Remodeled pulmonary arteries return to normal structural conditions after the increase in pulmonary artery flow resistance is reversed. We studied whether proteolysis of extracellular matrix proteins and apoptosis occur during reversal of remodeling produced by chronic hypoxia in the rat. Main pulmonary arteries were removed at different times during a 10-day period of exposure to 10% O2 and 14 days after return to air. Content and rates of degradation of collagen and elastin as well as immunoreactive collagenase in tissue and isolated mast cells were measured. Immunoblots for collagenase and tissue inhibitor of metalloproteinases (TIMP) were performed. Apoptosis was assessed by cleavage of DNA and TUNEL assay. Excess collagen and elastin present at 10 days of hypoxia decreased to near normal levels after 3-5 days of air. Transient increases in collagenolytic and elastolytic enzyme activities accompanied the rapid decrease in matrix proteins. Mast cells containing collagenase accumulated in remodeled pulmonary arteries, and the active form of collagenase appeared at the time of peak proteolytic activity. TIMP increased during remodeling. Apoptosis was maximal 3 days after return to air. Our results suggest that activation of enzymes, which degrade matrix proteins, and apoptosis play a role in resolution of vascular remodeling., D. J. Riley, S. Thakker-Varia, F. J. Wilson, G. J. Poiani, C. A. Tozzi., and Obsahuje bibliografii
Adjuvant therapy and radiotherapy improves the survival of patients with metastatic and locally advanced gastric cancer (GC). However, the resistance to radiotherapy limits its clinical usage. Rhotekin 2 (RTKN2) functions as an oncogene and confers resistance to ultraviolet B-radiation and apoptosis- inducing agents. Here, the role of RTKN2 in radiosensitivity of GC cell lines was investigated. RTKN2 was found to be elevated in GC tissues and cells. A series of functional assays revealed that overexpression of RTKN2 induced GC cell proliferation, promoted GC cell migration and invasion, while inhibiting GC cell apoptosis. However, silence of RTKN2 promoted GC cell apoptosis, while repressing GC cell proliferation, invasion and migration. GC cells were exposed to irradiation, and data from cell survival and apoptotic assays showed that knock-down of RTKN2 enhanced radiosensitivity of GC through up-regulation of apoptosis and down-regulation of proliferation in irradiation-exposed GC cells. Moreover, the protein expression of β-catenin and c-Myc in GC cells was enhanced by RTKN2 over-expression, but reduced by RTKN2 silence. Interference of RTKN2 down-regulated nuclear β-catenin expression, while up-regulating cytoplasmic β-catenin in GC. In conclusion, RTKN2 contributed to cell growth and radioresistance in GC through activation of Wnt/β-catenin signalling.
Many functions of the cardiovascular apparatus are affected by gender. The aim of our study was find out whether markers of cell death present in the donor myocardium differ in male and female hearts. The study involved 81 patients undergoing heart transplantation from September 2010 to January 2013. Patients were divided into two groups: male allograft (n=49), and female allograft (n=32). Two types of myocardial cell death were analyzed. High-sensitive cardiac troponin T as a necrosis marker and protein bcl-2, caspase 3 and TUNEL as apoptosis markers were measured. We observed a significantly higher level of high-sensitive cardiac troponin T after correcting for predicted ventricular mass in female donors before transplantation as well as in the female allograft group after transplantation throughout the monitored period (P=0.011). There were no differences in apoptosis markers (bcl-2, caspase 3, TUNEL) between male and female hearts before transplantation. Both genders showed a significant increase of TUNEL-positive myocytes one week after transplantation without differences between the groups. Moreover, there were no differences in caspase 3 and bcl-2
expression between the two groups. Our results demonstrated the presence of necrotic and apoptotic cell death in human heart allografts. High-sensitive cardiac troponin T adjusted for predicted ventricular mass as a marker of myocardial necrosis was higher in female donors, and this gender difference was even more pronounced after transplantation.
Obstructive sleep apnea (OSA) has been demonstrated to be
implicated in disorder of insulin secretion and diabetes mellitus.
In this study, we aimed to evaluate the protective role of tempol,
a powerful antioxidant, in chronic intermittent hypoxia
(IH)-induced pancreatic injury. The rat model of OSA was
established by IH exposure. The pathological changes, increased
blood-glucose level, and raised proinsulin/insulin ratio in
pancreatic tissues of rats received IH were effectively relieved
by tempol delivery. In addition, the enhanced levels of
pro-inflammatory cytokines, TNF-α, IL-1β, IL-6, and inflammatory
mediators, PGE2, cyclooxygenase-2 (COX-2), NO, and inducible
nitric oxide synthase (iNOS) in pancreatic tissue were suppressed
by tempol. Moreover, tempol inhibited IH-induced apoptosis in
pancreatic tissue as evidenced by upregulated Bcl-2 level, and
downregulated Bax and cleaved caspase-3 levels. Finally, the
abnormal activation of mitogen-activated protein kinase (MAPK)
and nuclear factor kappa-light-chain-enhancer of activated B cells
(NF-κB) signaling pathways induced by IH was restrained by
tempol administration. In summary, our study demonstrates that
tempol relieves IH-induced pancreatic injury by inhibiting
inflammatory response and apoptosis, which provides theoretical
basis for tempol as an effective treatment for OSA-induced
pancreatic injury.
There are several evolutionary grades of wing reduction in female bagworm moths of the family Psychidae. In this family, female adults of Taleporia trichopterella, Bacotia sakabei and Proutia sp. have vestigial wings, although as pupae they have small wings. Consequently, these species (usually called wingless-legged bagworm moths), are intermediate between the two extremes of females with normal wings and those with no wings. Using light and electron microscopy, the processes of wing development during the last-larval instar and wing degeneration during the pupal stage was investigated in these species. Female wing imaginal discs proliferated during the last-larval instar, but diminished due to apoptosis in the prepupal stage of the last instar. In the pupal stage, degenerate cells were observed between the epithelia of the degenerating wing discs of the female. The presence of these cells is associated with apoptotic cell death. These observations suggest that female-specific wing degeneration caused by apoptosis occurs in two steps in these bagworm moths, i.e. in the larval and pupal stages. Such a process of wing reduction has not been previously reported in holometabolous insects, and is reported here for the first time in bagworm moths.
The aim of our study was to test the in fluence of short exposure (6 h) of preimplantation rabbit embryos to elevated temperatures (41.5 ºC or 42.5 ºC) in vitro on their developmental capacity. Fertilized eggs recovered from female oviducts at the pronuclear stage (19 hpc) were cultured at standard temperature (37.5 ºC) until the morula stage (72 hpc). Afterwards, the embryos were divided into two groups, cultured for 6 h either at hyperthermic (41.5 ºC or 42.5 ºC) or standard temperature (control 37.5 ºC), post-incubated overnight (16-20 h) at 37.5 ºC and then evaluated for developmental stages, apoptosis (TUNEL), proliferation (cell number), actin cytoskeleton and presence of heat-shock proteins Hsp70. It was observed that hyperthermia at 41.5 ºC did not alter progression of embryos to higher preimplantation stages (expanded and hatching/hatched blastocysts), rate of apoptosis, total cell number of blastocysts and structure of actin filament compared to 37.5 ºC. We stern-blotting revealed the presence of heat stress-induced 72 kDa fraction of Hsp70 proteins in granulosa cells (exposed to 41 ºC) and embryos (exposed to 41.5 ºC). Following the elevation of temperature to 42.5 ºC embryo development was dramati cally compromised. The embryos were arrested at the morula or early blastocyst stage, showed an increased rate of apoptosis and decreased total cell number compared to control. The structure of actin filaments in most of blastomeres was damaged and such blastomeres often contained apoptotic nuclei. In this group a presence of heat-stress-induced fraction of Hsp70 proteins had not been confirmed. This is the first report demonstrating a threshold of thermotolerance of rabbit preimplantation embryos to hyperthermic exposure in vitro. A detrimental effect of higher temperature on the embryo is probably associated with the loss of their ability to produce Hsp70 de novo, which leads to cytoskeleton alterations and enhanced apoptosis., A. V. Makarevich, L. Olexiková, P. Chrenek, E. Kubovičová, K. Fréharová, J. Pivko., and Obsahuje bibliografii a bibliografické odkazy
In vitro models serve as a tool for studies of steatosis. Palmitic and oleic acids can induce steatosis in cultured hepatocytes. The aim of our study was to verify steatogenic and cytotoxic effects of palmitic acid (PA), oleic acid (OA) and their combinations as well as their impact on functional capacity of rat primary hepatocytes. Hepatocytes were exposed to OA or PA (0.125-2 mmol/l) or their combination at ratios of 3:1, 2:1 or 1:1 at the final concentrations of 0.5-1 mmol/l. Both OA and PA caused a dose-dependent increase in triacylglycerol content in hepatocytes. PA was more steatogenic at 0.25 and 0.5 mmol/l while OA at 0.75 and 1 mmol/l. PA exhibited a dose-dependent cytotoxic effect associated with ROS production, present markers of apoptosis and necrosis and a decrease in albumin production. OA induced a damage of the cytoplasmic membrane from 1 mM concentration. Mixture of OA and PA induced lower cytotoxicity with less weakened functional capacity than did PA alone. Extent of steatosis was comparable to that after exposure to OA alone. In conclusion, OA or combination of OA with PA is more suitable for simulation of simple steatosis than PA alone., A. Moravcová, Z. Červinková, O. Kučera, V. Mezera, D. Rychtrmoc, H. Lotková., and Obsahuje bibliografii
a1_We investigated the potential neuroprotective effect of transient hypertension on neuronal cell death induced by ischemia-reperfusion. Recovery of neurons, terminally differentiated cells, is almost entirely dependent upon active transcription and repair of DNA damage. We focused on the histochemical detection of distribution of NOR (argyrophylic nucleolar proteins) reflecting nucleolar integrity, immunohistochemical detection of PARP-1 (poly(ADP-ribose) polymerase-1), MADD (mitogen-activated death domain), a protein accumulated in nucleoli upon stimulation by ischemia, the active form of caspase-3, a universal proteolytic enzyme of apoptosis. The terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP-biotin nick-end-labeling method (TUNEL) proved the presence of in situ DNA fragmentation. We used the model of transient focal cerebral ischemia in rats with occlusion of middle cerebral artery. In experimental group of rats, the transient hypertension was induced by constriction of the abdominal aorta. The period of ischemia lasted 15, 30, 60 and 120 min followed by 48 h of reperfusion. We examined the frontal lobe of the ipsilateral hemisphere for apoptosis of neurons and compared it with the intact brain tissue. In normotensive rats with transient focal cerebral ischemia, we found disintegrated nucleoli of cortical as well as subcortical neurons at all investigated periods of ischemia, whereas the neurons of intact animals showed compact nucleoli with a few satellites. Nuclear positivity for MADD and PARP-1 was apparent in the neocortex after 15 min and peaked after 30 min of ischemia. On the other hand, the subcortical neurons showed nuclear positivity after 60 and 120 min. The immunohistochemical reaction for active caspase 3 was apparent after 30 min onwards predominantly in the cortex. The TUNEL staining was distinct after 60 and 120 min., a2_In hypertensive rats, we found nucleolar disintegration, positivity for MADD, PARP-1 and caspase 3 after 30 min cortically and subcortically, followed by TUNEL positive staining of cortical neurons after 60 and 120 min. In summary, we detected delayed activation of neuronal apoptosis in transiently hypertensive rats with focal cerebral ischemia compared to normotensive animals. The apoptotic phenotype was confirmed by a panel of complementary methods showing rapid proteolysis-nucleolar segregation, MADD, PARP-1 and caspase-3 positivity as well as ultimate DNA fragmentation proved by the TUNEL assay., M. Smrčka, M. Horký, F. Otevřel, Š. Kuchtíčková, V. Kotala, J. Mužík., and Obsahuje bibliografii
Tumour necrosis factor-stimulated gene 6 (TSG6) is a protective inflammatory reaction gene which is upregulated by inflammatory processes. Recent studies suggest that TSG-6 exhibits anti-scar-ring effects. However, the mechanism of TSG-6 action in the scar formation remains poorly understood. We investigated whether TSG-6 affects growth of the human hypertrophic scar fibroblasts (HSFs) via Fas/FasL signalling pathway. Cultured HSFs were transfected with a vector carrying the TSG6gene (pLVX-Puro-TSG-6) or with a vector not containing the TSG6 gene (pLVX-Puro). Untransfected HSFs served as a control group to both transfected HSFs. The expressions level of TSG-6 was up-regulated in the pLVX-Puro-TSG-6 group at the protein and mRNA level. MTT and flow cytometry were used to assess the effect of TSG-6 on the growth and apoptotic status of HSFs. Finally, qRT-PCR and western blot were used to measure the expression levels of Fas, FasL, FADD, caspase-3 and caspase-8 in each group. The apoptosis rate was significantly enhanced and the growth rate reduced in the HSFs transfected with the TSG6 gene vector. The expression levels of Fas, FasL, FADD, caspase-3 and caspase-8 were significantly raised in the TSG-6 overexpressing HSFs. It is concluded that increased expression of TSG-6 may induce apoptosis of human hypertrophic scar fibroblasts via activation of the Fas/FasL signalling pathway. and Corresponding author: Xiao-Jing Li
Ulinastatin [or called as urinary trypsin inhibitor (UTI)] plays a role in regulating neurological deficits evoked by transient cerebral ischemia. However, the underlying mechanisms still need to be determined. The present study was to examine the effects of UTI on autophagy, Nrf2-ARE and apoptosis signal pathway in the hippocampus in the process of neurological functions after cerebral ischemia using a rat model of cardiac arrest (CA). CA was induced by asphyxia followed by cardiopulmonary resuscitation (CPR) in rats. Western blot analysis was employed to determine the expression of representative autophagy (namely, Atg5, LC3, Beclin 1), p62 protein (a maker of autophagic flux), and Nrf2-ARE pathways. Neuronal apoptosis was assessed by determining expression levels of Caspase-3 and Caspase-9, and by examining terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL). The modified neurological severity score (mNSS) and spatial working memory performance were used to assess neurological deficiencies in CA rats. Our results show that CA amplified autophagy and apoptotic Caspase-3/Caspase-9, and downregulated Nrf2-ARE pathway in the hippocampus CA1 region. Systemic administration of UTI attenuated autophagy and apoptosis, and largely restored Nrf2-ARE signal pathway following cerebral ischemia and thereby alleviated neurological deficits with increasing survival of CA rats. Our data suggest that UTI improves the worsened protein expression of autophagy and apoptosis, and restores Nrf2-ARE signals in the hippocampus and this is linked to inhibition of neurological deficiencies in transient cerebral ischemia. UTI plays a beneficial role in modulating neurological deficits induced by transient cerebral ischemia via central autophagy, apoptosis and Nrf2-ARE mechanisms., Xiao-Ming Jiang, Jing-Hai Hu, Lu-Lu Wang, Chi Ma, Xu Wang, Xiao-Liang Liu., and Obsahuje bibliografii