calpain-inhibitor-iii and Hypoxia

Calpain is a family of calcium-dependent nonlysosomal neutral cysteine endopeptidases. Akt is a serine/threonine kinase that belongs to AGC kinases and plays important roles in cell survival, growth, proliferation, angiogenesis, and cell metabolism. Both calpain and Akt are the downstream signaling molecules of platelet-derived growth factor (PDGF) and mediate PDGF-induced collagen synthesis and proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. We found that inhibitions of calpain-2 by using calpain inhibitor MDL28170 and calpain-2 small interfering RNA attenuated Akt phosphorylations at serine-473 (S473) and threonine-308 (T308), as well as collagen synthesis and cell proliferation of PASMCs induced by PDGF. Overexpression of calpain-2 in PASMCs induced dramatic increases in Akt phosphorylations at S473 and T308. Moreover, knockout of calpain attenuated Akt phosphorylations at S473 and T308 in smooth muscle of pulmonary arterioles of mice with chronic hypoxic pulmonary hypertension. The cell-permeable-specific transforming growth factor (TGF)-β receptor inhibitor SB431542 attenuated Akt phosphorylations at both S473 and T308 induced by PDGF and by overexpressed calpain-2 in PASMCs. Furthermore, SB-431452 and knocking down activin receptor-like kinase-5 significantly reduced PDGF-induced collagen synthesis and cell proliferation of PASMCs. Nevertheless, neutralizing extracellular TGF-β1 using a cell-impermeable TGF-β1 neutralizing antibody did not affect PDGF-induced Akt phosphorylations at S473 and T308. Furthermore, inhibition of mammalian target of rapamycin complex 2 (mTORC2) by knocking down its component protein Rictor prevented Akt phosphorylations at S473 and T308 induced by PDGF and by overexpressed calpain-2. These data provide first evidence supporting that calpain-2 upregulates PDGF-induced Akt phosphorylation in pulmonary vascular remodeling via an intracrine TGF-β1/mTORC2 mechanism.

Topics: Animals; Becaplermin; Benzamides; Calpain; Cell Proliferation; Cells, Cultured; Collagen; Cysteine Proteinase Inhibitors; Dioxoles; Dipeptides; Disease Models, Animal; Enzyme Activation; Humans; Hypertension, Pulmonary; Hypoxia; Mechanistic Target of Rapamycin Complex 2; Mice, Knockout; Multiprotein Complexes; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-sis; Pulmonary Artery; Receptors, Transforming Growth Factor beta; Ribonucleosides; RNA Interference; Signal Transduction; Time Factors; TOR Serine-Threonine Kinases; Transfection; Transforming Growth Factor beta1; Vascular Remodeling

To explore the role of calpain in pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension and the underlying mechanisms.
. Sprague-Dawley rats were randomly divided into the hypoxia group and the normoxia control group. Right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) were monitored by a method with right external jugular vein cannula. Right ventricular hypertrophy index was presented as the ratio of right ventricular weight to left ventricular weight (left ventricle plus septum weight). Levels of calpain-1, -2 and -4 mRNA in pulmonary artery were determined by real-time PCR. Levels of calpain-1, -2 and -4 protein were determined by Western blot. Primary rat pulmonary arterial smooth muscle cells (PASMCs) were divided into 4 groups: a normoxia control group, a normoxia+MDL28170 group, a hypoxia group and a hypoxia+MDL28170 group. Cell proliferation was detected by MTS and flow cytometry. Levels of Ki-67 and proliferating cell nuclear antigen (PCNA) mRNA were determined by real-time PCR.
. RVSP, mPAP and right ventricular remodeling index were significantly elevated in the hypoxia group compared to those in the normoxia group. In the hypoxia group, pulmonary vascular remodeling was significantly developed, accompanied by up-regulation of calpain-1, -2 and -4. MDL28170 significantly inhibited hypoxia-induced proliferation of PASMCs concomitant with the suppression of Ki-67 and PCNA mRNA expression.
. Calpain mediates vascular remodeling via promoting proliferation of PASMCs in hypoxia-induced pulmonary hypertension.. 目的：研究钙蛋白酶(calpain)在低氧诱导肺动脉高压肺血管重构中的作用及机制。方法：SD大鼠随机分为低氧模型组和常氧对照组。插管法测定大鼠右心室收缩压及平均肺动脉压，右心室/(左心室+室间隔)比值评价右心肥厚指数，HE染色检测血管重构情况，分别采用实时荧光定量PCR和Western印迹检测肺动脉calpain-1，-2和-4 mRNA和蛋白的表达。原代培养的大鼠肺动脉平滑肌细胞分为4组：常氧对照组、常氧对照+
MDL28170(calpain抑制剂)组、低氧模型组、低氧模型+MDL28170组。MTS及流式细胞术观察细胞增殖情况，实时荧光定量PCR检测Ki-67及增殖细胞核抗原(proliferating cell nuclear antigen，PCNA) mRNA表达情况。结果：与常氧对照组大鼠比较，低氧模型组大鼠右心室收缩压、平均肺动脉压及右心肥厚指数显著增加，肺动脉血管显著重构；同时，低氧模型组大鼠肺动脉中calpain-1，-2，-4 mRNA和蛋白表达也显著上调。与常氧对照组细胞比较，低氧模型组细胞显著增殖，MDL28170可显著抑制低氧诱导的肺动脉平滑肌细胞增殖，同时逆转低氧诱导的Ki-67和PCNA mRNA表达上调。结论：calpain介导低氧诱导的肺动脉高压肺血管重构，其机制可能与介导低氧诱导的肺动脉平滑肌细胞增殖有关。.

Topics: Animals; Calpain; Cell Proliferation; Dipeptides; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Ki-67 Antigen; Myocytes, Smooth Muscle; Proliferating Cell Nuclear Antigen; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Up-Regulation; Vascular Remodeling

Calpains are Ca(2+)-activated enzymes which cleave cytoskeletal and other proteins, contributing to neuronal damage in conditions of pathological intracellular Ca(2+) elevation, including stroke. However, the consequences of calpain overactivation have typically been observed hours after insult. To identify the earliest events attributable to calpain activation, and thus potentially isolate calpain substrates involved in acute neuronal damage, we dynamically recorded the effects of calpain inhibition in an in vitro model of stroke. Extracellular DC potentials and fEPSPs were monitored together with changes of light transmittance (as a measure of cell and mitochondrial swelling) and Rh 123 fluorescence (to monitor mitochondrial membrane potential; DeltaPsi(m)) in hippocampal slices obtained from P12-P17 rats. No differences were observed in the latencies of fEPSP disruption or onset of extracellular DC shifts associated with hypoxic spreading depression (HSD) evoked by oxygen-glucose deprivation (OGD) under control conditions or in the presence of calpain inhibitor III (MDL 28170). However, a significant difference was observed in transmitted light signals during OGD with calpain inhibition. Given the potential contribution of mitochondrial swelling to changes in light transmittance, these experiments were also conducted in the presence of cyclosporin A to block opening of the mitochondrial permeability transition pore (MPTP). Our results indicate that differences in OGD-induced changes of light transmittance in the presence of MDL 28170 are not likely the result of MPTP blockade or changes in dendritic beading. We propose that calpain inhibition may alter changes in light transmittance by limiting conformational changes of mitochondria.

Topics: Animals; Animals, Newborn; Brain Injuries; Calpain; Cortical Spreading Depression; Cysteine Proteinase Inhibitors; Dipeptides; Edema; Electric Stimulation; Excitatory Postsynaptic Potentials; Glucose; Hippocampus; Hypoxia; Membrane Potential, Mitochondrial; Rats; Rats, Wistar; Tissue Culture Techniques

Activation of calpain results in the breakdown of alpha II spectrin (alpha-fodrin), a neuronal cytoskeleton protein, which has previously been detected in various in vitro and in vivo neuronal injury models. In this study, a 150 kDa spectrin breakdown product (SBDP150) was found to be released into the cell-conditioned media from SH-SY5Y cells treated with the calcium channel opener maitotoxin (MTX). SBDP150 release can be readily quantified on immunoblot using an SBDP150-specific polyclonal antibody. Increase of SBDP150 also correlated with cell death in a time-dependent manner. MDL28170, a selective calpain inhibitor, was the only protease inhibitor tested that significantly reduced MTX-induced SBDP150 release. The cell-conditioned media of cerebellar granule neurons challenged with excitotoxins (NMDA and kainate) also exhibited a significant increase of SBDP150 that was attenuated by pretreatment with an NMDA receptor antagonist, R(-)-3-(2-carbopiperazine-4-yl)-propyl-1-phosphonic acid (CPP), and MDL28170. In addition, hypoxic/hypoglycemic challenge of cerebrocortical cultures also resulted in SBDP150 liberation into the media. These results support the theory that an antibody-based detection of SBDP150 in the cell-conditioned media can be utilized to quantify injury to neural cells. Furthermore, SBDP150 may potentially be used as a surrogate biomarker for acute neuronal injury in clinical settings.

Topics: Analysis of Variance; Animals; Blotting, Western; Calpain; Cell Death; Cells, Cultured; Cerebellum; Cerebral Cortex; Dipeptides; Dose-Response Relationship, Drug; Glucose; Humans; Hypoxia; Marine Toxins; Nerve Tissue Proteins; Neuroblastoma; Neurons; Neurotoxins; Oxocins; Rats; Rats, Sprague-Dawley; Spectrin; Time Factors

Calpains are ubiquitous Ca(2+)-activated neutral proteases that have been implicated in ischemic and traumatic CNS injury. Ischemia and trauma of central white matter are dependent on Ca2+ accumulation, and calpain overactivation likely plays a significant role in the pathogenesis. Adult rat optic nerves, representative central white matter tracts, were studied in an in vitro anoxic model. Functional recovery following 60 min of anoxia and reoxygenation was measured electrophysiologically. Calpain activation was assessed using western blots with antibodies against calpain-cleaved spectrin breakdown products. Sixty minutes of in vitro anoxia increased the amount of spectrin breakdown approximately 20-fold over control, with a further increase after reoxygenation to >70 times control, almost as much as 2 h of continuous anoxia. Blocking voltage-gated Na+ channels with tetrodotoxin or removing bath Ca2+ was highly neuroprotective electrophysiologically and resulted in a marked reduction of spectrin degradation. The membrane-permeable calpain inhibitors MDL 28,170 and calpain inhibitor-I (10-100 microM) were effective at reducing spectrin breakdown in anoxic and reoxygenated optic nerves, but no electrophysiological improvement was observed. We conclude that calpain activation is an important step in anoxic white matter injury, but inhibition of this Ca(2+)-dependent process in isolation does not improve functional outcome, probably because other deleterious Ca(2+)-activated pathways proceed unchecked.

Topics: Animals; Calcium; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Electrophysiology; Glycoproteins; Hypoxia; Ion Channel Gating; Male; Optic Nerve; Oxygen; Rats; Rats, Long-Evans; Sodium Channel Blockers; Sodium Channels; Spectrin; Tetrodotoxin; Time Factors

It has been suggested that, after ischaemia, activation of proteases such as calpains could be involved in cytoskeletal degradation leading to neuronal cell death. In vivo, calpain inhibitors at high doses have been shown to reduce ischaemic damage and traumatic brain injury, however, the relationship between calpain activation and cell death remains unclear. We have investigated the role of calpain activation in a model of ischaemia based on organotypic hippocampal slice cultures using the appearance of spectrin breakdown products (BDPs) as a measure of calpain I activation. Calpain I activity was detected on Western blot immediately after a 1-h exposure to ischaemia. Up to 4 h post ischaemia, BDPs were found mainly in the CA1 region and appeared before uptake of the vital dye propidium iodide (PI). 24 h after the insult, BDPs were detected extensively in CA1 and CA3 pyramidal cells, all of which was PI-positive. However, there were many more PI-positive cells that did not have BDPs, indicating that the appearance of BDPs does not necessarily accompany ischaemic cell death. Inhibition of BDP formation by the broad-spectrum protease inhibitor leupeptin was not accompanied by any neuroprotective effects. The more specific and more cell-permeant calpain inhibitor MDL 28170 had a clear neuroprotective effect when added after the ischaemic insult. In contrast, when MDL 28170 was present throughout the entire pre- and post-incubation phases, PI labelling actually increased, indicating a toxic effect. These results suggest that calpain activation is not always associated with cell death and that, while inhibition of calpains can be neuroprotective under some conditions, it may not always lead to beneficial outcomes in ischaemia.

Topics: Animals; Calpain; Coloring Agents; Cysteine Proteinase Inhibitors; Dipeptides; Glucose; Hippocampus; Hypoxia; Immunohistochemistry; In Vitro Techniques; Propidium; Rats; Rats, Wistar; Spectrin; Staining and Labeling

Stroke patients often experience a significant temporal delay between the onset of ischemia and the time to initiation of therapy. Thus, there is a need for neuroprotectants with a long therapeutic window of opportunity. The efficacy of a potent, central nervous system-penetrating calpain inhibitor (MDL 28,170) was evaluated in a temporary model of focal cerebral ischemia to determine the window of opportunity for intracellular protease inhibition.. An ex vivo brain protease inhibition assay established pharmacodynamic dosing parameters for MDL 28,170. Middle cerebral artery (MCA) occlusion was accomplished by advancing a monofilament through the internal carotid artery to the origin of the MCA. Postmortem infarct volumes were determined by quantitative image analysis of triphenyltetrazolium-stained brain sections.. Maximal inhibition of brain protease activity was observed 30 minutes after injection of MDL 28,170 with an estimated pharmacodynamic half-life of 2 hours. MDL 28,170 caused a dose-dependent reduction in infarct volume when administered 30 minutes after MCA occlusion. A window of opportunity study was conducted to determine the maximal delay between the onset of ischemia and the initiation of efficacious therapy. MDL 28,170 reduced infarct volume when therapy was delayed for 0.5, 3, 4, and 6 hours after the initiation of ischemia. The protective effect of MDL 28,170 was lost after an 8-hour delay.. These data indicate that the therapeutic window of opportunity for calpain inhibition is at least 6 hours in a reversible focal cerebral ischemia model. This protection is observed despite the lethal hypoxic and excitotoxic challenge, suggesting that calpain activation may be an obligatory, downstream event in the ischemic cell death cascade.

Topics: Animals; Brain; Calpain; Carotid Artery, Internal; Cell Death; Cerebral Arterial Diseases; Cerebral Infarction; Coloring Agents; Cysteine Proteinase Inhibitors; Dipeptides; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Half-Life; Hypoxia; Image Processing, Computer-Assisted; Ischemic Attack, Transient; Male; Neuroprotective Agents; Neurotoxins; Rats; Rats, Wistar; Tetrazolium Salts; Time Factors

The role of calcium-activated proteolysis in hypoxic neuronal injury was investigated using an in vitro slice model of moderate hypoxia that mimics many features of an ischemic penumbra. The calpain inhibitor, MDL28170, significantly improved the recovery of synaptic responses in hippocampal slices following prolonged, moderate hypoxia without hypoxic depolarization. This finding further implicates calpain-mediated proteolysis in the development of neuronal injury following moderate metabolic challenge such as occurs in regions of partial ischemia.

Topics: Animals; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Excitatory Postsynaptic Potentials; Hippocampus; Hypoxia; Male; Neurons; Rats; Rats, Sprague-Dawley; Synapses

In vitro hippocampal slices from adult rats were subjected to transient hypoxia in the presence of a cell-penetrating, calpain inhibitor (Cbz-Val-Phe-H; MDL-28170). The posthypoxic recovery of synaptic potentials was greatly improved in protease inhibitor-treated slices relative to control slices. These findings support a role for calcium-activated proteolysis in the process of hypoxic pathophysiology.

Topics: Animals; Calpain; Cell Death; Cell Membrane Permeability; Dipeptides; Hippocampus; Hypoxia; Rats; Rats, Inbred Strains