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Background: Granulocyte macrophage colony-stimulating factor (GM-CSF) deficiency affects the production and fiber assembly/organization of the vascular collagenous matrix; structural alterations to the elastic system were observed. The present study elaborates the effect of GM-CSF deficiency on the vascular elastin system. Methods and Results: Histological examination of the aorta of GM-CSF-deficient mice revealed structurally altered elastic fibers. The elastic fiber area was significantly enhanced, whereas the remaining medial area was not affected. Aortic size was significantly increased. Reverse transcription polymerase chain reaction demonstrated decreased expression levels of tropoelastin, lysyl oxidase and bone morphogenetic protein 1 (BMP-1). Cell culture studies on vascular smooth muscle cells showed that after clearance of GM-CSF with GM-CSF antibodies, the tropoelastin mRNA expression was markedly reduced. Concomitantly, lysyl oxidase and BMP-1 mRNA levels were decreased. Treatment with GM-CSF stimulated the expression of these mRNAs. Conclusions: Our studies demonstrate that disorganization of elastic lamellae as induced by GM-CSF deficiency is associated with adaptive vascular remodeling. The decreased tropoelastin expression observed is associated with elastic fiber hypertrophy. This paradox effect may be explained by decreased expression levels of lysyl oxidase and BMP-1, both mediating cross-linkage and thus assembly and organization of elastic fibers. From our data, we conclude that GM-CSF is a prerequisite for the maintenance of structural integrity of the vessel wall.

OBJECTIVES To assess the potential of the donor heart to respond to interleukin-6 (IL6), the present study investigated the expression of IL6 receptor components in the myocardium of donor hearts before transplantation. BACKGROUND Donor heart dysfunction early after transplantation has been associated with the cytokine storm after donor brain death. Proinflammatory cytokines are thought to play a central role in this process. Interleukin-6 is of specific interest because it has been associated with cardiac allograft dysfunction and is related to an impaired prognosis. Its action requires expression of the specific IL6 receptor (IL6R), and the common signal transducer of the IL6 family glycoprotein 130 (gp130) in the donor heart. METHODS The activation of IL6, IL6R and gp130 messenger ribonucleic acid (mRNA) and protein was studied via reverse transcription-polymerase chain reaction (RT-PCR) and immunohistology in donor hearts (n = 6) and compared with patients undergoing evaluation of ventricular arrhythmias (control, n = 9) or with advanced heart failure (n = 20). RESULTS Messenger RNA of IL6, IL6R and gp130 was strongly expressed in all chambers of donor hearts, whereas right ventricles of control patients did not show any expression (donor vs. control: p < 0.005). Right ventricles of failing hearts showed IL6, IL6R and gp130 mRNA levels comparable with those found in donor hearts. Immunohistochemistry paralleled the RT-PCR data on the protein level. While IL6 was mainly expressed by myocytes, both receptor components were preferentially found mainly on interstitial cells. CONCLUSIONS The expression of the IL6 receptor components in the donor heart before transplantation establishes the condition sine qua non for the response of the donor heart to circulating IL6. This mechanism may explain the close association of elevated IL6 serum levels to acute cardiac allograft dysfunction in the early perioperative period.

Vascular remodeling is influenced by trauma and proatherogenic factors such as cholesterol. It has been shown that cholesterol exerts a direct effect on vessel wall structure. In this study we evaluated the effects of vascular trauma and cholesterol treatment on vascular remodeling and plaque integrity in carotid ligated ApoE-deficient mice. The right carotid artery was ligated in mice fed regular chow or cholesterol and fat containing diet. After 4 weeks left (non-ligated) and right (ligated) carotids were prepared. For studying vascular remodeling the vascular areas were evaluated morphometrically by calculating the areas from circumference measurements on Verhoff-van Gieson stains. The cellular and structural features of the plaque were analyzed by histological staining and immunohistochemistry. Under regular chow total vessel area decreased by 35% (p0.001); cholesterol-rich diet led to an increase by 20% (p0.05). In both feeding groups ligated carotids presented neointima development. The medial area increased only in mice fed regular chow. The luminal area was reduced by 80% (regular chow: p0.001) and by 90% (cholesterol-rich diet: p0.01). Regular chow led to structured plaques showing the typical features of stable plaques. Under cholesterol diet well defined plaque structures were missing. These lesions were characterized by numerous macrophages, few mostly PCNA positive smooth muscle cell (SMC) and less collagen particularly in the shoulder region. Our data indicate that in ApoE-deficient mice both direction of the remodeling response and lesion integrity are due to the diet applied: regular chow led to constrictive remodeling, whereas cholesterol and fat containing diet was associated with an adaptive response. Our data further indicate that the direction of response is not only related to the macrophage content but also to a proliferative intimal SMC-phenotype. Our data implicate that high serum cholesterol levels are not only inducers of plaque instability but also of the so far "positively recorded" compensatory remodeling.

Background— LOX-1, a receptor for oxidized low-density lipoprotein (OxLDL), seems to play a critical role in foam cell formation of macrophages (Mφs) and smooth muscle cells (SMC). Inhibition of LOX-1 expression reduces foam cell formation and might influence lipid core formation in atherosclerotic lesions. Because statins are able to downregulate LOX-1 expression in vitro, we examined if pravastatin can be used to reduce LOX-1 expression and lipid core formation in lesions of Watanabe heritable hyperlipidemic (WHHL) rabbits. Methods and Results— Pravastatin downregulated LOX-1 expression in cultured human Mφs and in cultured human aortic SMCs. Homozygous WHHL rabbits were treated with 50 mg kg −1 d −1 pravastatin for 32 weeks. Immunohistochemical studies revealed that LOX-1 was expressed in intimal Mφs and SMCs of atherosclerotic lesions. The pravastatin-treated rabbits showed, compared with untreated rabbits, a significantly reduced LOX-1 protein and mRNA expression in the aortic arch. Lipid labeling of this aorta region also demonstrated a strong reduction of the ratio of lipid core area/total lesion area in pravastatin-treated rabbits. Conclusions— The in vivo inhibition of LOX-1 expression by pravastatin demonstrated here represents a new pleiotropic effect of pravastatin. This in vivo inhibition of LOX-1 might be one mechanism for the lipid core reducing effect of pravastatin in atherogenesis.

Increased expression of connexin43 gap junctions in smooth muscle cells (SMC) is implicated in the response to primary arterial injury and in the early stages of human coronary atherosclerosis, but the relevance of these findings to restenosis is unknown. Here we investigated the expression of connexin43 gap junctions in restenotic aortas of cholesterol-fed double injured rabbits. Immunofluorescence confocal microscopy was used to evaluate temporal and spatial expression patterns and to characterize the major expressing cell type. Parallel studies were conducted by electron microscopy, in situ hybridization and Northern blot analysis. Connexin43 gap junctions- and connexin43 mRNA-expressing cells were abundant in the media of non-injured control aorta. Following primary injury and 6 weeks cholesterol diet, connexin43 gap junctions were found distributed throughout the primary intimal layer; although medial expression was reduced, the overall mRNA expression level remained similar to that of non-injured controls. After secondary injury, no major change in distribution pattern of connexin43 gap junctions occurred up to day 10, when marked neointimal labeling was observed. This overall pattern persisted, though with some diminution, at later stages. On the mRNA level total connexin43 mRNA expression declined to about 40% of control values within 4 days after secondary injury (P < 0.05), but subsequently increased four-fold, attaining levels double that of non-injured controls in the 10-day group (P < 0.005 versus control and 4 days). At later stages mRNA expression levels returned to values similar to those of non-injured controls. At all stages, connexin43 gap junctions were localized to the SMC, not to macrophages. We conclude that the enhanced gap junction formation may contribute to the coordination of the response of SMC after secondary injury, particularly in the early phase of restenosis.

Objective : Basic fibroblast growth factor (bFGF)-stimulated proliferation of coronary smooth muscle cells (cSMC) contributes to the pathogenesis of arteriosclerosis and restenosis. However, the molecular mechanisms involved are not fully understood. We have shown previously that protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) are required for the bFGF-stimulated mitogenic process in bovine cSMC. In this study, we determined the PKC isoform(s) involved and investigated their functional role in the bFGF-stimulated signaling and cell cycle progression in human and bovine cSMC. Methods and Results : Downregulation of PKC by phorbol 12-myristate 13-acetate (PMA) inhibited bFGF-induced DNA synthesis, the activation of MAPK, and the expression of c-myc, demonstrating the involvement of PMA-sensitive PKC isoforms in growth factor-induced proliferation and the MAPK pathway. The PMA-sensitive classical PKC isoforms α, β, γand novel PKC isoforms δ and e were found in human cSMC. Whereas blocking of the classical PKC isoforms had no influence, the suppression of PKC δ by genetic and pharmacological approaches inhibited the bFGF-stimulated c-Raf1–MEK–MAPK–c-myc signaling and DNA synthesis in cSMC. In contrast to PKC e, our results showed that bFGF activated PKC δ by phosphorylation in a time-dependent manner. In addition, inhibition of PKC δ induced a hypophosphorylation of the retinoblastoma protein and suppression of the cyclins D1 and A, demonstrating the importance of PKC δ for bFGF-induced cell cycle progression through the G1 phase in cSMC. Conclusions : Our results show that PKC δ is required for the bFGF-stimulated c-Raf1–MEK–MAPK–c-myc signaling pathway involved in the proliferation of cSMC. Therefore, it may be an interesting therapeutic target for preventing proliferative vascular disorders.

To the Editor: We read with interest the report by Podewski et al1 on the expression of interleukin-6 (IL-6) and related cytokines, their receptors, and molecules of the Janus kinase (JAK)–signal transducers and activators of transcription (STAT) signal-transduction cascade in patients with idiopathic dilated cardiomyopathy. We agree that in patients with dilated cardiomyopathy, the protein expression of IL-6–related molecules and their signal-transduction cascade have not been evaluated before in such detail. However, we have some concerns. IL-6 and its receptor subunits have been extensively investigated before in failing hearts.2,3 In one of our studies on the cardiac IL-6/IL-6 receptor system, we particularly emphasized the significance of the type of controls used.4,5 Nonfailing donor hearts showed expression levels of IL-6 receptor and glycoprotein (gp) 130 similar to those observed in failing hearts. Because these hearts were nonfailing donor hearts, just as those used by Podewski et al,1 the lack of B-type natriuretic peptide upregulation apparently does not rule out upregulation of the cardiac IL-6 system, presumably related to brain death. In …

GM-CSF takes part in the cytokine network regulating the metabolism of extracellular matrix (ECM) during atherogenesis. Since data also point to an effect of GM-CSF on the vascular ECM in general, the vascular collagenous matrix was studied in wild-type and GM-CSF-deficient mice. Histological examination revealed a disorganized vascular ECM in GM-CSF-deficient mice involving the collagenous matrix and elastic fiber system. As shown by electron microscopy, collagen bundles were disrupted and reduced. The diameter of fibrils varied widely. mRNA expression of collagens and related molecules was studied. Fibrillar collagens were markedly reduced, alpha1(I)procollagen to 16.5% of control levels alpha1(III)procollagen was abolished whereas the expression level of network-forming alpha1(VIII)procollagen was not altered. As shown by in situ hybridization, the number of collagen-expressing cells was reduced. Matrix metalloproteinases and their inhibitor 1 were not affected by GM-CSF deficiency. Our studies demonstrate that GM-CSF plays a major role in the cytokine network regulating the metabolism of vascular collagens. GM-CSF deficiency leads to an altered composition of the vascular collagenous matrix, i.e., reduced amount of fibrillar collagen, altered ratio of fibrillar and network-forming collagen, and failures in the fibrillogenesis. We suggest that GM-CSF is a basic requirement for the maintenance of vessel wall integrity and resilience.

Autocrine stimulation and paracrine interaction between coronary smooth muscle cells (cSMC) and endothelial cells (EC) act as regulators of the vascular angiogenesis. Basic fibroblast growth factor (bFGF), its receptor FGF-R1, and coreceptor heparansulfate proteoglycan (HSPG) are important components involved in this angiogenic process. We investigated the influence of angiotensin (Ang) II on this trimolecular bFGF complex, the underlying signaling and the proliferative process in human cSMC. Ang II induces an AT1 receptor-dependent expression of bFGF and also upregulates the FGF-R1 and HSPG expression which is suppressed by losartan, the AT1 receptor blocker. AT1 receptor signaling which is characterized by phosphorylation of p42-mitogen-activated protein kinase (MAPK) is involved in Ang II-induced bFGF, FGF-R1 and HSPG upregulation and DNA synthesis in human cSMC. In contrast, inhibition of the AT2 receptor by PD123,319 has no influence on these Ang II-stimulated and via the MAPK cascade-mediated proangiogenic effects. Finally, our data show that the Ang II-induced DNA synthesis in cSMC is mediated via the bFGF expression. In conclusion, our results suggest that the Ang II-induced angiogenic effects in the vessel wall are supported by the AT1 receptor-stimulated and MAPK pathway-mediated upregulation of the autocrine/paracrine trimolecular bFGF complex in cSMC.

We read with great interest the report by Indolfi et al. [(1)][1]. The data reported are very interesting because, to the best of our knowledge, this is the first report demonstrating simultaneously that: 1) a hydroxymethylglutaryl Coenzyme A (HMG-CoA) reductase inhibitor blocks smooth muscle cell (