[DEVELOPMENT OF FUNCTIONAL AND MORPHOLOGICAL CHANGES OF A PANCREAS DEPENDING ON THE DURATION OF OBSTRUCTIVE CHOLESTASIS].

PURPOSE OF THE STUDY: To study the severity and timing of the development of functional (reversible) and morphological (irreversible) disturbances in the pancreas, depending on the duration of obstructive cholestasis. MATERIALS AND METHODS: Obstructive jaundice in the experiment 18 dogs modeled by applying the loop stranglehold on the common bile duct, followed by observation for 30 days. We measured total bilirubin and fractions aspartate aminotransferase activity (AST) and alanine aminotransferase (ALT), the contents alpha-amylase, pancreatic lipase, glucose, histological examination of the pancreas (magnification of 100 times and 400). RESULTS: On day 3 the common bile duct obstruction bilirubin increased from 7.1 to 286.8 µmol/l, ALT--from 0.17 to 4.18 µmol*h/l, alpha-amylase from 89 to 186 U/L and lipase--to 68 to 179 U/L. Then there was a slight decrease in the parameters studied with repeated their increase to 15 hours. Morphological changes in the first three days were characterized by reversible (swelling), impaired organ that 14-16 days passed in organic (irreversible) changes. CONCLUSION: Dynamics of fluctuations in the level of liver enzymes in the pancreas and obstructive cholestasis correlates with morphological abnormalities in the pancreas and fit into the concept of general biological organism's reaction to injury.

Stem cell-mediated natural tissue engineering.

Recently, we demonstrated that a fully differentiated tissue developed on a ventricular septal occluder that had been implanted due to infarct-related septum rupture. We suggested that this tissue originated from circulating stem cells. The aim of the present study was to evaluate this hypothesis and to investigate the physiological differentiation and transdifferentiation potential of circulating stem cells. We developed an animal model in which a freely floating membrane was inserted into each the left ventricle and the descending aorta. Membranes were removed after pre-specified intervals of 3 days, and 2, 6 and 12 weeks; the newly developed tissue was evaluated using quantitative RT-PCR, immunohistochemistry and in situ hybridization. The contribution of stem cells was directly evaluated in another group of animals that were by treated with granulocyte macrophage colony-stimulating factor (GM-CSF) early after implantation. We demonstrated the time-dependent generation of a fully differentiated tissue composed of fibroblasts, myofibroblasts, smooth muscle cells, endothelial cells and new blood vessels. Cells differentiated into early cardiomyocytes on membranes implanted in the left ventricles but not on those implanted in the aortas. Stem cell mobilization with GM-CSF led to more rapid tissue growth and differentiation. The GM-CSF effect on cell proliferation outlasted the treat ment period by several weeks. Circulating stem cells contributed to the development of a fully differentiated tissue on membranes placed within the left ventricle or descending aorta under physiological conditions. Early cardiomyocyte generation was identified only on membranes positioned within the left ventricle.

Calcium-dependent signalling is essential during collateral growth in the pig hind limb-ischemia model.

We investigated the effect of pharmacological activation of the Ca(2+)-channel transient receptor potential cation channel, subfamily V, member 4 (TRPV4) on collateral growth in a pig hind limb-ischemia model thereby identifying subcellular mechanisms. Domestic pigs received femoral artery ligature and were randomly assigned to one of the following groups (each n=6): (1) 4alpha-phorbol 12,13-didecanoate (4alphaPDD) treatment; (2) treatment with an arterio-venous shunt (AV-shunt) distal to the occlusion; or (3) implantation of NaCl-filled minipump. Six sham-operated pigs acted as controls. Aortic and peripheral mean arterial pressure (MAP) measurements were performed to assess the collateral flow index (CFI). Tissue was isolated from M. quadriceps for immunohistochemistry and from isolated collateral arteries for quantitative real time PCR (qRT-PCR). Shortly after ligature the CFI dropped from 0.96+/-0.02 to 0.21+/-0.02 in all ligature-treated groups. In ligature-only-treated pigs CFI increased to 0.56+/-0.03 after 7days. Treatment with 4alphaPDD led to an enhancement of CFI compared with ligature alone (0.73+/-0.03). CD31-staining showed improved arteriolar density. Increased Ki67 staining in collaterals indicated proliferation. qRT-PCR and Western blot analysis showed upregulation or modulation of Ca(2+)-dependent transcription factors nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), Kv channel interacting protein 3, calsenilin (KCNIP3/CSEN/DREAM), and myocyte enhancer factor 2C (MEF2C) in 4alphaPDD- and AV-shunt-treated pigs compared with controls. Improved CFI after 4alphaPDD treatment identifies TRPV4 as an initial fluid shear-stress sensor and collateral remodelling and growth trigger. Subcellularly, modulation of Ca(2+)-dependent transcription factors indicates a pivotal role for Ca(2+)-signalling during arteriogenesis.

Telocytes in human isolated atrial amyloidosis: ultrastructural remodelling.

The human heart can be frequently affected by an organ-limited amyloidosis called isolated atrial amyloidosis (IAA). IAA is a frequent histopathological finding in patients with long-standing atrial fibrillation (AF). The aim of this paper was to investigate the ultrastructure of cardiomyocytes and telocytes in patients with AF and IAA. Human atrial biopsies were obtained from 37 patients undergoing cardiac surgery, 23 having AF (62%). Small fragments were harvested from the left and right atrial appendages and from the atrial sleeves of pulmonary veins and processed for electron microscopy (EM). Additional fragments were paraffin embedded for Congo-red staining. The EM examination certified that 17 patients had IAA and 82% of them had AF. EM showed that amyloid deposits, composed of characteristic 10-nm-thick filaments were strictly extra-cellular. Although, under light microscope some amyloid deposits seemed to be located within the cardiomyocyte cytoplasm, EM showed that these deposits are actually located in interstitial recesses. Moreover, EM revealed that telopodes, the long and slender processes of telocytes, usually surround the amyloid deposits limiting their spreading into the interstitium. Our results come to endorse the presumptive association of AF and IAA, and show the exclusive, extracellular localization of amyloid fibrils. The particular connection of telopodes with amyloid deposits suggests their involvement in isolated atrial amyloidosis and AF pathogenesis.

Telocytes in human epicardium.

The existence of the epicardial telocytes was previously documented by immunohistochemistry (IHC) or immunofluorescence. We have also demonstrated recently that telocytes are present in mice epicardium, within the cardiac stem-cell niches, and, possibly, they are acting as nurse cells for the cardiomyocyte progenitors. The rationale of this study was to show that telocytes do exist in human (sub)epicardium, too. Human autopsy hearts from 10 adults and 15 foetuses were used for conventional IHC for c-kit/CD117, CD34, vimentin, S-100, τ, Neurokinin 1, as well as using laser confocal microscopy. Tissue samples obtained by surgical biopsies from 10 adults were studied by digital transmission electron microscopy (TEM). Double immunolabelling for c-kit/CD34 and, for c-kit/vimentin suggests that in human beings, epicardial telocytes share similar immunophenotype features with myocardial telocytes. The presence of the telocytes in human epicardium is shown by TEM. Epicardial telocytes, like any of the telocytes are defined by telopodes, their cell prolongations, which are very long (several tens of µm), very thin (0.1-0.2 µm, below the resolving power of light microscopy) and with moniliform configuration. The interconnected epicardial telocytes create a 3D cellular network, connected with the 3D network of myocardial telocytes. TEM documented that telocytes release shed microvesicles or exocytotic multivesicular bodies in the intercellular space. The human epicardial telocytes have similar phenotype (TEM and IHC) with telocytes located among human working cardiomyocyte. It remains to be established the role(s) of telocytes in cardiac renewing/repair/regeneration processes, and also the pathological aspects induced by their 'functional inhibition', or by their variation in number. We consider telocytes as a real candidate for future developments of autologous cell-based therapy in heart diseases.

[Qualitative and quantitative changes of circulating in blood endotheliocyte precursor cells in patients with hypercholesterolemia].

Aim of this study was elucidation of diagnostic value of qualitative and quantitative parameters of circulating endotheliocyte precursor cells (EPC), in the state of apoptosis (apoptotic endotheliocytes A AE), as well as effect of main risk factors on the studied parameters in patients with coronary atherosclerosis and hypercholesterolemia. We examined 24 patients with ischemic heart disease (14 men, 10 women, mean age 58.4 +/- 2.3 years) with stenoses >75% in coronary arteries according to angiography data and hypercholesterolemia (main group). Control group comprised 15 healthy volunteers. Circulating EPC in peripheral blood were evaluated by cytofluorimetry. Functional parameters of isolated EPC were assessed after cultivation in human fibronectin. Adhesive properties were studied in cell culture by immunehistochemical method after 7 days of incubation. Proliferation capacity was assessed by number of EPC in 1 mm3 of medium at day 7 of incubation. Determination and counting of AE was carried out using cytofluorimetry with immunostaining of cells by anexin B and CDA31 after preliminary treatment of endotheliocyte culture with H2O2 and comparing the results with control culture of endotheliocytes. Number of circulating EPC in patients with IHD was 58.% less than in healthy persons. Adhesive capacity of EPC in patients with IHD was lowered by 43%. Number of EPC surviving in cell culture was 80.9% less than in control group. At the same time number of circulating AE in patients with IHD was 2.5 times greater. Reduction of number of EDC, adhesive capacity, and rise of number of AE correlated significantly with number of involved coronary arteries, presence of diabetes, and smoking status. Presence of arterial hypertension and blood cholesterol level did not correlate with quantitative and qualitative parameters of EPC. Thus qualitative and qualitative parameters of circulating EPC and apoptotic cells can be considered as markers of dysfunction of endothelium and predictors of atherosclerotic vascular lesions in patients with hypercholesterolemia.

A distinct type of cell in myocardium: interstitial Cajal-like cells (ICLCs).

The existence of a novel type of interstitial cells in the heart, interstitial Cajal-like cells (ICLCs), had been described for the first time in 2005. Their identification was mainly based on ultrastructural criteria: very long (tens up to hundreds of micrometres) and moniliform prolongations, which are extremely thin (less than 0.2 microm), below the resolving power of light microscopy. Myocardial ICLCs were also identified by methylene-blue vital staining, silver impregnation, and immunoreactivity for CD 34, vimentin, CD117/c-kit, etc. Although a series of studies provided evidence for the existence of ICLCs in human atria and rat ventricles, further investigations in other laboratories, using additional techniques, are required to substantiate the consistency of these findings. Here we provide further evidence for the existence of ICLCs in human and mammalian hearts (by transmission and scanning electron microscopy, as well as confocal laser scanning microscopy). Noteworthy, we confirm that ICLCs communicate with neighbouring cells via shedding (micro)vesicles. Although these so-called ICLCs represent a distinct type of cells, different from classical interstitial cells of Cajal, or fibroblasts, their role(s) in myocardium remain(s) to be established. Several hypotheses are proposed: (i) adult stromal (mesenchymal) stem cells, which might participate in cardiac repair/remodelling; (ii) intercellular signalling (e.g. via shedding microvesicles); (iii) chemo-mechanical transducers and (iv) players in pacemaking and/or arrhytmogenesis, and so on.

Classically and alternatively activated macrophages contribute to tissue remodelling after myocardial infarction.

An important goal in cardiology is to minimize myocardial necrosis and to support a discrete but resilient scar formation after myocardial infarction (MI). Macrophages are a type of cells that influence cardiac remodelling during MI. Therefore, the goal of the present study was to investigate their transcriptional profile and to identify the type of activation during scar tissue formation. Ligature of the left anterior descending coronary artery was performed in mice. Macrophages were isolated from infarcted tissue using magnetic cell sorting after 5 days. The total RNA of macrophages was subjected to microarray analysis and compared with RNA from MI and LV-control. mRNA abundance of relevant targets was validated by quantitative real-time PCR 2, 5 and 10 days after MI (qRT-PCR). Immunohistochemistry was performed to localize activation type-specific proteins. The genome scan revealed 68 targets predominantly expressed by macrophages after MI. Among these targets, an increased mRNA abundance of genes, involved in both the classically (tumour necrosis factor alpha, interleukin 6, interleukin 1beta) and the alternatively (arginase 1 and 2, mannose receptor C type 1, chitinase 3-like 3) activated phenotype of macrophages, was found 5 days after MI. This observation was confirmed by qRT-PCR. Using immunohistochemistry, we confirmed that tumour necrosis factor alpha, representing the classical activation, is strongly transcribed early after ligature (2 days). It was decreased after 5 and 10 days. Five days after MI, we found a fundamental change towards alternative activation of macrophages with up-regulation of arginase 1. Our results demonstrate that macrophages are differentially activated during different phases of scar tissue formation after MI. During the early inflammatory phase, macrophages are predominantly classically activated, whereas their phenotype changes during the important transition from inflammation to scar tissue formation into an alternatively activated type.

Impulse conduction and gap junctional remodelling by endothelin-1 in cultured neonatal rat ventricular myocytes.

Endothelin-1 (ET-1) is an important contributor to ventricular hypertrophy and failure, which are associated with arrhythmogenesis and sudden death. To elucidate the mechanism(s) underlying the arrhythmogenic effects of ET-1 we tested the hypothesis that long-term (24 hrs) exposure to ET-1 impairs impulse conduction in cultures of neonatal rat ventricular myocytes (NRVM). NRVM were seeded on micro-electrode-arrays (MEAs, Multi Channel Systems, Reutlingen, Germany) and exposed to 50 nM ET-1 for 24 hrs. Hypertrophy was assessed by morphological and molecular methods. Consecutive recordings of paced activation times from the same cultures were conducted at baseline and after 3, 6 and 24 hrs, and activation maps for each time period constructed. Gap junctional Cx43 expression was assessed using Western blot and confocal microscopy of immunofluorescence staining using anti-Cx43 antibodies. ET-1 caused hypertrophy as indicated by a 70% increase in mRNA for atrial natriuretic peptide (P < 0.05), and increased cell areas (P < 0.05) compared to control. ET-1 also caused a time-dependent decrease in conduction velocity that was evident after 3 hrs of exposure to ET-1, and was augmented at 24 hrs, compared to controls (P < 0.01). ET-1 increased total Cx43 protein by approximately 40% (P < 0.05) without affecting non- phosphorylated Cx43 (NP-Cx43) protein expression. Quantitative confocal microscopy showed a approximately 30% decrease in the Cx43 immunofluorescence per field in the ET-1 group (P < 0.05) and a reduced field stain intensity (P < 0.05), compared to controls. ET-1-induced hypertrophy was accompanied by reduction in conduction velocity and gap junctional remodelling. The reduction in conduction velocity may play a role in ET-1 induced susceptibility to arrhythmogenesis.

Desynchronization: a novel model to induce heart failure.

BACKGROUND: Previous large animal heart failure models led to inhomogeneous results. Therefore, we developed a novel model combining rapid pacing with forced ventricular desynchronization. METHODS: Heart failure was induced in 20 pigs during a pacing period of 21 days. Group A (n = 10) received one right ventricular lead (220 bpm). In group B (n = 10), two leads were implanted in different right ventricular regions with beat-to-beat alternation of activation sites (each lead 110 bpm). Sham-operated pigs (n = 6) served as controls. Hemodynamics were invasively evaluated and tissue was analyzed by immunohistochemistry and zymography. RESULTS: Hemodynamics were significantly more impaired in group B with an increase of pulmonary capillary wedge and central venous pressure and a reduction of cardiac index (control 4.3 +/- 0.1 l/min/m (2); A 3.6 +/- 0.2; B 2.9 +/- 0.2, P < 0.05). Heart-to-body weight ratio was significantly higher in group B. Histological analyses showed a significant increase of cell diameters and interstitial fibrosis with significantly higher collagen contents in group B. CONCLUSION: The new model with a combination of rapid pacing and forced desynchronization of the ventricular contraction is superior to traditional heart failure models induced solely by rapid pacing.

Negative inotropic effect of rapamycin on isolated human cardiomyocytes.

Rapamycin is an increasingly important immunosuppressive drug and reduces restenosis after coronary stenting, but its effects on cardiac contractility are largely unknown. We investigated the acute inotropic effects of rapamycin on isolated human cardiomyocytes. Cardiomyocytes were enzymatically isolated from right atrial appendages obtained during routine coronary artery bypass surgery. Cell morphology was examined by confocal microscopy. Cell contraction was recorded after electrical stimulation. Rapamycin elicited a concentration-dependent decrease in fractional cell shortening ranging from 14.3 +/- 2.6% at 10(-8) M rapamycin to 26.4 +/- 4.2% at 10(-5) M. Rapamycin also caused a concentration-dependent decrease in diastolic cell length. Contractile performance of isolated cardiomyocytes was well preserved, as evidenced by the profound positive inotropic effects of high extracellular calcium concentration and the beta-adrenoreceptor agonist isoproterenol. The acute negative inotropic effect of rapamycin on human cardiomyocytes might be due to altered calcium homeostasis through the binding of rapamycin to FKBP12.6 and its regulatory function on the ryanodine receptor, with increased calcium leakage from the sarcoplasmic reticulum.

Tissue-specific patterns of Gap junctions in adult rat atrial and ventricular cardiomyocytes in vivo and in vitro.

To verify the hypothesis that tissue-specific patterns of gap junctions (GJs) are determined by intrinsic factors within myocytes forming different cardiac tissues, we have compared by quantitative transmission electron microscopy (TEM) the structural features of GJs in adult rat atrial myocytes (AMs) and ventricular myocytes (VMs) in vivo with those in developing GJs in cultured AMs and VMs in vitro. Quantitative TEM data revealed a 3-fold increase in the number of developing GJs per intercalated disk in both AMs and VMs from 6 to 15 days in culture. However, at days 12 and 15, the percentage of GJ length per intercalated disk and mean GJ length were 2-fold higher in VMs than in AMS: Measurements of connexin43 GJs by confocal microscopy confirmed TEM data and demonstrated respectively 2- and 4.5-fold greater mean values of GJ length and area in VMs than in AMS: These differences are attributable to the development of large GJs (>3 micrometer) in VMs, closely resembling those observed in VMs in vivo. Although large GJs in cultured VMs comprised approximately 14% of the total number of GJs, their contribution to total GJ length and area constituted >60% and 85%, respectively. In marked contrast, the number of large GJs in AMs both in vitro and in vivo was <1% from the total number of GJS: These data confirm our hypothesis and provide the first evidence that tissue-specific patterns of GJs in AMs and VMs are determined primarily by intrinsic factors within cardiac myocytes and are developmentally regulated.

Tissue engineering of a differentiated cardiac muscle construct.

Cardiac tissue engineering is an emerging field. The suitability of engineered heart tissue (EHT) for both in vitro and in vivo applications will depend on the degree of syncytoid tissue formation and cardiac myocyte differentiation in vitro, contractile function, and electrophysiological properties. Here, we demonstrate that cardiac myocytes from neonatal rats, when mixed with collagen I and matrix factors, cast in circular molds, and subjected to phasic mechanical stretch, reconstitute ring-shaped EHTs that display important hallmarks of differentiated myocardium. Comparative histological analysis of EHTs with native heart tissue from newborn, 6-day-old, and adult rats revealed that cardiac cells in EHTs reconstitute intensively interconnected, longitudinally oriented, cardiac muscle bundles with morphological features resembling adult rather than immature native tissue. Confocal and electron microscopy demonstrated characteristic features of native differentiated myocardium; some of these features are absent in myocytes from newborn rats: (1) highly organized sarcomeres in registry; (2) adherens junctions, gap junctions, and desmosomes; (3) a well-developed T-tubular system and dyad formation with the sarcoplasmic reticulum; and (4) a basement membrane surrounding cardiac myocytes. Accordingly, EHTs displayed contractile characteristics of native myocardium with a high ratio of twitch (0.4 to 0.8 mN) to resting tension (0.1 to 0.3 mN) and a strong beta-adrenergic inotropic response. Action potential recordings demonstrated stable resting membrane potentials of -66 to -78 mV, fast upstroke kinetics, and a prominent plateau phase. The data indicate that EHTs represent highly differentiated cardiac tissue constructs, making EHTs a promising material for in vitro studies of cardiac function and tissue replacement therapy.

Prolonged myocardial hibernation exacerbates cardiomyocyte degeneration and impairs recovery of function after revascularization.

OBJECTIVES: We sought to define the effects of time on contractile function, morphology and functional recovery after coronary revascularization in patients with dysfunctional but viable (hibernating) myocardium. BACKGROUND: Functional recovery after coronary artery bypass graft surgery in patients with chronic myocardial hibernation is incomplete or delayed. The proposed cause is a progressive temporal degeneration of cardiomyocytes. METHODS: In 32 patients with multivessel coronary disease, regional wall motion analysis was performed in hypoperfused but metabolically active areas before and 6 months after bypass surgery. During bypass surgery, transmural biopsy samples were obtained from the center of the hypokinetic zone for light and electron microscopic analyses. The proposed duration of myocardial hibernation was retrospectively assessed. RESULTS: Patients with a subacute hibernating condition (<50 days) demonstrated a higher preoperative ejection fraction (EF, 50+/-8%), and a better preserved wall motion (WM) in the supraapical wall (-1.4+/-0.4) than did patients with intermediate-term (>50 days, EF 37+/-9%, p < 0.05; WM -2.4+/-1.5, p = 0.08) or chronic (>6 months, EF 40+/-14%, WM -2.7+/-0.9, p < 0.005) ischemia. Structural degeneration correlated with the duration of ischemia (r = 0.56, p < 0.05). Postoperative recovery of function was enhanced in patients with a short history of hibernation compared with patients with an intermediate-term or chronic condition (EF 60+/-10% vs. 40+/-10%, p < 0.001, and vs. 47+/-14%, p < 0.05). CONCLUSIONS: Hibernating myocardium exhibits time-dependent deterioration due to progressive structural degeneration with enhanced fibrosis. Early revascularization should be attempted to salvage the jeopardized tissue and improve postoperative outcome.

Characterization of nebulette and nebulin and emerging concepts of their roles for vertebrate Z-discs.

Nebulin is an 800 kDa large actin-binding protein specific to skeletal muscle and thought to act as a molecular template that regulates the length of thin filaments. Recently, a 100 kDa nebulin-like protein has been described in the avian cardiac muscle and referred to as nebulette. We have determined the full-length (8 kb) cDNA sequence of the human nebulette. Its open reading frame (3044 bp) encodes a 109 kDa protein that shares extensive similarity with the C-terminal region of human nebulin. The C-terminal regions of nebulin and nebulette are identical in domain organization and share a family of highly related C-terminal repeats, a serine-rich domain with potential phosphorylation sites, and an SH3 domain. Immunoelectron-microscopy suggests that the C-terminal 30 kDa of nebulin and nebulette filaments integrate into the Z-disc lattice, whereas their N termini appear to project into the I-band. Gene mapping studies assign the human nebulette gene to chromosome 10p12, whereas the nebulin gene has been previously assigned to 2q21. Evolutionary constraints appear to have maintained identical modular arrangements in these two independent genes. Comparison of nebulin and nebulette cDNAs demonstrates that a subgroup of repeats within the C-terminal regions is regulated tissue-specifically and stage-dependently during development of both molecules. This leads to a substantial diversity of nebulin and nebulette isoforms. Their further study is likely to provide insights into how they contribute to the molecular diversity of Z-discs from different muscle tissues and fiber types.

The role of the cytoskeleton in heart failure.

The cytoskeleton of cardiac myocytes consists of actin, the intermediate filament desmin and of alpha- and beta-tubulin that form the microtubules by polymerization. Vinculin, talin, dystrophin and spectrin represent a separate group of membrane-associated proteins. In numerous experimental studies, the role of cytoskeletal alterations especially of microtubules and desmin, in cardiac hypertrophy and failure (CHF) has been described. Microtubules were found to be accumulated thereby posing an increased load on myocytes which impedes sarcomere motion and promotes cardiac dysfunction. Other groups were unable to confirm microtubular densification. The possibility exists that these changes are species, load and chamber dependent. Recently, damage of the dystrophin molecule and MLP (muscle LIM protein) were identified as possible causes of CHF. Our own studies in human hearts with chronic CHF due to dilated cardiomyopathy (DCM) showed that a morphological basis of reduced contractile function exists: the cytoskeletal and membrane-associated proteins are disorganized and increased in amount confirming experimental reports. In contrast, the contractile myofilaments and the proteins of the sarcomeric skeleton including titin, alpha-actinin, and myomesin are significantly decreased. These changes can be assumed to occur in stages and are here presented as a testable hypothesis: (1) The early and reversible stage as present in animal experiments is characterized by accumulation of cytoskeletal proteins to counteract an increased strain without loss of contractile material. (2) Further accumulation of microtubules and desmin to compensate for the increasing loss of myofilaments and titin represents the late clinical and irreversible state. We suggest, based on a structural basis for heart failure, an integrative view which closes the gap between changes within cardiac myocytes and the involvement of the extracellular matrix, including the development of fibrosis. These factors contribute significantly to structural ventricular remodeling and dilatation finally resulting in reduced cardiac function.

Effects of local MCP-1 protein therapy on the development of the collateral circulation and atherosclerosis in Watanabe hyperlipidemic rabbits.

OBJECTIVE: The objective of our study was to quantify the arteriogenic potency of Monocyte Chemoattractant Protein-1 (MCP-1) under hyperlipidemic conditions. Additionally, we aimed to determine the effects of locally applied MCP-1 on systemic serum lipid levels as well as on atherosclerosis. METHODS: A total of sixty-four Watanabe rabbits was treated with either low dose MCP-1 (1 microg/kg/week), high dose MCP-1 (3.3 microg/kg/week) or PBS as a control substance. Substances were applied directly into the collateral circulation via an osmotic minipump with the catheter placed in the proximal stump of the ligated femoral artery. Either 1 week or 6 months after initiation of the treatment X-ray angiography was performed as well as measurements of collateral conductance using fluorescent microspheres. The extent of atherosclerosis was quantified in whole aortas using Sudan IV staining. RESULTS: One week after ligation of the femoral artery a significant increase in collateral conductance was observed in animals treated with high dose MCP-1 (control: 2.2+/-0.8 ml/min/100 mmHg vs. MCP-1 high dose: 8.9+/-2.0 ml/min/100 mmHg, P<0.05). Six months after femoral artery ligation no differences were found between the treated and the control group (PBS; 44.9+/-11.6 ml/min/100 mmHg, MCP-1; 47.8+/-11.5 ml/min/100 mmHg, P=NS). No influence was found on serum lipids or on the development of atherosclerosis in the present model. CONCLUSION: MCP-1 accelerates arteriogenesis upon femoral artery ligation under hyperlipidemic conditions. Six months after treatment these pro-arteriogenic effects of MCP-1 can no longer be observed. The present data do not show an effect of local MCP-1 treatment on serum lipids or on atherosclerosis. It should be noted however that a high standard deviation was observed for the data on atherosclerotic surface area, necessitating additional experiments in a different model of atherosclerosis.

GM-CSF: a strong arteriogenic factor acting by amplification of monocyte function.

We investigated the role of the colony stimulating factor for monocytes (GM-CSF) to test the hypothesis whether prolongation of the monocyte's life cycle will support arteriogenesis (rapid growth of preexisting collateral arteries). This appeared logical in view of our discovery that circulating monocytes play an important part in the positive remodeling of small preexisting arterioles into arteries to compensate for arterial occlusions (arteriogenesis) and especially following our findings that MCP-1 markedly increases the speed of arteriogenesis. The continuous infusion of GM-CSF for 7 days into the proximal stump of the acutely occluded femoral artery of rabbits by osmotic minipump produced indeed a marked arteriogenic response as demonstrated by an increase (2-fold) in number and size of collateral arteries on postmortem angiograms and by the increase of maximal blood flow during vasodilation measured in vivo by blood pump perfusion of the hindquarter (5-fold). When GM-CSF and MCP-1 were simultaneously infused the effects on arteriogenesis were additive on angiograms as well as on conductance. GM-CSF was also able to widen the time window of MCP-1 activity: MCP-1 treatment alone was ineffective when given after the third week following occlusion. When administered together with GM-CSF about 80% of normal maximal conductance of the artery that was replaced by collaterals were achieved, a result that was not reached before by any other experimental treatment. Experiments with cells isolated from treated animals showed that monocyte apoptosis was markedly reduced. In addition we hypothesize that GM-CSF may aid in releasing pluripotent monocyte (stem-) cells from the bone marrow into the circulation. In contrast to MCP-1, GM-CSF showed no activity on monocyte transmigration through- and also no influence on monocyte adhesion to cultured endothelial cells. In conclusion we have discovered a new function of the hemopoietic stem cell factor GM-CSF, which is also a powerful arteriogenic peptide that acts via prolongation of the life cycle of monocytes/macrophages.

Myocardial alterations induced by prolonged noradrenaline administration in various doses.

Prolonged noradrenaline administration to rats in steadily increasing dosages for a period of one to four weeks (cumulative doses 25-35 mg/kg) resulted in the development of focal necrotic areas with abundant collagen fibers and marked hypertrophy of cardiomyocytes. Cellular diameter was higher by 37-44% and extracellular space area by 70-100%. A combination of overcontracted and overdistended sarcomeres in some cardiomyocytes and a twofold rise in serum creatine kinase presumably reflected cellular calcium overload. Myocardial high energy phosphate content was depleted to 50-62% of the control level. The extent of this depletion positively correlated with a decrease in heart rate and cardiac output of the isolated heart. The latter may be attributed to limited left ventricular filling caused by elevated LV diastolic pressure and stiffness. Minimal metabolic and functional changes were observed after lowest noradrenaline dose (5 mg/kg for nine days) that was followed by only moderate depletion of myocardial phosphocreatine content and moderate rise in LV diastolic stiffness. Results suggest that energy-deficient increase in myofibrillar stiffness may form the basis for decreased myocardial distensibility and cardiac pump failure.

Functional disturbances due to structural remodeling in the failing human heart.

In severely hypertrophied hearts structural remodeling occurs continuously and finally leads to heart failure. The remodeling process involves all structural components of the cardiomyocyte and all protein families and it consists of cellular enlargement accompanied by degeneration in addition to the occurrence of fibrosis. Nuclei are increased in size but the nuclear volume/cell volume ratio is reduced. Transcription and translation are downregulated for contractile and sarcomeric skeleton proteins but both are upregulated for cytoskeletal and membrane-associated proteins. The connexin43 content is significantly reduced. Chronic degeneration finally leads to cell death by ubiquitin-related autophagy, and acute ischemic cell death (oncosis) is also observed. Apoptosis seems to be of minor importance. The morphological alterations described here are the structural correlate of the typical clinical characteristics of heart failure in human patients: of reduced contractile function, of increased ventricular stiffness represented by an increased left end-diastolic pressure and of ventricular arrhythmia.