microRNA-352 regulates collateral vessel growth induced by elevated fluid shear stress in the rat hind limb.

Although collateral vessel growth is distinctly enhanced by elevated fluid shear stress (FSS), the underlying regulatory mechanism of this process remains incompletely understood. Recent studies have shown that microRNAs (miRNAs) play a pivotal role in vascular development, homeostasis and a variety of diseases. Therefore, this study was designed to identify miRNAs involved in elevated FSS-induced collateral vessel growth in rat hind limbs. A side-to-side arteriovenous (AV) shunt was created between the distal stump of one of the bilaterally occluded femoral arteries and the accompanying vein. The miRNA array profile showed 94 differentially expressed miRNAs in FSS-stressed collaterals including miRNA-352 which was down-regulated. Infusion of antagomir-352 increased the number and proliferation of collateral vessels and promoted collateral flow restoration in a model of rat hind limb ligation. In cell culture studies, the miR-352 inhibitor increased endothelial proliferation, migration and tube formation. In addition, antagomir-352 up-regulated the expression of insulin-like growth factor II receptor (IGF2R), which may play a part in the complex pathway leading to arterial growth. We conclude that enhanced collateral vessel growth is controlled by miRNAs, among which miR-352 is a novel candidate that negatively regulates arteriogenesis, meriting additional studies to unravel the pathways leading to improved collateral circulation.

The Formation of Aberrant Collateral Vessels during Coronary Arteriogenesis in Dog Heart.

We previously reported excessive growth of collateral vessels in the dog heart during arteriogenesis induced by implantation of an ameroid constrictor around the circumflex branch of the left coronary artery. In the present study, using histology and immunocofocal microscopy, we further investigated how these aberrant collateral vessels form. By comparison with mature collateral vessels the following findings were made: perivascular space was very narrow where damage of the perivascular myocardium occurred; the neointima was very thick, resulting in a very small lumen; elastica van Gieson staining revealed the absence of the internal elastic lamina and of elastic fibers in the adventitia, but abundant collagen in the adventitia as well as in the neointima; smooth muscle cells of the neointima expressed less α-SM actin and little desmin; expression of the fibroblast growth factors aFGF, bFGF and platelet-derived growth factor (PDGF)-AB was observed mainly in the endothelial cells and abluminal region, but transforming growth factor-ß1 was only present in the adventitia and damaged myocardium; angiogenesis in the neointima was observed in some collateral vessels expressing high levels of eNOS, and cell proliferation was mainly present in the abluminal region, but apoptosis was in the deep neointima. In conclusion, these data for the first time reveal that the formation of the aberrant collateral vessels in the dog heart involves active extracellular proteolysis and a special expression profile of growth factors, eNOS, cell proliferation and apoptosis. The finding of a narrow perivascular space and perivascular myocardial damage suggests that anatomical constraint is most likely the cause for exacerbated inward remodeling in aberrant collateral vessels in dog heart.

Nitric Oxide Increases Arterial Endotheial Permeability through Mediating VE-Cadherin Expression during Arteriogenesis.

Macrophage invasion is an important event during arteriogenesis, but the underlying mechanism is still only partially understood. The present study tested the hypothesis that nitric oxide (NO) and VE-cadherin, two key mediators for vascular permeability, contribute to this event in a rat ischemic hindlimb model. In addition, the effect of NO on expression of VE-caherin and endothelial permeability was also studied in cultured HUVECs. We found that: 1) in normal arteriolar vessels (NAV), eNOS was moderately expressed in endothelial cells (EC) and iNOS was rarely detected. In contrast, in collateral vessels (CVs) induced by simple femoral artery ligation, both eNOS and iNOS were significantly upregulated (P<0.05). Induced iNOS was found mainly in smooth muscle cells, but also in other vascular cells and macrophages; 2) in NAV VE-cadherin was strongly expressed in EC. In CVs, VE-cadherin was significantly downregulated, with a discontinuous and punctate pattern. Administration of nitric oxide donor DETA NONOate (NONOate) further reduced the amounts of Ve-cadherin in CVs, whereas NO synthase inhibitor L-NAME inhibited downregulation of VE-cadherin in CVs; 3) in normal rats Evans blue extravasation (EBE) was low in the musculus gracilis, FITC-dextron leakage was not detected in the vascular wall and few macrophages were observed in perivascular space. In contrast, EBE was significantly increased in femoral artery ligation rats, FITC-dextron leakage and increased amounts of macrophages were detected in CVs, which were further enhanced by administration of NONOate, but inhibited by L-NAME supplement; 4) in vitro experiments confirmed that an increase in NO production reduced VE-cadherin expression, correlated with increases in the permeability of HUVECs. In conclusion, our data for the first time reveal the expression profile of VE-cadherin and alterations of vascular permeability in CVs, suggesting that NO-mediated VE-cadherin pathway may be one important mechanism responsible, at least in part, for macrophage invasion during arteriogenesis.

Collateral vessel growth induced by femoral artery ligature is impaired by denervation.

Innervation plays an important role in development and remodeling of blood vessels. However, very little is known whether innervation is involved in arteriogenesis. In the present study, we tested the hypothesis that innervation may contribute to the process of arteriogenesis induced by ligature of femoral artery in rat/rabbit hind limb with or without denervation. We found that: (1) angiography showed more collateral vessels in the ligature side than that in ligature plus denervation side; (2) collateral vessels in denervation side was characterized by an inward remodeling; (3) in both collateral vessels (CVs) from only femoral ligature side as well as the ligature plus denervation side, ICAM-1 and VCAM-1 expression was up-regulated but increased VCAM-1 was more evident in the adventitia of collateral vessels of only femoral ligature side; (4) 7 days after surgery, in CVs from the femoral ligature side only, numerous macrophages (RAM11 positive cells) and high cell proliferation ratio (ki67 positive cells) were detected, but they were less in the denervation side. In conclusion, our data demonstrate for the first time that neural regulation is one of the factors that contributes to collateral vessel growth in rat/rabbit hind limb ischemic model by showing collateral vessel growth induced by femoral artery ligature is impaired by denervation.

A common MLP (muscle LIM protein) variant is associated with cardiomyopathy.

RATIONALE: We previously discovered the human 10T-->C (Trp4Arg) missense mutation in exon 2 of the muscle LIM protein (MLP, CSRP3) gene. OBJECTIVE: We sought to study the effects of this single-nucleotide polymorphism in the in vivo situation. METHODS AND RESULTS: We now report the generation and detailed analysis of the corresponding Mlp(W4R/+) and Mlp(W4R/W4R) knock-in animals, which develop an age- and gene dosage-dependent hypertrophic cardiomyopathy and heart failure phenotype, characterized by almost complete loss of contractile reserve under catecholamine induced stress. In addition, evidence for skeletal muscle pathology, which might have implications for human mutation carriers, was observed. Importantly, we found significantly reduced MLP mRNA and MLP protein expression levels in hearts of heterozygous and homozygous W4R-MLP knock-in animals. We also detected a weaker in vitro interaction of telethonin with W4R-MLP than with wild-type MLP. These alterations may contribute to an increased nuclear localization of W4R-MLP, which was observed by immunohistochemistry. CONCLUSIONS: Given the well-known high frequency of this mutation in Caucasians of up to 1%, our data suggest that (W4R-MLP) might contribute significantly to human cardiovascular disease.

Bone marrow-derived cells contribute to infarct remodelling.

OBJECTIVE: The paradigm that cardiac myocytes are non-proliferating and terminally differentiated cells has recently been challenged by several studies reporting the ability of bone marrow-derived cells (BMC) to transdifferentiate into cardiomyocytes. However, these results are controversial and could not be reproduced by others. Therefore, we studied the contribution and potential transdifferentiation of BMC into different cell types during the remodelling process in mouse hearts with experimental myocardial infarction. METHODS: Mice (C57BL/6J) were sublethally irradiated, and BM from enhanced green fluorescent protein (eGFP)-transgenic mice was transplanted. Coronary artery ligation was performed 3 months later. The hearts were studied 7 days (n=13) and 21 days (n=12) after infarction. Immunohistochemical staining was performed using antibodies against titin, connexin 43, vimentin, SMemb alpha-smooth muscle actin, CD45, CD34, F4/80, BS-1, CD31, and eGFP. Sections were analyzed using fluorescence and confocal laser microscopy. RESULTS: Success of BM transplantation was confirmed by FACS analysis. Occlusion of the coronary artery resulted in infarct sizes of 41+/-6% of the left ventricle. CD45+/eGFP+ inflammatory cells were found frequently after 7 days and to a lesser degree after 21 days. In 25 examined hearts, only 3 eGFP-positive cardiomyocytes were found. However, numerous BMC-derived fibroblasts and myofibroblasts were found in the infarct area. BMC contributed to scar tissue neoangiogenesis but not to angiogenesis in the periinfarct and remote zones. CONCLUSION: Transdifferentiation of BMC into viable cardiomyocytes is a negligible event in normal repair processes after myocardial damage. BMC-derived fibroblasts and myofibroblasts as well as neoangiogenesis significantly contribute to post-infarction scar formation and might be important in scar tissue remodelling.

Reduced sarcoplasmic reticulum Ca2+ -ATPase activity and dephosphorylated phospholamban contribute to contractile dysfunction in human hibernating myocardium.

Human hibernating myocardium (HHM) is characterized by reversible contractile dysfunction during chronic ischemia. A disturbed calcium-homeostasis is a decisive factor for reduced functional capacity in heart diseases. We therefore investigated calcium-handling proteins in HHM. In 12 patients suffering from multi-vessel coronary artery disease and contractile dysfunction with indication for bypass surgery, HHM was detected preoperatively by thallium scintigraphy, radionuclide ventriculography and dobutamine echocardiography. Transmural biopsies of these regions were taken and analyzed by immunohistochemistry and electron microscopy. Furthermore, SR-calcium ATPase (SERCA2a), phospholamban (PLN), the phosphorylated forms of PLN (PLN-Ser16, PLN-Thr17) as well as sodium-calcium exchanger (NCX) and ryanodine receptor (RyR2) were investigated by RT-PCR and Western-blotting. Additionally, SERCA2a activity was measured by an enzyme-coupled assay. In all patients complete functional recovery could be documented 3 months after revascularization by repeating all preoperative investigations. In HHM maximal SERCA2a activity was significantly reduced (HHM: 424.5 +/- 33.9, control: 609.0 +/- 48.5 nmol ATP mg protein(-1) min(-1), p

Carvedilol improves myocardial contractility compared with metoprolol in patients with chronic hibernating myocardium after revascularization.

BACKGROUND: We tested the hypothesis of whether carvedilol delays morphologic degeneration and improves functional outcome compared with metoprolol tartrate in patients with hibernating myocardium undergoing surgical revascularization. We have previously shown that patients with chronic hibernating myocardium undergo progressive cellular degeneration and fibrosis. METHODS: Twenty patients with multivessel coronary artery disease revascularization and hibernating myocardium as assessed by technetium-99m perfusion scintigraphy and fluorine-18-fluorodeoxyglucose positron emission tomography were randomized to receive either carvedilol or metoprolol tartrate for at least 2 months before surgery, and this was continued for 7 months postoperatively. Left ventricular ejection fraction and regional wall motion abnormalities were assessed by left ventriculography at baseline and 7 months postoperatively. Intraoperative transmural needle biopsy samples were obtained for microscopic analysis. RESULTS: Postoperatively, the ejection fraction increased from 31% +/- 5% to 44% +/- 4% (P < .005) in the carvedilol group (n = 10), and from 30% +/- 6% to 40% +/- 6% in the metoprolol tartrate group (P < .05 vs preoperatively and vs carvedilol). Wall motion abnormalities in the carvedilol group improved from -2.1 +/- 0.4 to -0.6 +/- 0.5 (P < .05) and from -2.3 +/- 0.5 to -1.6 +/- 0.6 in the metoprolol tartrate group (P < .05 vs preoperatively and vs carvedilol). Microscopic analysis after 72 +/- 18 days of either treatment showed mild cardiomyocyte degeneration and moderate-to-severe fibrosis (28% +/- 7%) in the carvedilol group compared with moderate cardiomyocyte degeneration and moderate-to-severe fibrosis (33% +/- 6%) in the metoprolol tartrate group. Apoptosis, as assessed by the terminal deoxynucleotidyl transferase nick end labeling method, was observed in only 1 patient in each group. CONCLUSIONS: Carvedilol treatment of hibernating myocardium results in improved functional recovery after revascularization compared with metoprolol tartrate, and this might partially be related to reduced cardiomyocyte degeneration.

Expression of endothelial nitric oxide synthase in the vascular wall during arteriogenesis.

Nitric oxide (NO) has been demonstrated to play an important role in angiogenesis, and also to be involved in collateral vessel growth. The expression of endothelial NO synthase (eNOS) is moderated partly by blood flow-induced mechanical factors, i.e., shear stress. The purpose of this study was to evaluate how the expression of eNOS correlates with the development of collateral vessels in dog heart, induced by chronic occlusion of the left circumflex artery. Immunoconfocal microscopy using an antibody against eNOS was used to detect expression of eNOS in different stages of arteriogenesis. Collateral vessels were classified into normal, growing and mature vessels by using the cytoskeleton marker desmin. Expression of the growth factors bFGF and metallproteinase-2 (MMP-2) was also examined. The data show that in normal arteriolar vessels, expression of eNOS is very low, but in growing collateral vessel there is a 6.2-fold increase, which, however, returned to normal levels in mature collateral vessels. The expression of eNOS was localized only in endothelium, either in normal or growing vessels. bFGF was very weakly stained in normal vessels, but highly expressed in growing collateral vessels. MMP-2 was strongly stained in neointima, but very weak in endothelium. In addition, we also examined expression of iNOS because iNOS may be induced in vessel injury or in disease states, but it was not detected in either normal or growing collateral vessels. Our findings indicate that the expression pattern of eNOS is closely associated with the development of collateral vessels, suggesting that eNOS plays an important role in arteriogenesis.

Matrix metalloproteinases and their tissue inhibitors in pressure-overloaded human myocardium during heart failure progression.

OBJECTIVES: We studied the role of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in fibrosis formation in the transition from hypertrophy to heart failure (HF) as well as the cellular source of MMPs and TIMPs. BACKGROUND: Human pressure-overloaded hearts are characterized by a significant increase in cardiac fibrosis. However, the contribution of the proteolytic/antiproteolytic system in aortic stenosis (AS) during hypertrophy progression has not yet been elucidated. METHODS: Three groups of AS patients (I: EF >50%, n = 12; II: EF 50% to 30%, n = 10; III: EF <30%, n = 12) undergoing aortic valve replacement and seven controls were studied. Tissue samples were investigated by immunoconfocal microscopy, Western blotting, and zymography. RESULTS: Quantitative analysis by immunoconfocal microscopy and Western blotting showed an upregulation of MMP-1, -2, -3, -9, -13, and -14 in group I and further increases in later stages. Tissue inhibitors of metalloproteinase-1 and -2 were enhanced and TIMP-4 was decreased in comparison to control. Gelatinolytic activity of MMP-2 significantly (p < 0.05) increased 1.2-fold (group I), 1.5-fold (group II), and 1.6-fold (group III) over control. The level of collagen I was significantly upregulated in all AS groups. Immunoconfocal microscopy showed that MMPs and TIMPs are produced predominantly by fibroblasts. The number of proliferating fibroblasts was significantly elevated during the transition to HF (0.67 n/mm(2)-control, 5.03-group III, p < 0.05). CONCLUSIONS: In human hearts a continuous turnover of the extracellular matrix occurs during the progression from compensated hypertrophy to HF that is characterized by the upregulation of MMPs and inadequate inhibition by TIMPs. The altered balance between proteolysis/antiproteolysis with accompanying proliferation of fibroblasts results in fibrosis progression.

Human hibernating myocardium is jeopardized by apoptotic and autophagic cell death.

OBJECTIVES: The aim of the present study was to objectify the loss of myocytes and the mechanism by which myocytes die in human hibernating myocardium (HHM). BACKGROUND: Intracellular degeneration, reduced cellular protein synthesis, and the replacement fibrosis contribute to structural disintegration of HHM. METHODS: In 14 patients, HHM was diagnosed by dobutamine echocardiography, radionuclide ventriculography, and thallium-201 scintigraphy. Functional recovery was documented by repeating the preoperative clinical investigations three months after successful coronary artery bypass graft surgery (CABG). During CABG, transmural biopsies were taken from the center of HHM regions and studied by electron microscopy, immunohistochemistry, the terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) method, reverse transcription-polymerase chain reaction, and Western blotting. Control samples were taken from nondiseased human myocardium. RESULTS: All patients showed significant improvement or normalization of the regional function of HHM. Ubiquitin-related autophagic cell death was evident ultrastructurally by the occurrence of autophagic vacuoles, cellular degeneration, and nuclear disassembly. Ubiquitin-protein complexes were found in 0.03 +/- 0.008% (control: 0%, p < 0.005) of all myocytes. The proteasome 20S subunit/total myocytes were reduced from 63.3 +/- 9.6% in control myocardium to 36.9 +/- 8.4% in HHM. Complement-9, indicating oncosis, was found in only one of 14 biopsies. TUNEL-positive myocytes were 0.002 +/- 0.0003%. Electron microscopy showed apoptotic cells in 3 of 14 samples. However, the bcl-2/bax ratio was significantly reduced. Moreover, caspase-3 messenger ribonucleic acid was 8.5 times upregulated, and caspase-3 was activated. Cell death was absent in controls. CONCLUSIONS: In HHM, ubiquitin-related autophagic cell death and apoptosis cause a loss of myocytes. This plays an important role in progressive tissue damage and causes a reduction of the extent of functional recovery of HHM.

Morphological changes of the aortic valve leaflets in non-compliant aortic roots: in-vivo experiments.

BACKGROUND AND AIM OF THE STUDY: Age-related loss of elasticity of the naturally compliant aortic root disrupts the coordinated function of the valve leaflets. Morphological changes that developed over time in the aortic valve leaflets of non-compliant aortic roots were studied. METHODS: Stiffening of the aortic roots was achieved in vivo by applying Super Glue around the sinus of Valsalva in 27 New Zealand White rabbits. In nine animals, glue was applied only partially, and eight untreated rabbits served as controls. Histological evaluation of the aortic valves was performed at 8-11 months after surgery, and included immunohistochemistry and confocal microscopy with quantitative tissue assessment. Levels of collagen I, as a main component of fibrosis, and matrix metalloproteinases (MMP)-1 and MMP-9 and angiotensin-converting enzyme (ACE), as regulators of fibrosis, were analyzed. The morphological structure of the aortic valve leaflets was studied, and the length, thickness and area of leaflets were measured. RESULTS: Leaflects in all groups were found to be composed of a continuous layer of collagen fibers at the mural side, and loose connective tissue containing fibroblasts and few capillaries on the aortic luminal aspect. In stiffened aortic roots, the length and area of the leaflets were increased. The area occupied by collagen was elevated in non-compliant aortic root leaflets, but collagen fluorescence intensity was decreased, indicating less densely packed collagen fibers. Degradation and synthesis of collagen as reflected by MMP-1, MMP-9 and ACE levels was up-regulated. CONCLUSION: Loss of compliance in aortic roots leads to elongation of the leaflets which, combined with a decrease in collagen density, may render leaflets more susceptible to mechanical stress. In time, this may promote the development of degenerative changes in the aortic valve.

Translocation and cleavage of myocardial dystrophin as a common pathway to advanced heart failure: a scheme for the progression of cardiac dysfunction.

Advanced heart failure (HF) is the leading cause of death in developed countries. The mechanism underlying the progression of cardiac dysfunction needs to be clarified to establish approaches to prevention or treatment. Here, using TO-2 hamsters with hereditary dilated cardiomyopathy, we show age-dependent cleavage and translocation of myocardial dystrophin (Dys) from the sarcolemma (SL) to the myoplasm, increased SL permeability in situ, and a close relationship between the loss of Dys and hemodynamic indices. In addition, we observed a surprising correlation between the amount of Dys and the survival rate. Dys disruption is not an epiphenomenon but directly precedes progression to advanced HF, because long-lasting transfer of the missing delta-SG gene to degrading cardiomyocytes in vivo with biologically nontoxic recombinant adenoassociated virus (rAAV) vector ameliorated all of the pathological features and changed the disease prognosis. Furthermore, acute HF after isoproterenol toxicity and chronic HF after coronary ligation in rats both time-dependently cause Dys disruption in the degrading myocardium. Dys cleavage was also detected in human hearts from patients with dilated cardiomyopathy of unidentified etiology, supporting a scheme consisting of SL instability, Dys cleavage, and translocation of Dys from the SL to the myoplasm, irrespective of an acute or chronic disease course and a hereditary or acquired origin. Hereditary HF may be curable with gene therapy, once the responsible gene is identified and precisely corrected.

The carboxyl-terminal ahnak domain induces actin bundling and stabilizes muscle contraction.

Ahnak, a 700 kDa protein, is expressed in a variety of cells and has been implicated in different cell-type-specific functions. In the human heart, we observed an endogenous carboxyl-terminal 72 kDa ahnak fragment that copurified with myofibrillar proteins. Immunocytochemistry combined with confocal microscopy localized this fragment to the intercalated discs and close to the Z-line of cardiomyocytes. No endogenous myofibrillar ahnak fragment was observed in the skeletal muscle. We elucidated the role of the recombinant carboxyl-terminal ahnak fragment (ahnak-C2) in actin filament organization and in the function of muscle fibers. Addition of ahnak-C2 to actin filaments induced filament bundling into paracrystalline-like structures as revealed by electron microscopy. Incubation of demembranated skeletal muscle fibers with ahnak-C2 attenuated the decline in isometric force development upon repeated contraction-relaxation cycles. Our results suggest that the carboxyl-terminal ahnak domain exerts a stabilizing effect on muscle contractility via its interaction with actin of thin filaments.

Increased glycolysis as protective adaptation of energy depleted, degenerating human hibernating myocardium.

In the current study on human hibernating myocardium (HHM), we tested the hypothesis that increased glycolysis might exert a positive effect during a supply-demand balance situation by augmentation of myocardial energy formation. In 14 patients HHM was preoperatively detected by clinical methods and validated by the recovery of contractile function three months following revascularization. During open-heart surgery, transmural biopsies were removed from the hibernating areas and analyzed using biochemical and morphologic methods. Metabolite contents were normalized for the degree of fibrosis (control: 9.8 +/- 0.5%, HHM 28.1 +/- 3.0%; p < 0.05), providing values for cardiomyocytes only. In energy depleted HHM, severe intracellular degeneration, glycogen accumulation and myocyte loss were found. Elevated lactate levels (2.22 +/- 0.26 vs. 25.38 +/- 3.53 micromol/wet wt, p < 0.001) were indicative of an increased anaerobic glycolytic flux. In conclusion the presence of abundant intracellular glycogen and an increased anaerobic glycolysis in HHM is indicative of a protective adaptation of this myocardium, which might balance energy deficit and may limit structural damage.

Myocytes die by multiple mechanisms in failing human hearts.

We tested the hypothesis that myocyte loss in failing human hearts occurs by different mechanisms: apoptosis, oncosis, and autophagic cell death. Explanted hearts from 19 patients with idiopathic dilated cardiomyopathy (EF< or =20%) and 7 control hearts were analyzed. Myocyte apoptosis revealed by caspase-3 activation and TUNEL staining occurred at a rate of 0.002+/-0.0005% (P<0.05 versus control) and oncosis assessed by complement 9 labeling at 0.06+/-0.001% (P<0.05). Cellular degeneration including appearance of ubiquitin containing autophagic vacuoles and nuclear disintegration was present at the ultrastructural level. Nuclear and cytosolic ubiquitin/protein accumulations occurred at 0.08+/-0.004% (P<0.05). The ubiquitin-activating enzyme E1 and the ligase E3 were not different from control. In contrast, ubiquitin mRNA levels were 1.8-fold (P<0.02) elevated, and the conjugating enzyme E2 was 2.3-fold upregulated (P<0.005). The most important finding, however, is the 2.3-fold downregulation of the deubiquitination enzyme isopeptidase-T and the 1.5-fold reduction of the ubiquitin-fusion degradation system-1, which in conjunction with unchanged proteasomal subunit levels and proteasomal activity results in massive storage of ubiquitin/protein complexes and in autophagic cell death. A 2-fold decrease of cathepsin D might be an additional factor responsible for the accumulation of ubiquitin/protein conjugates. It is concluded that in human failing hearts apoptosis, oncosis, and autophagy act in parallel to varying degrees. A disturbed balance between a high rate of ubiquitination and inadequate degradation of ubiquitin/protein conjugates may contribute to autophagic cell death. Together, these different types of cell death play a significant role for myocyte disappearance and the development of contractile dysfunction in failing hearts.

Cardiac overexpression of monocyte chemoattractant protein-1 in transgenic mice mimics ischemic preconditioning through SAPK/JNK1/2 activation.

OBJECTIVE AND METHODS: Although a beneficial association between innate immunity and ischemic preconditioning has recently been proposed, the mechanisms responsible for this link are poorly understood. To test the hypothesis that pro-inflammatory cytokines have a beneficial role in the activation of the cell survival pathway mediated by ischemic preconditioning, we have studied transgenic mice with cardiac myocyte specific overexpression of murine monocyte chemoattractant protein-1 (MCP-1). The resistance to ischemia was studied by performing 45-min (with or without injection of the SAPK/JNKs inhibitor D-JNKI1) and 3-day left coronary artery occlusions as well as 45-min left coronary artery occlusion followed by 3 days of reperfusion. In addition, quantitative Western blot analyses for TNF-alpha, and SAPK/JNK1/2, ERK1/2 and p38 activity were performed. RESULTS: Infarct size, expressed in percent of either the risk area or the left ventricle, was reduced in transgenic mice when compared with control after both, 45-min (14.7+/-2.6% vs. 52.0+/-2.4%; P<0.05) and 45-min occlusion followed by 3 days of reperfusion (23.2+/-1.8% vs. 30.0+/-1.8%; P<0.05) but it was not significantly different for 3-day occlusion. Western blot analyses showed significantly increased levels of TNF-alpha (1.8-fold) and phosphorylated-SAPK/JNK1/2 (1.5-fold) in transgenic hearts. Phosphorylated-ERK1/2, and phosphorylated-p38 levels were unchanged. Immunohistochemistry revealed that in transgenic mice monocytes/macrophages, lymphocytes, and fibroblasts are the source of TNF-alpha, whereas myocytes have increased phosphorylated-SAPK/JNK1/2 levels. In addition, injection of the SAPK/JNKs inhibitor D-JNKI1 partially abrogated the cardioprotective effect observed in untreated transgenic mice. CONCLUSION: Overexpression of MCP-1 by cardiomyocytes causes chronic infiltration and activation of leukocytes, resulting in elevated TNF-alpha secretion and SAPK/JNK1/2 activation. The activation of this pathway is in part responsible for the preconditioning effect of MCP-1 overexpression. These results show a possible beneficial link between innate immunity and ischemic preconditioning through MAP-kinase activation.

Regulation of EMAP II by hypoxia.

Endothelial-monocyte-activating polypeptide II (EMAP II) is a proinflammatory cytokine and a chemoattractant for monocytes and granulocytes. We have previously shown that EMAP II mRNA is strongly expressed at sites of apoptosis in the mouse embryo and that the mature protein is cleaved from its cellular precursor (proEMAP II/p43) by caspase activation to become released from cells. Here we demonstrate in vivo that EMAP II mRNA expression is strongly increased in tumor necrosis factor alpha (TNF)-treated murine meth A fibrosarcomas and in B16 melanomas, especially in close proximity to areas of tissue necrosis. Furthermore, by means of confocal microscopy, high level expression of proEMAP II/p43 protein correlated predominantly with hypoxic but also with apoptotic cells. In vitro, EMAP II mRNA levels were not increased by hypoxia. However, high amounts of mature EMAP II protein were detected in the supernatants of hypoxic tumor cells. Unlike in apoptotic cells, neither a broad-range caspase inhibitor nor an inhibitor specific for the internal cleavage site was able to inhibit processing of proEMAP II/p43 to the mature EMAP II protein. In conclusion, these data suggest that hypoxia and apoptosis provide two alternative mechanisms of EMAP II generation by tumor cells.

Remodeling of the adventitia during coronary arteriogenesis.

We studied the role of the adventitia in adaptive arteriogenesis during the phase of active growth of coronary collateral vessels (CV) induced by chronic occlusion of the left circumflex coronary artery in canine hearts. We used electron microscopy and immunoconfocal (IF) labeling for bFGF, matrix metalloproteinase (MMP)-2, MMP-9, tissue-type plasminogen activator (tPA), its inhibitor (PAI-1), fibronectin (FN), and Ki-67. Proliferation of smooth muscle cells and adventitial fibroblasts was evident. Quantitative IF showed that adventitial MMP-2, MMP-9, and FN were 9.2-, 7.5-, and 8.6-fold, bFGF was 5.1-fold, and PAI-1 was 3.4-fold higher in CV than in normal vessels (NV). The number of fibroblasts was 5-fold elevated in CV, but the elastic fiber content was 25-fold greater in NV than in CV. Perivascular myocyte damage and induction of endothelial nitric oxide synthase in peri-CV capillaries indicate expansion of CV. It was concluded that adventitial activation is associated with the development of CV through cell proliferation, production of growth factors, and induction of extracellular proteolysis thereby contributing to remodeling during adaptive arteriogenesis.