Epithelial cell senescence impairs repair process and exacerbates inflammation after airway injury

Background

Genotoxic stress, such as by exposure to bromodeoxyuridine (BrdU) and cigarette smoke, induces premature cell senescence. Recent evidence indicates that cellular senescence of various types of cells is accelerated in COPD patients. However, whether the senescence of airway epithelial cells contributes to the development of airway diseases is unknown. The present study was designed to test the hypothesis that premature senescence of airway epithelial cells (Clara cells) impairs repair processes and exacerbates inflammation after airway injury.

Methods

C57/BL6J mice were injected with the Clara-cell-specific toxicant naphthalene (NA) on days 0, 7, and 14, and each NA injection was followed by a daily dose of BrdU on each of the following 3 days, during which regenerating cells were allowed to incorporate BrdU into their DNA and to senesce. The p38 MAPK inhibitor SB202190 was injected 30 minutes before each BrdU dose. Mice were sacrificed at different times until day 28 and lungs of mice were obtained to investigate whether Clara cell senescence impairs airway epithelial regeneration and exacerbates airway inflammation. NCI-H441 cells were induced to senesce by exposure to BrdU or the telomerase inhibitor MST-312. Human lung tissue samples were obtained from COPD patients, asymptomatic smokers, and nonsmokers to investigate whether Clara cell senescence is accelerated in the airways of COPD patients, and if so, whether it is accompanied by p38 MAPK activation.

Results

BrdU did not alter the intensity of the airway epithelial injury or inflammation after a single NA exposure. However, after repeated NA exposure, BrdU induced epithelial cell (Clara cell) senescence, as demonstrated by a DNA damage response, p21 overexpression, increased senescence-associated β-galactosidase activity, and growth arrest, which resulted in impaired epithelial regeneration. The epithelial senescence was accompanied by p38 MAPK-dependent airway inflammation. Senescent NCI-H441 cells impaired epithelial wound repair and secreted increased amounts of pro-inflammatory cytokines in a p38 MAPK-dependent manner. Clara cell senescence in COPD patients was accelerated and accompanied by p38 MAPK activation.

Conclusions

Senescence of airway epithelial cells impairs repair processes and exacerbates p38 MAPK-dependent inflammation after airway injury, and it may contribute to the pathogenesis of COPD.     

Nogo-A expressed in Schwann cells impairs axonal regeneration after peripheral nerve injury

Injured axons in mammalian peripheral nerves often regenerate successfully over long distances, in contrast to axons in the brain and spinal cord (CNS). Neurite growth-inhibitory proteins, including the recently cloned membrane protein Nogo-A, are enriched in the CNS, in particular in myelin. Nogo-A is not detectable in peripheral nerve myelin. Using regulated transgenic expression of Nogo-A in peripheral nerve Schwann cells, we show that axonal regeneration and functional recovery are impaired after a sciatic nerve crush. Nogo-A thus overrides the growth-permissive and -promoting effects of the lesioned peripheral nerve, demonstrating its in vivo potency as an inhibitor of axonal regeneration.     

HDAC9 exacerbates endothelial injury in cerebral ischaemia/reperfusion injury

Histone deacetylase (HDAC) 9, a member of class II HDACs, regulates a wide variety of normal and abnormal physiological functions, which is usually expressed at high levels in the brain and skeletal muscle. Although studies have highlighted the importance of HDAC‐mediated epigenetic processes in the development of ischaemic stroke and very recent genome‐wide association studies have identified a variant in HDAC9 associated with large‐vessel ischemic stroke, the molecular events by which HDAC9 induces cerebral injury keep unclear. In this study, we found that HDAC9 was up‐regulated in the ischaemic cerebral hemisphere after cerebral ischaemia/reperfusion (I/R) injury in rats and in vivo gene silencing of HDAC9 by recombinated lentivirus infection in the brain reduced cerebral injury in experimental stroke. We further demonstrated that HDAC9 contributed to oxygen‐glucose deprivation‐induced brain microvessel endothelial cell dysfunction as demonstrated by the increased inflammatory responses, cellular apoptosis and endothelial cell permeability dysfunction accompanied by reduced expression of tight‐junction proteins. We further found that HDAC9 suppressed autophagy, which was associated with endothelial dysfunction. This study for the first time provides direct evidence that HDAC9 contributes to endothelial cell injury and demonstrates that HDAC9 is one of critical components of a signal transduction pathway that links cerebral injury to epigenetic modification in the brain.     

Intramural delivery of Sirolimus prevents vascular remodeling following balloon injury

OBJECTIVE: Several studies have demonstrated that Sirolimus-eluting stents reduce restenosis in patients with coronary artery disease. Here, we tested whether direct delivery of Sirolimus into the vessel wall during balloon angioplasty can modify vascular remodeling over several weeks. METHODS AND RESULTS: During angioplasty of the rabbit iliac artery we administered an intramural infusion of Sirolimus or its vehicle directly through a balloon catheter into the vessel wall. After 3 weeks neointimal formation was decreased (0.71+/-0.1 vs. 1.4+/-0.12 intima/media ratio), and this process was attributed to the inhibitory properties of Sirolimus on ECM deposition and smooth muscle cell proliferation. Sirolimus also significantly reduced the deposition of elastin, collagen III and fibronectin within the vascular wall. In parallel, proteomic profiles of arterial wall segments were obtained and 485 protein spots were consistently matched between non-dilated and dilated vessels. Differential expression of 12 proteins were observed between the groups and direct sequencing of digested peptides was performed. Local delivery of sirolimus during angioplasty attenuated the expression of structural proteins that included lamin A, vimentin, alpha-1-antitrypsin, and alpha-actin. CONCLUSIONS: Local administration of Sirolimus during angioplasty prevents smooth muscle cell proliferation associated with vascular remodeling as well as the expression of extracellular matrix and structural proteins. Therefore, local injection of Sirolimus during balloon inflation may be an alternative therapeutic approach for preventing restenosis in small stenotic vessels (i.e., <2.5 mm).     

Splenectomy exacerbates lung injury after ischemic acute kidney injury in mice

Patients with acute kidney injury (AKI) have increased serum proinflammatory cytokines and an increased occurrence of respiratory complications. The aim of the present study was to examine the effect of renal and extrarenal cytokine production on AKI-mediated lung injury in mice. C57Bl/6 mice underwent sham surgery, splenectomy, ischemic AKI, or ischemic AKI with splenectomy and kidney, spleen, and liver cytokine mRNA, serum cytokines, and lung injury were examined. The proinflammatory cytokines IL-6, CXCL1, IL-1β, and TNF-α were increased in the kidney, spleen, and liver within 6 h of ischemic AKI. Since splenic proinflammatory cytokines were increased, we hypothesized that splenectomy would protect against AKI-mediated lung injury. On the contrary, splenectomy with AKI resulted in increased serum IL-6 and worse lung injury as judged by increased lung capillary leak, higher lung myeloperoxidase activity, and higher lung CXCL1 vs. AKI alone. Splenectomy itself was not associated with increased serum IL-6 or lung injury vs. sham. To investigate the mechanism of the increased proinflammatory response, splenic production of the anti-inflammatory cytokine IL-10 was determined and was markedly upregulated. To confirm that splenic IL-10 downregulates the proinflammatory response of AKI, IL-10 was administered to splenectomized mice with AKI, which reduced serum IL-6 and improved lung injury. Our data demonstrate that AKI in the absence of a counter anti-inflammatory response by splenic IL-10 production results in an exuberant proinflammatory response and lung injury.     

Gene transfer of dominant negative Rho kinase suppresses neointimal formation after balloon injury in pigs

Restenosis after angioplasty still remains a major problem for which neointimal formation appears to play an important role. Recent studies in vitro suggested that Rho kinase, a target protein of Rho, is important in various cellular functions. We thus examined whether Rho kinase is involved in the restenotic changes after balloon injury. In vivo gene transfer was performed immediately after balloon injury in both sides of the porcine femoral arteries with adenoviral vector encoding either a dominant negative form of Rho kinase (AdDNRhoK) or beta-galactosidase (AdLacZ) as a control. One week after the transfer, immunohistochemistry confirmed the successful gene expression in the vessel wall, whereas 2 wk after the transfer, Western blotting showed the functional upregulation of Rho kinase at the AdLacZ site and its suppression at the AdDNRhoK site. Angiography showed the development of a stenotic lesion at the AdLacZ site where histological neointimal formation was noted, whereas those changes were significantly suppressed at the AdDNRhoK site. These results indicate that Rho kinase is involved in the pathogenesis of neointimal formation after balloon injury in vivo.     

Deficiency of TIMP-1 exacerbates LV remodeling after myocardial infarction in mice

Recent studies have been directed at modulating the heart failure process through inhibition of activated matrix metalloproteinases (MMPs). We hypothesized that a loss of MMP inhibitory control by tissue inhibitor of MMP (TIMP)-1 deficiency alters the course of postinfarction chamber remodeling and induced chronic myocardial infarction (MI) in wild-type (WT) and TIMP-1(-/-) mice. Left ventricular (LV) pressure-volume loops obtained from WT and TIMP-1(-/-) mice demonstrated that LV end-diastolic volume [52 +/- 4 (WT) vs. 71 +/- 6 (TIMP-1(-/-)) microl] and LV end-diastolic pressure [9.0 +/- 1.2 (WT) vs. 12.7 +/- 1.4 (TIMP-1(-/-)) mmHg] were significantly increased in the TIMP-1(-/-) mice 2 wk after MI. LV contractility was reduced to a similar degree in the WT and TIMP-1(-/-) groups after MI, as indicated by a significant fall in the LV end-systolic pressure-volume relationship. Ventricular weight and cross-sectional areas of LV myocytes were significantly increased in TIMP-1(-/-) mice, indicating that the hypertrophic response was more pronounced. The observed significant loss of fibrillar collagen in the TIMP-1(-/-) controls may have been an important contributory factor for the observed LV alterations in the TIMP-1(-/-) mice after MI. These findings demonstrate that TIMP-1 deficiency amplifies adverse LV remodeling after MI in mice and emphasizes the importance of local endogenous control of cardiac MMP activity by TIMP-1.     

Neutrophil adhesion to endothelial cells impairs the effects of catalase and glutathione in preventing endothelial injury

We studied the effects of the CuZn superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) on endothelial permeability to ¹²⁵I-albumin after activation of neutrophils (PMN) with phorbol 12-myristate-13-acetate (PMA; 10^?8M). PMN were either in direct contact with the endothelial cell monolayer grown on a porous gelatin-coated microporous 10-μm-thick polycarbonate filter (upright system) or separated from the endothelium by a similar filter (inverted system). Transendothelial ¹²⁵I-albumin clearance rates were measured as an index of endothelial permeability. In the absence of antioxidants, activation of PMN increased transendothelial ¹²⁵I-albumin clearnace rates in both systems from 0.041 ± 0.006 μl/min (baseline) to 0.262 ± 0.18 μl/min (upright system) and from 0.063 ± 0.02 μl/min to 0.244 ± 0.06 μl/min (inverted system). PMA induced 80–90% of PMN to adhere to either gelatin-coated filters or to endothelial cells, from the basal PMN adhesion value of 5.3 ± 2.2% and 4.3 ± 1.1%, respectively. SOD, which dismutates superoxide anion to hydrogen peroxide (H₂O₂), did not alter the transendothelial ¹²⁵I-albumin clearance rates in either systm at any concerntration from 10–300 U/ml. CAT (100–1,000 U/ml) and GSH (0.5–10 mM), which remove the H₂O₂ generated during PMN activation, did not alter the increase in transendothelial ¹²⁵I-clearance rates after PMN activation in the upright system, but both agents prvented the increase in transendothelial ¹²⁵I-clearance rates in the inverted system. We conclude that PMN activation with PMA causes endothelial injury irrespective of PMN contact to the endothelial monolayer. Moreover, H₂O₂, a release product of PMN activation, is a critical mediator of PMN-dependent endothelial injury. Finally, the results indicate that CAT and GSH prevent endothelial injury only in the absence of direct PMN contact with endothelial cells, suggesting that antioxidants such as GSH and CAT are excluded from sites of PMN-endothelial contact and thus are ineffective antioxidants. © 1993 Wiley-Liss, Inc.     

Granulocyte colony-stimulating factor exacerbates hematopoietic stem cell injury after irradiation

Background

Exposure to a moderate to high dose of ionizing radiation (IR) not only causes acute radiation syndrome but also induces long-term (LT) bone marrow (BM) injury. The latter effect of IR is primarily attributed to the induction of hematopoietic stem cell (HSC) senescence. Granulocyte colony-stimulating factor (G-CSF) is the only treatment recommended to be given to radiation victims soon after IR. However, clinical studies have shown that G-CSF used to treat the leukopenia induced by radiotherapy or chemotherapy in patients can cause sustained low white blood cell counts in peripheral blood. It has been suggested that this adverse effect is caused by HSC and hematopoietic progenitor cell (HPC) proliferation and differentiation stimulated by G-CSF, which impairs HSC self-renewal and may exhaust the BM capacity to exacerbate IR-induced LT-BM injury.

Methods

C57BL/6 mice were exposed to 4 Gy γ-rays of total body irradiation (TBI) at a dose-rate of 1.08 Gy per minute, and the mice were treated with G-CSF (1 μg/each by ip) or vehicle at 2 and 6 h after TBI on the first day and then twice every day for 6 days. All mice were killed one month after TBI for analysis of peripheral blood cell counts, bone marrow cellularity and long-term HSC (CD34-lineage-sca1+c-kit+) frequency. The colony-forming unit-granulocyte and macrophage (CFU-GM) ability of HPC was measured by colony-forming cell (CFC) assay, and the HSC self-renewal capacity was analyzed by BM transplantation. The levels of ROS production, the expression of phospho-p38 mitogen-activated protein kinase (p-p38) and p16^INK4a (p16) mRNA in HSCs were measured by flow cytometry and RT-PCR, respectively.

Results

The results of our studies show that G-CSF administration mitigated TBI-induced decreases in WBC and the suppression of HPC function (CFU-GM) (p < 0.05), whereas G-CSF exacerbated the suppression of long-term HSC engraftment after transplantation one month after TBI (p < 0.05); The increase in HSC damage was associated with increased ROS production, activation of p38 mitogen-activated protein kinase (p38), induction of senescence in HSCs.

Conclusion

Our findings suggest that although G-CSF administration can reduce ARS, it can also exacerbate TBI-induced LT-BM injury in part by promoting HSC senescence via the ROS-p38-p16 pathway.

     

Vascular remodeling after laser photocoagulation: concurrent regeneration and atrophy.

Laser photocoagulation is associated with paradoxical results: it causes obliteration of vessels, but leads also to the formation of new ones. In an attempt to better understand this dual vascular response we conducted an ultrastructure study of the choroidal vascular repair following krypton laser injury in rats. Three processes were observed: recanalization, neovascularization, and atrophy of both recanalized and newly formed capillaries. Post-lasering repair of the choroidal vasculature can therefore be described as a remodeling process, characterized by both regeneration and involution. The latter appears to be a secondary process of atrophy, contributing to permanent vascular obliteration. These mechanisms might explain why, in spite of initial vascular regeneration, laser photocoagulation treatment has a beneficial effect on choroidal subretinal neovascularization.     

Diphenylpiperazines enhance regeneration after facial nerve injury

Immature rat facial motoneurons are very sensitive to injury with nearly 80% dying during the first week after axotomy. This motoneuron death is apoptotic, similar to that induced in neurons after tropic factor withdrawal. The diphenylpiperazines, flunarizine and cinnarizine, protect dorsal root ganglion neurons from death after withdrawal of trophic support, i.e., nerve growth factor withdrawal, in vitro. Similarly, the monoamine oxidase inhibitor, deprenyl, promotes survival of facial motoneurons after axotomy. These pharmacological agents were assessed both alone and in combination for their ability to prevent death in non-nerve growth factor dependent CNS motoneurons after facial nerve axotomy in newborn rats. Long-term experiments were done with the diphenylpiperazines to evaluate potential enhancement of regeneration. Facial nerve transection resulted in 78% neuronal loss in the injured compared with the contralateral, uninjured nucleus. Systemic administration of diphenylpiperazines for 1 week after facial nerve transection doubled the number of surviving motoneurons from 23% to 47%. Similar results were obtained with deprenyl. Combinations of diphenylpiperazines and deprenyl provide a similar degree of neuronal protection 1 week after injury as that obtained by either agent alone. We assessed the ability of diphenylpiperazines to protect facial motoneurons from death over a prolonged period and enhance subsequent regeneration. Motor neuron counts in rats treated with diphenylpiperazines for 1 month after injury and assessed 2 months later demonstrated long-term enhancement of neuronal protection with an increase of 45% in the number of horseradish peroxidase-labelled motoneurons. The diphenylpiperazines group had ～80% more regenerated myelinated axons in the distal facial nerve than the control group. Thus, diphenylpiperazine treatment during the first month after injury provides long-term protection of non-nerve growth factor dependent CNS motoneurons with subsequent potentiation of long-term facial nerve regeneration.     

Busulphan-cyclophosphamide cause endothelial injury, remodeling of resistance arteries and enhanced expression of endothelial nitric oxide synthase

Stem cell transplantation (SCT) is a curative treatment for malignant and non malignant diseases. However, transplantation-related complications including cardiovascular disease deteriorate the clinical outcome and quality of life. We have investigated the acute effects of conditioning regimen on the pharmacology, physiology and structure of large elastic arteries and small resistance-sized arteries in a SCT mouse model. Mesenteric resistance arteries and aorta were dissected from Balb/c mice conditioned with busulphan (Bu) and cyclophosphamide (Cy). In vitro isometric force development and pharmacology, in combination with RT-PCR, Western blotting and electron microscopy were used to study vascular properties. Compared with controls, mesenteric resistance arteries from the Bu-Cy group had larger internal circumference, showed enhanced endothelium mediated relaxation and increased expression of endothelial nitric oxide synthase (eNOS). Bu-Cy treated animals had lower mean blood pressure and signs of endothelial injury. Aortas of treated animals had a higher reactivity to noradrenaline. We conclude that short-term consequences of Bu-Cy treatment divergently affect large and small arteries of the cardiovascular system. The increased noradrenaline reactivity of large elastic arteries was not associated with increased blood pressure at rest. Instead, Bu-Cy treatment lowered blood pressure via augmented microvascular endothelial dependent relaxation, increased expression of vascular eNOS and remodeling toward a larger lumen. The changes in the properties of resistance arteries can be associated with direct effects of the compounds on vascular wall or possibly indirectly induced via altered translational activity associated with the reduced hematocrit and shear stress. This study contributes to understanding the mechanisms that underlie the early effects of conditioning regimen on resistance arteries and may help in designing further investigations to understand the late effects on vascular system.     

Lymphatic vessels in bone support regeneration after injury

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Nitroxyl exacerbates ischemic cerebral injury and oxidative neurotoxicity

Nitroxyl (HNO) donor compounds function as potent vasorelaxants, improve myocardial contractility and reduce ischemia-reperfusion injury in the cardiovascular system. With respect to the nervous system, HNO donors have been shown to attenuate NMDA receptor activity and neuronal injury, suggesting that its production may be protective against cerebral ischemic damage. Hence, we studied the effect of the classical HNO-donor, Angeli's salt (AS), on a cerebral ischemia/reperfusion injury in a mouse model of experimental stroke and on related in vitro paradigms of neurotoxicity. I.p. injection of AS (40 μmol/kg) in mice prior to middle cerebral artery occlusion exacerbated cortical infarct size and worsened the persistent neurological deficit. AS not only decreased systolic blood pressure, but also induced systemic oxidative stress in vivo indicated by increased isoprostane levels in urine and serum. In vitro , neuronal damage induced by oxygen-glucose-deprivation of mature neuronal cultures was exacerbated by AS, although there was no direct effect on glutamate excitotoxicity. Finally, AS exacerbated oxidative glutamate toxicity – that is, cell death propagated via oxidative stress in immature neurons devoid of ionotropic glutamate receptors. Taken together, our data indicate that HNO might worsen cerebral ischemia-reperfusion injury by increasing oxidative stress and decreasing brain perfusion at concentrations shown to be cardioprotective in vivo .     

The nucleotide analogue 3-deazaadenosine prevents neointima-formation after balloon injury

We have recently shown that 3-deazaadenosine (c3Ado) inhibits atherogenesis in mice. We studied whether its anti-inflammatory capacity would also affect neointima-formation after balloon injury. Sprague Dawley rats underwent balloon angioplasty. C3Ado was administered orally, starting 5 days prior to the balloon injury and continued for 2 weeks. Fourteen days after balloon injury the intima/media ratio in the c3Ado-treated group was reduced by 67% (p < 0.001) and luminal stenosis by 50% (p < 0.001). Neointimal cellular density was decreased by 25% (p < 0.001) and the induction of c-Jun and ki67 was markedly lower. The reduction of the intima/media ratio was still observed 3 months after balloon injury. Furthermore, a c3Ado-dependent inhibition of PDGF-mediated ERK-activation and proliferation could be demonstrated.Short-term administration of C3Ado inhibits neointima-formation in rats for at least 3 months after injury. The present findings implicate that c3Ado may be useful as an inhibitor of restenosis-formation after balloon angioplasty in humans.     

Mouse gastrocnemius muscle regeneration after mechanical or cardiotoxin injury

The goal of our study was to compare the skeletal muscle regeneration induced by two types of injury: either crushing, that causes muscle degeneration as a result of mechanical devastation of myofibers, or the injection of a cardiotoxin that is a myotoxic agent causing myolysis of myofibers leading to muscle degeneration. Regenerating muscles were analyzed at selected intervals, until the 14th day following the injury. We analyzed their weight and morphology. We also studied the expression of different myosin heavy chain isoforms as a molecular marker of the regeneration progress. Histological analysis revealed that inflammatory response and myotube formation in crushed muscles was delayed compared to cardiotoxin-injected ones. Moreover, the expression of myosin heavy chain isoforms was observed earlier in cardiotoxin-injured versus crushed muscles. We conclude that the dynamics of skeletal muscle regeneration depends on the method of injury.