Metabolic Syndrome and Cardiac Vessel Remodeling Associated with Vessel Rarefaction: A Possible Underlying Mechanism May Result from a Poor Angiogenic Response to Altered VEGF Signaling Pathways.

BACKGROUND: Elevated mortality rates in patients with metabolic syndrome (MetS) are partly due to adverse remodeling of multiple organs, which may lead to cardiovascular disease, nonalcoholic fatty liver disease, kidney failure, or other conditions. MetS symptoms, such as obesity, hypertension, hyperglycemia, dyslipidemia, associated with insulin and leptin resistance, are recognized as major cardiovascular risk factors that adversely affect the heart. SUMMARY: Pathological cardiac remodeling is accompanied by endothelial cell dysfunction which may result in diminished coronary flow, dysregulated oxygen demand/supply balance, as well as vessel rarefaction. The reduced number of vessels and delayed or inhibited formation of collaterals after myocardial infarction in MetS heart may be due to unfavorable changes in endothelial cell metabolism but also to altered expression of vascular endothelial growth factor molecules, their receptors, and changes in signal transduction from the cell membrane, which severely affect angiogenesis. KEY MESSAGES: Given the established role of cardiac vessel endothelial cells in maintaining tissue homeostasis, defining the molecular background underlying vessel dysfunction associated with impaired angiogenesis is of great importance for future therapeutic purposes. Therefore, the aim of this paper was to present current information regarding vascular endothelial growth factor signaling in the myocardium of MetS individuals.

Cells with hematopoietic potential reside within mouse proepicardium.

During embryonic development, hematopoietic cells are present in areas of blood-vessel differentiation. These hematopoietic cells emerge from a specific subpopulation of endothelial cells called the hemogenic endothelium. We have previously found that mouse proepicardium contained its own population of endothelial cells forming a network of vascular tubules. We hypothesize that this EC population contains cells of hematopoietic potential. Therefore, we investigated an in vitro hematopoietic potential of proepicardial cell populations. The CD31+/CD45-/CD71- cell population cultured for 10 days in MethocultTM gave numerous colonies of CFU-GEMM, CFU-GM, and CFU-E type. These colonies consisted of various cell types. Flk-1+/CD31-/CD45-/CD71-, and CD45+ and/or CD71+ cell populations produced CFU-GEMM and CFU-GM, or CFU-GM and CFU-E colonies, respectively. Immunohistochemical evaluations of smears prepared from colonies revealed the presence of cells of different hematopoietic lineages. These cells were characterized by labeling with various combinations of antibodies directed against CD31, CD41, CD71, c-kit, Mpl, Fli1, Gata-2, and Zeb1 markers. Furthermore, we found that proepicardium-specific marker WT1 co-localized with Runx1 and Zeb1 and that single endothelial cells bearing CD31 molecule expressed Runx1 in the proepicardial area of embryonic tissue sections. We have shown that cells of endothelial and/or hematopoietic phenotypes isolated from mouse proepicardium possess hematopoietic potential in vitro and in situ. These results are supported by RT-PCR analyses of proepicardial extract, which revealed the expression of mRNA for crucial regulatory factors for hemogenic endothelium specification, i.e., Runx1, Notch1, Gata2, and Sox17. Our data are in line with previous observation on hemangioblast derivation from the quail PE.

SK053 triggers tumor cells apoptosis by oxidative stress-mediated endoplasmic reticulum stress.

Thioredoxins (Trx) together with thioredoxin reductases (TrxR) participate in the maintenance of protein thiol homeostasis and play cytoprotective roles in tumor cells. Therefore, thioredoxin-thioredoxin reductase system is considered to be a promising therapeutic target in cancer treatment. We have previously reported that SK053, a peptidomimetic compound targeting the thioredoxin-thioredoxin reductase system, induces oxidative stress and demonstrates antitumor activity in mice. In this study, we investigated the mechanisms of SK053-mediated tumor cell death. Our results indicate that SK053 induces apoptosis of Raji cells accompanied by the activation of the endoplasmic reticulum (ER) stress and induction of unfolded protein response. Incubation of tumor cells with SK053 induces increase in BiP, CHOP, and spliced XBP-1 levels, which precede induction of apoptosis. CHOP-deficient (CHOP(-/-)) mouse embryonic fibroblasts are more resistant to SK053-induced apoptosis as compared with normal fibroblasts indicating that the apoptosis of tumor cells depends on the expression of this transcription factor. Additionally, the ER-stress-induced apoptosis, caused by SK053, is strongly related with Trx expression levels. Altogether, our results indicate that SK053 induces ER stress-associated apoptosis and reveal a link between thioredoxin inhibition and induction of UPR in tumor cells.

Comparison of airway wall remodeling in asthma and COPD: biopsy findings.

BACKGROUND: Bronchial remodeling is currently known to affect not only patients with asthma, but also COPD patients. Some studies have demonstrated that basement membrane thickening and destruction of the bronchial epithelium are also found in COPD. The aim of the study was to compare the basement membrane thickness (BMT) and epithelial damage in biopsy specimens from patients with asthma and COPD. METHODS: The study was performed in 20 subjects with asthma and 12 subjects with COPD, who had not been treated with corticosteroids for at least 3 months before study enrollment. Subjects' characteristics were based on the results of clinical assessment, allergic skin-prick tests, lung function testing, and methacholine bronchial challenge. All subjects underwent bronchoscopy with forceps biopsies of bronchial mucosa. Light-microscope and semi-automatic software were used to measure BMT in hematoxylin-eosin stained sections. Total (denudation) and partial epithelial damage were assessed independently by 2 pathologists. RESULTS: The mean BMT in subjects with asthma was 12.54 ± 2.8 µm, and only 7.81 ± 2.0 µm in COPD patients (P < .001). Overall percentage of the basement membrane length lined with damaged epithelium was 45 ± 20% in the asthma group and 47 ± 22% in the COPD group (difference not significant). Complete and partial epithelial damage did not differ between the groups. CONCLUSIONS: BMT might be a histopathological parameter helpful in distinguishing asthma and COPD patients, whereas the extent and pattern of epithelial damage is not.

Cardiotoxicity of the anticancer therapeutic agent bortezomib.

Recent case reports provided alarming signals that treatment with bortezomib might be associated with cardiac events. In all reported cases, patients experiencing cardiac problems were previously or concomitantly treated with other chemotherapeutics including cardiotoxic anthracyclines. Therefore, it is difficult to distinguish which components of the therapeutic regimens contribute to cardiotoxicity. Here, we addressed the influence of bortezomib on cardiac function in rats that were not treated with other drugs. Rats were treated with bortezomib at a dose of 0.2 mg/kg thrice weekly. Echocardiography, histopathology, and electron microscopy were used to evaluate cardiac function and structural changes. Respiration of the rat heart mitochondria was measured polarographically. Cell culture experiments were used to determine the influence of bortezomib on cardiomyocyte survival, contractility, Ca(2+) fluxes, induction of endoplasmic reticulum stress, and autophagy. Our findings indicate that bortezomib treatment leads to left ventricular contractile dysfunction manifested by a significant drop in left ventricle ejection fraction. Dramatic ultrastructural abnormalities of cardiomyocytes, especially within mitochondria, were accompanied by decreased ATP synthesis and decreased cardiomyocyte contractility. Monitoring of cardiac function in bortezomib-treated patients should be implemented to evaluate how frequently cardiotoxicity develops especially in patients with pre-existing cardiac conditions, as well as when using additional cardiotoxic drugs.

[Relationship between airway basement membrane thickness and lung function tests in patients with asthma]. / Zaleznosc pomiedzy gruboscia blony podstawnej oskrzeli i wybranymi parametrami czynnosci ukladu oddechowego u chorych na astme.

INTRODUCTION: Airway remodeling is a characteristic feature of asthma. It is believed that airway remodeling affects lung function and bronchial hyper-responsiveness. Therefore, the relationship between remodeling and lung function is still a matter of extensive research. However, the results of many studies are inconsistent. The aim of the study was to assess the relationship between lung function parameters and basement membrane (BM) thickness in patients with asthma. MATERIAL AND METHODS: Twenty asthma patients were chosen for the study (ten male, ten female, mean age 37 +/- 15 yrs). Ten were newly diagnosed, steroid-naive patients and the other ten were patients known to have asthma who had not been treated with steroids for at least three months. The study group was selected based on the results of: clinical assessment, allergic skin-prick tests, lung function testing and bronchial challenge with methacholine. Nine (45%) patients had chronic mild, nine (45%) had moderate and two (10%) had intermittent asthma. Mean FEV1% pred. was 83 +/- 18, mean FEV1%VC 69 +/- 9, mean FVC% pred. 101 +/- 14. All patients underwent research fiberoptic bronchoscopy with BAL and bronchial mucosal biopsies. Light-microscopic measurements of BM thickness were performed in hematoxylin-eosin stained slides of bronchial wall specimens with semi-automatic software analysis MultiScan Base 08.98. RESULTS: Mean BM thickness was 12.8 +/- 2.8 microm (range: 8.5-20.7 microm). No significant correlations between BM thickness and FEV1% pred., FEV1%VC, FVC% pred., RV% pred., TLC% pred., Raw (pre- and post-bronchodilator) and PC20 were observed. CONCLUSIONS: In our group of asthma patients, mean BM was significantly thickened. No relationship between BM thickness and lung function tests, including hyper-responsiveness, was found.

Proteasome inhibition potentiates antitumor effects of photodynamic therapy in mice through induction of endoplasmic reticulum stress and unfolded protein response.

Photodynamic therapy (PDT) is an approved therapeutic procedure that exerts cytotoxic activity toward tumor cells by inducing production of reactive oxygen species such as singlet oxygen. PDT leads to oxidative damage of cellular macromolecules, including proteins that undergo multiple modifications such as fragmentation, cross-linking, and carbonylation that result in protein unfolding and aggregation. Because the major mechanism for elimination of carbonylated proteins is their degradation by proteasomes, we hypothesized that a combination of PDT with proteasome inhibitors might lead to accumulation of carbonylated proteins in endoplasmic reticulum (ER), aggravated ER stress, and potentiated cytotoxicity toward tumor cells. We observed that Photofrin-mediated PDT leads to robust carbonylation of cellular proteins and induction of unfolded protein response. Pretreatment of tumor cells with three different proteasome inhibitors, including bortezomib, MG132, and PSI, gave increased accumulation of carbonylated and ubiquitinated proteins in PDT-treated cells. Proteasome inhibitors effectively sensitized tumor cells of murine (EMT6 and C-26) as well as human (HeLa) origin to PDT-mediated cytotoxicity. Significant retardation of tumor growth with 60% to 100% complete responses was observed in vivo in two different murine tumor models (EMT6 and C-26) when PDT was combined with either bortezomib or PSI. Altogether, these observations indicate that combination of PDT with proteasome inhibitors leads to potentiated antitumor effects. The results of these studies are of immediate clinical application because bortezomib is a clinically approved drug that undergoes extensive clinical evaluations for the treatment of solid tumors.

Decreased ER-associated degradation of alpha-TCR induced by Grp78 depletion with the SubAB cytotoxin.

HeLa cells stably expressing the alpha chain of T-cell receptor (alphaTCR), a model substrate of ER-associated degradation (ERAD), were used to analyze the effects of BiP/Grp78 depletion by the SubAB cytotoxin. SubAB induced XBP1 splicing, followed by JNK phosphorylation, eIF2alpha phosphorylation, upregulation of ATF3/4 and partial ATF6 cleavage. Other markers of ER stress, including elements of ERAD pathway, as well as markers of cytoplasmic stress, were not induced. SubAB treatment decreased absolute levels of alphaTCR, which was caused by inhibition of protein synthesis. At the same time, the half-life of alphaTCR was extended almost fourfold from 70 min to 210 min, suggesting that BiP normally facilitates ERAD. Depletion of p97/VCP partially rescued SubAB-induced depletion of alphaTCR, confirming the role of VCP in ERAD of alphaTCR. It therefore appears that ERAD of alphaTCR is driven by at least two different ATP-ase systems located at two sides of the ER membrane, BiP located on the lumenal side, while p97/VCP on the cytoplasmic side. While SubAB altered cell morphology by inducing cytoplasm vacuolization and accumulation of lipid droplets, caspase activation was partial and subsided after prolonged incubation. Expression of CHOP/GADD153 occurred only after prolonged incubation and was not associated with apoptosis.

[The relationship between markers of airway inflammation and thickness of the basement membrane in patients with asthma]. / Zwiazek pomiedzy wybranymi wskaznikami zapalenia dróg oddechowych a gruboscia blony podstawnej u chorych na astme.

INTRODUCTION: Airway inflammation and remodeling are well recognized features of asthma. Remodeling is usually regarded as a consequence of chronic inflammation, however there are also data suggesting that remodeling is a relatively independent process in asthma. Neither inflammation nor remodeling is a uniform process. Thus the precise relationship between markers of inflammation and different patterns of remodeling are still matter of investigations. The aim of the study was to assess the relationship between total and differential cell count in induced sputum (IS) and BALF, and thickness of the basement membrane (BM) in patients with stable asthma. MATERIAL AND METHODS: 18 patients with asthma (M/F 9/9, mean age 36 +/- 15 yrs). Duration of symptoms amounted to 12.7 +/- 11.5 years. Patients who have not been treated with steroids for at least 3 months were enrolled to the study. All patients underwent sputum induction and fiberoptic bronchoscopy with BAL and bronchial biopsies. Total and differential cell counts were measured in induced sputum and BALF. Light-microscopic measurements of BM thickness were performed in hematoxylin-eosin stained slides of bronchial wall specimens with semi-automatic software analysis. RESULTS: Mean BM thickness was 12.9 +/- 2.8 microm (range: 8.5-20.7 microm). Total sputum cell count was 3.4 +/- 2.7 x 106 cells/ml, whereas in BALF 9.7 +/- 10.2 x 106 cells/ml. There was no correlation between differential cell count in induced sputum and BALF. No significant correlations between BM thickness and total and differential cell count in IS and BALF were observed. There also was no correlation between BM thickness and length of asthma duration or degree of the disease. CONCLUSIONS: There was no relationship between BM thickness and total number of cells nor number of eosinophils in BALF and/or IS.

Hoc protein regulates the biological effects of T4 phage in mammals.

We previously investigated the biological, non-antibacterial effects of bacteriophage T4 in mammals (binding to cancer cells in vitro and attenuating tumour growth and metastases in vivo); we selected the phage mutant HAP1 that was significantly more effective than T4. In this study we describe a non-sense mutation in the hoc gene that differentiates bacteriophage HAP1 and its parental strain T4. We found no substantial effects of the mutation on the mutant morphology, and its effects on electrophoretic mobility and hydrodynamic size were moderate. Only the high ionic strength of the environment resulted in a size difference of about 10 nm between T4 and HAP1. We compared the antimetastatic activity of the T2 phage, which does not express protein Hoc, with those of T4 and HAP1 (B16 melanoma lung colonies). We found that HAP1 and T2 decreased metastases with equal effect, more strongly than did T4. We also investigated concentrations of T4 and HAP1 in the murine blood, tumour (B16), spleen, liver, or muscle. We found that HAP1 was rapidly cleared from the organism, most probably by the liver. Although HAP1 was previously defined to bind cancer cells more effectively (than T4), its rapid elimination precluded its higher concentration in tumours.

Valosin-containing protein (p97) is a regulator of endoplasmic reticulum stress and of the degradation of N-end rule and ubiquitin-fusion degradation pathway substrates in mammalian cells.

Valosin-containing protein (VCP; p97; cdc48 in yeast) is a hexameric ATPase of the AAA family (ATPases with multiple cellular activities) involved in multiple cellular functions, including degradation of proteins by the ubiquitin (Ub)-proteasome system (UPS). We examined the consequences of the reduction of VCP levels after RNA interference (RNAi) of VCP. A new stringent method of microarray analysis demonstrated that only four transcripts were nonspecifically affected by RNAi, whereas approximately 30 transcripts were affected in response to reduced VCP levels in a sequence-independent manner. These transcripts encoded proteins involved in endoplasmic reticulum (ER) stress, apoptosis, and amino acid starvation. RNAi of VCP promoted the unfolded protein response, without eliciting a cytosolic stress response. RNAi of VCP inhibited the degradation of R-GFP (green fluorescent protein) and Ub-(G76V)-GFP, two cytoplasmic reporter proteins degraded by the UPS, and of alpha chain of the T-cell receptor, an established substrate of the ER-associated degradation (ERAD) pathway. Surprisingly, RNAi of VCP had no detectable effect on the degradation of two other ERAD substrates, alpha1-antitrypsin and deltaCD3. These results indicate that VCP is required for maintenance of normal ER structure and function and mediates the degradation of some proteins via the UPS, but is dispensable for the UPS-dependent degradation of some ERAD substrates.

Temporal/spatial expression and efflux activity of ABC transporter, P-glycoprotein/Abcb1 isoforms and Bcrp/Abcg2 during early murine development.

ABC transporters pump out from cells a large number of endo- and xenobiotics including signal molecules and toxins; they are molecular markers of stem/progenitor cells as well. Here, we present the study of temporal/spatial patterns of Abcb1 isoforms and Abcg2 transporter expression and efflux activity in pre- and early postimplantation murine embryos. We found in 2-cell embryos abcb1a, abcb1b and abcg2 mRNAs which were believed to be maternally inherited. The expression of abcb1b and abcg2 genes was found in blastocysts and in 7 days postcoitum (dpc) embryos, while in 9dpc embryos beside of abcb1b/abcg2, the abcb1a gene was expressed. The abcb2 mRNA was detectable neither in pre- nor in postimplantation embryos. Moreover, we analysed temporal/spatial patterns of rhodamine 123/Hoechst 33342 efflux, which mirrors the ABC transporter phenotype, from individual cells of pre- and postimplantation murine embryos. The blastomeres of 2-, 4- and 8-cell embryos had efflux-inactive phenotype. Single, efflux-active cells emerged first in the morulae and their number increased in blastocyst inner cell mass. In 6 and 7 dpc embryos, all embryonic cells hold the efflux-active phenotype. Proximal embryonic endoderm of 6-8 dpc embryos contained two sub-domains: one consisted of efflux-active cells and another one of efflux-inactive cells reflecting polarity of an embryo. Between 7 and 8 dpc, at the onset of organogenesis, the vehement surge of efflux-inactive embryonic cells occurred, and their number increased in 9 dpc embryos, which consequently contained few efflux-active cells.

Quantitative ultrastructural changes in satellite cells of rats immobilized after soleus muscle denervation.

Quantitative ultrastructural evaluation of satellite cells (SCs) of rats immobilized for 7, 14, and 36 days after soleus muscle denervation was performed. Alterations in SCs of experimental animals concerned mainly the decrease in the size of cells and their nuclei, in the volume fractions of the nucleus and nucleolus, as well as in the number of ribosome-like structures. They suggest that immobilization which proceeded denervation caused a decline in cell activity. An increase in the volume fraction and number of endosome/lysosome-like structures, suggesting elevated processes of degradation was also observed. The changes occurred mainly in the period between 7 and 14 days after muscle denervation and immobilization. In all groups of experimental animals an increase in number of caveolae-like structures on both, inner (muscle fiber-facing) and outer (basal lamina-facing) sides of SCs was found. Thus, it is likely that SCs of denervated and immobilized rats are affected by signal molecules released by muscle fibers and/or other cell types present in muscle. A tendency in changes in SCs, observed in the present study, was similar to those which we noticed previously in denervated soleus muscle. However, immobilization after denervation aggravated some of the ultrastructural alterations or the changes appeared earlier.

Morphometric ultrastructural analysis of satellite cells in denervated rat soleus muscle.

Morphometric analysis of the ultrastructural changes of satellite cells (SCs) of rat soleus muscle 7, 14, and 36 days post denervation was performed. Denervation caused decrease of surface area cross-section of SCs and their nuclei, volume fractions of nucleus and Golgi complex elements and ribosomes number. In contrast, the increase of surface area/volume ratio of SCs and nucleus, volume fraction and number of endosome/lysosome-like structures, and number of caveolae-like structures was noticed. Ultrastructural changes of SCs in denervated muscles strongly suggest decline of cell activity accompanied by increased processes of degradation of material of endo-, and/or egzogenous origin.

Morphometric changes in the corpus luteum of pregnant hypokinetic rats.

The aim of this work was to study the influence of hypokinetic conditions on the ovary and corpus luteum of pregnant rats. The rats were kept in hypokinetic conditions for 5 days in the period between the 13th and 18th days of pregnancy. A three-dimensional reconstruction of the ovary and corpora lutea and also a stereological evaluation of the luteal cells and their nuclei were performed using serially cut material. Hypokinesia caused a decrease in the mean volume of the ovary and individual corpus luteum and in the total volume of corpora lutea per ovary in immobilised animals as compared to the control. Moreover, a decrease was observed in the mean number of luteal cells and an increase in the size of these cells, as well as in the mean volume fraction of their nuclei. These results indicate that immobilisation of pregnant rats for 5 days considerably influences the morphology of the corpus luteum and luteal cells.