muscle growth in the SHR.

1. This study examined the effects of transforming growth factor-beta 1 (TGF-beta 1) on platelet-derived growth factor-BB (PDGF-BB)-stimulated proliferation of vascular smooth muscle cells (VSMC) isolated from aortic tissue of 1, 4 and 12 week old spontaneously hypertensive rats (SHR). 2. In 1 week old SHR, TGF-beta 1 inhibited by about 60% VSMC proliferation stimulated by PDGF-BB; this inhibitory action of TGF-beta 1 was absent in VSMC isolated from 4 week old, prehypertensive SHR. In contrast, TGF-beta 1 potentiated by about 125% the mitogenic activity of PDGF-BB in VSMC cultures from adult SHR. 3. Age-dependent alterations in the action of TGF-beta 1 suggests an important role for TGF-beta 1 in the development of vascular hypertrophy from adolescence onwards in the SHR.

Chronic angiotensin II type 1 receptor antagonism in genetic hypertension: effects on vascular structure and reactivity.

OBJECTIVE AND DESIGN: The aim of the study was to assess the role of angiotensin II (Ang II) in the maintenance of cardiovascular hypertrophy and the abnormal vascular amplifier properties in spontaneously hypertensive rats (SHR) with established hypertension. Losartan, a type 1 Ang II receptor antagonist, was administered to SHR and Wistar-Kyoto (WKY) rats, and its effects on blood pressure, cardiac hypertrophy, vascular morphology and hindquarter vascular amplifier properties assessed at the end of treatment and 3 months later. METHODS: Losartan was administered for 6 weeks to 14-week-old SHR (60 mg/kg per day orally). A bio-equivalent dose (20 mg/kg per day orally) was administered to age-matched WKY rats. Systolic blood pressure (SBP) was measured in conscious rats by tail-cuff plethysmography. Morphological changes were assessed both in the heart, from the ratio of the weight of the left ventricular wall plus septum to body weight, and in blood vessels from the medial cross-sectional areas of the abdominal aorta and mesenteric arteries. Vascular amplifier properties were measured by perfusion of the rat hindquarters under conditions of full dilation (papaverine hydrochloride) and incremental constriction with methoxamine hydrochloride. RESULTS: Losartan lowered SBP in SHR to normotensive WKY rat levels during treatment. Left ventricular hypertrophy and aortic cross-sectional area were reduced at the end of treatment to WKY rat levels; mesenteric artery cross-sectional area was reduced to a lesser extent. The abnormal hindquarter vascular amplifier properties of the SHR were normalized by losartan. Three months after treatment ended, SBP had returned to untreated SHR levels. Left ventricular hypertrophy and the abnormal hindquarter vascular amplifier properties had also partially redeveloped. CONCLUSIONS: Our findings support the hypothesis that Ang II contributes to the maintenance of cardiovascular hypertrophy and the abnormal vascular amplifier properties in SHR with established hypertension. However, its role appears to be variable and to depend on the type of vascular bed. Other, pressure-independent, factors may also contribute to vascular hypertrophy.