Endothelial cell apoptosis in chronically obstructed and reperfused pulmonary artery

<p>Abstract</p> <p>Background</p> <p>Endothelial dysfunction is a major complication of pulmonary endarterectomy (PTE) that can lead to pulmonary edema and persistent pulmonary hypertension. We hypothesized that endothelial dysfunction is related to increased endothelial-cell (EC) death.</p> <p>Methods</p> <p>In piglets, the left pulmonary artery (PA) was ligated to induce lung ischemia then reimplanted into the main PA to reperfuse the lung. Animals sacrificed 5 weeks after ligation (n = 5), 2 days after reperfusion (n = 5), or 5 weeks after reperfusion (n = 5) were compared to a sham-operated group (n = 5). PA vasoreactivity was studied and eNOS assayed. EC apoptosis was assessed by TUNEL in the proximal and distal PA and by caspase-3 activity assay in the proximal PA. Gene expression of pro-apoptotic factors (thrombospondin-1 (Thsp-1) and plasminogen activator inhibitor 1 (PAI-1)) and anti-apoptotic factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) was investigated by QRT-PCR.</p> <p>Results</p> <p>Endothelium-dependent relaxation was altered 5 weeks after ligation (<it>p </it>= 0.04). The alterations were exacerbated 2 days after reperfusion (p = 0.002) but recovered within 5 weeks after reperfusion. EC apoptosis was increased 5 weeks after PA ligation (<it>p </it>= 0.02), increased further within 2 days after reperfusion (<it>p </it>< 0.0001), and returned to normal within 5 weeks after reperfusion. Whereas VEGF and bFGF expressions remained unchanged, TSP and PAI-1 expressions peaked 5 weeks after ligation (<it>p </it>= 0.001) and returned to normal within 2 days after reperfusion.</p> <p>Conclusion</p> <p>Chronic lung ischemia induces over-expression of pro-apoptotic factors. Lung reperfusion is followed by a dramatic transient increase in EC death that may explain the development of endothelial dysfunction after PE. Anti-apoptotic agents may hold considerable potential for preventing postoperative complications.</p

Delayed union of femoral fractures in older rats:decreased gene expression

BACKGROUND: Fracture healing slows with age. While 6-week-old rats regain normal bone biomechanics at 4 weeks after fracture, one-year-old rats require more than 26 weeks. The possible role of altered mRNA gene expression in this delayed union was studied. Closed mid-shaft femoral fractures were induced followed by euthanasia at 0 time (unfractured) or at 1, 2, 4 or 6 weeks after fracture in 6-week-old and 12-15-month-old Sprague-Dawley female rats. mRNA levels were measured for osteocalcin, type I collagen α1, type II collagen, bone morphogenetic protein (BMP)-2, BMP-4 and the type IA BMP receptor. RESULTS: For all of the genes studied, the mRNA levels increased in both age groups to a peak at one to two weeks after fracture. All gene expression levels decreased to very low or undetectable levels at four and six weeks after fracture for both age groups. At four weeks after fracture, the younger rats were healed radiographically, but not the older rats. CONCLUSIONS: (1) All genes studied were up-regulated by fracture in both age groups. Thus, the failure of the older rats to heal promptly was not due to the lack of expression of any of the studied genes. (2) The return of the mRNA gene expression to baseline values in the older rats prior to healing may contribute to their delayed union. (3) No genes were overly up-regulated in the older rats. The slower healing response of the older rats did not stimulate a negative-feedback increase in the mRNA expression of stimulatory cytokines

Primary cilia disappear in rat podocytes during glomerular development

Most tubular epithelial cell types express primary cilia, and mutations of primary-cilium-associated proteins are well known to cause several kinds of cystic renal disease. However, until now, it has been unclear whether mammalian podocytes express primary cilia in vivo. In this study, we determined whether primary cilia are present in the podocytes of rat immature and mature glomeruli by means of transmission electron microscopy of serial ultrathin sections. In immature glomeruli of fetal rats, podocytes express the primary cilia with high percentages at the S-shaped body (88 ± 5%, n = 3), capillary loop (95 ± 4%, n =  4), and maturing glomerulus (76 ± 13%, n = 5) stages. The percentage of ciliated podocytes was significantly lower at the maturing glomerulus stage than at the former two stages. In mature glomeruli of adult rats, ciliated podocytes were not found at all (0 ± 0%, n = 11). These findings indicate that the primary cilia gradually disappear in rat podocytes during glomerular development. Since glomerular filtration rate increases during development, the primary cilia on the podocytes are subjected to a stronger bending force. Thus, the disappearance of the primary cilia presumably prevents the entry of excessive calcium-ions via the cilium-associated polycystin complexes and the disturbance of intracellular signaling cascades in mature podocytes

Vasoactive agents affect growth and protein synthesis of cultured rat mesangial cells

Vasoactive agents affect growth and protein synthesis of cultured rat mesangial cells. Mesangial cell (MC) proliferation and extracellular matrix (ECM) formation are hallmarks of chronic glomerular disease. The present in vitro study examined the effects of the vasoactive agents angiotensin II (Ang II), arginine vasopressin (AVP), and serotonin (5-HT) on growth and protein biosynthesis of cultured rat MCs after 72 hours of incubation. AVP and 5-HT (10-6 M) significantly increased DNA synthesis and growth of quiescent subconfluent MCs to levels of 25 and 45%, respectively, of the optimal stimulatory effect of 10% fetal calf serum (FCS) (both P < 0.001). The mitogenic effect of Ang II was 10% of the 10% FCS effect (P < 0.01). ECM production was studied by ELISA assay for fibronectin (FN) secreted into the culture medium (SeFN) and cell-associated FN, that is, intra- and pericellular FN (CaFN). In all incubations, highly significant negative linear relationships were found between the numbers of MCs per well and quantities of both SeFN and CaFN after normalization of the data by logarithmic transformation (SeFN: r values > -0.9705; CaFN: r < -0.9620; P < 0.001). Thus, increasing cell densities progressively suppressed ECM formation by MCs. The ECM production was found to be independent of growth activity. AVP significantly increased SeFN (P < 0.05) and decreased CaFN (P < 0.001) in subconfluent cultures; Ang II and 5-HT had no effect. Metabolic labeling with 35S-methionine (18 hr, 200 µCi/ml medium) and 2-D electrophoresis of MC lysates resulted in resolution of >500 different radiolabeled intracellular proteins in molecular weight from 110 to 20 Kd over an isoelectric interval of 5.0 to 7.0. Computerized video densitometry and scintillation counting of excised spots revealed prominent upregulation of 10 different MC proteins in response to AVP, and enhanced expression of five proteins in response to 5-HT, events characteristic of cellular activation. Ang II caused weakly increased expression of only one protein. The stimulatory effects of AVP and 5-HT on growth and protein synthesis of MCs in-vitro imply a possible in vivo role for these factors in glomerular disease

BCL10 is rarely mutated in human prostate carcinoma, small-cell lung cancer, head and neck tumours, renal carcinoma and sarcomas

We have used single-strand conformation polymorphism (SSCP) analysis to screen for mutations in the BCL 10 gene in 81 primary prostate carcinomas, 20 squamous cell cancers of the head and neck, 15 small-cell lung cancer cell lines, 24 renal carcinoma cell lines and 13 sarcoma cell lines. We failed to find evidence of somatically acquired mutations of the BCL10 gene suggesting that BCL 10 does not play a major role in the development of these malignancies

Deadly liaisons: fatal attraction between CCN matricellular proteins and the tumor necrosis factor family of cytokines

Recent studies have revealed an unexpected synergism between two seemingly unrelated protein families: CCN matricellular proteins and the tumor necrosis factor (TNF) family of cytokines. CCN proteins are dynamically expressed at sites of injury repair and inflammation, where TNF cytokines are also expressed. Although TNFα is an apoptotic inducer in some cancer cells, it activates NFκB to promote survival and proliferation in normal cells, and its cytotoxicity requires inhibition of de novo protein synthesis or NFκB signaling. The presence of CCN1, CCN2, or CCN3 overrides this requirement and unmasks the apoptotic potential of TNFα, thus converting TNFα from a proliferation-promoting protein into an apoptotic inducer. These CCN proteins also enhance the cytotoxicity of other TNF cytokines, including LTα, FasL, and TRAIL. Mechanistically, CCNs function through integrin α6β1 and the heparan sulfate proteoglycan (HSPG) syndecan-4 to induce reactive oxygen species (ROS) accumulation, which is essential for apoptotic synergism. Mutant CCN1 proteins defective for binding α6β1-HSPGs are unable to induce ROS or apoptotic synergism with TNF cytokines. Further, knockin mice that express an α6β1-HSPG-binding defective CCN1 are blunted in TNFα- and Fas-mediated apoptosis, indicating that CCN1 is a physiologic regulator of these processes. These findings implicate CCN proteins as contextual regulators of the inflammatory response by dictating or enhancing the cytotoxicity of TNFα and related cytokines

Enterocyte Shedding and Epithelial Lining Repair Following Ischemia of the Human Small Intestine Attenuate Inflammation

BACKGROUND: Recently, we observed that small-intestinal ischemia and reperfusion was found to entail a rapid loss of apoptotic and necrotic cells. This study was conducted to investigate whether the observed shedding of ischemically damaged epithelial cells affects IR induced inflammation in the human small gut. METHODS AND FINDINGS: Using a newly developed IR model of the human small intestine, the inflammatory response was studied on cellular, protein and mRNA level. Thirty patients were consecutively included. Part of the jejunum was subjected to 30 minutes of ischemia and variable reperfusion periods (mean reperfusion time 120 (+/-11) minutes). Ethical approval and informed consent were obtained. Increased plasma intestinal fatty acid binding protein (I-FABP) levels indicated loss in epithelial cell integrity in response to ischemia and reperfusion (p<0.001 vs healthy). HIF-1alpha gene expression doubled (p = 0.02) and C3 gene expression increased 4-fold (p = 0.01) over the course of IR. Gut barrier failure, assessed as LPS concentration in small bowel venous effluent blood, was not observed (p = 0.18). Additionally, mRNA expression of HO-1, IL-6, IL-8 did not alter. No increased expression of endothelial adhesion molecules, TNFalpha release, increased numbers of inflammatory cells (p = 0.71) or complement activation, assessed as activated C3 (p = 0.14), were detected in the reperfused tissue. CONCLUSIONS: In the human small intestine, thirty minutes of ischemia followed by up to 4 hours of reperfusion, does not seem to lead to an explicit inflammatory response. This may be explained by a unique mechanism of shedding of damaged enterocytes, reported for the first time by our group

PI3Kγ Protects from Myocardial Ischemia and Reperfusion Injury through a Kinase-Independent Pathway

BACKGROUND: PI3Kgamma functions in the immune compartment to promote inflammation in response to G-protein-coupled receptor (GPCR) agonists and PI3Kgamma also acts within the heart itself both as a negative regulator of cardiac contractility and as a pro-survival factor. Thus, PI3Kgamma has the potential to both promote and limit M I/R injury. METHODOLOGY/PRINCIPAL FINDINGS: Complete PI3Kgamma-/- mutant mice, catalytically inactive PI3KgammaKD/KD (KD) knock-in mice, and control wild type (WT) mice were subjected to in vivo myocardial ischemia and reperfusion (M I/R) injury. Additionally, bone-marrow chimeric mice were constructed to elucidate the contribution of the inflammatory response to cardiac damage. PI3Kgamma-/- mice exhibited a significantly increased infarction size following reperfusion. Mechanistically, PI3Kgamma is required for activation of the Reperfusion Injury Salvage Kinase (RISK) pathway (AKT/ERK1/2) and regulates phospholamban phosphorylation in the acute injury response. Using bone marrow chimeras, the cardioprotective role of PI3Kgamma was mapped to non-haematopoietic cells. Importantly, this massive increase in M I/R injury in PI3Kgamma-/- mice was rescued in PI3Kgamma kinase-dead (PI3KgammaKD/KD) knock-in mice. However, PI3KgammaKD/KD mice exhibited a cardiac injury similar to wild type animals, suggesting that specific blockade of PI3Kgamma catalytic activity has no beneficial effects. CONCLUSIONS/SIGNIFICANCE: Our data show that PI3Kgamma is cardioprotective during M I/R injury independent of its catalytic kinase activity and that loss of PI3Kgamma function in the hematopoietic compartment does not affect disease outcome. Thus, clinical development of specific PI3Kgamma blockers should proceed with caution