Imbalances in protein homeostasis caused by mutant desmin.

AIMS: We investigated newly generated immortalized heterozygous and homozygous R349P desmin knock-in myoblasts in conjunction with the corresponding desminopathy mice as models for desminopathies to analyse major protein quality control processes in response to the presence of R349P mutant desmin. METHODS: We used hetero- and homozygous R349P desmin knock-in mice for analyses and for crossbreeding with p53 knock-out mice to generate immortalized R349P desmin knock-in skeletal muscle myoblasts and myotubes. Skeletal muscle sections and cultured muscle cells were investigated by indirect immunofluorescence microscopy, proteasomal activity measurements and immunoblotting addressing autophagy rate, chaperone-assisted selective autophagy and heat shock protein levels. Muscle sections were further analysed by transmission and immunogold electron microscopy. RESULTS: We demonstrate that mutant desmin (i) increases proteasomal activity, (ii) stimulates macroautophagy, (iii) dysregulates the chaperone assisted selective autophagy and (iv) elevates the protein levels of αB-crystallin and Hsp27. Both αB-crystallin and Hsp27 as well as Hsp90 displayed translocation patterns from Z-discs as well as Z-I junctions, respectively, to the level of sarcomeric I-bands in dominant and recessive desminopathies. CONCLUSIONS: Our findings demonstrate that the presence of R349P mutant desmin causes a general imbalance in skeletal muscle protein homeostasis via aberrant activity of all major protein quality control systems. The augmented activity of these systems and the subcellular shift of essential heat shock proteins may deleteriously contribute to the previously observed increased turnover of desmin itself and desmin-binding partners, which triggers progressive dysfunction of the extrasarcomeric cytoskeleton and the myofibrillar apparatus in the course of the development of desminopathies.

Deuterium oxide-based University of Wisconsin solution improves viability of hypothermically stored vascular tissue.

BACKGROUND: Preservation of vascular function largely determines the outcome of transplantation. We have investigated replacing the water (H2O) in University of Wisconsin (UW) solution with deuterium oxide (D2O) in an attempt to improve vascular function after hypothermic storage. METHODS: Rat aortic segments were stored in UW solutions based on 100% H2O, 25% D2O, 50% D2O, and 100% D2O at 4 degrees C for 24, 48, or 72 hr. Vascular function was measured via contraction and endothelium-dependent relaxation after stimulation with phenylephrine and acetylcholine. RESULTS: UW solution with 25% D2O gave a significant (P<0.05) improvement of contraction and relaxation in comparison with H2O-based UW solution and other concentrations of D2O. CONCLUSIONS: Low concentrations (25%) of D2O-UW solution are significantly superior to the H2O-based (i.e., commonly used) equivalent at up to 72 hr. These results suggest that low concentrations of D2O-UW solution can improve the quality of hypothermic storage.

[Whiplash injuries of the cervical spine. Neurologic contribution to diagnosis. Therapy and expert evaluation]. / Beschleunigungsverletzungen der Halswirbelsäule. Beitrag der Neurologie zu Diagnostik, Therapie und Begutachtung.

The cervical spine is an extremely complex functional unit and has always been the subject of impassioned discussions among surgeons, orthopedic specialists and neurologists. In the presence of injuries affecting this section of the spine, which sometimes have grave consequences--including implications for legal insurance aspects, neurologists are usually at the end of the line when it comes to diagnosis and treatment. In fact, in view of the clinical and technical neurophysiological options available to neurologists, it would be desirable for them to be involved as early as possible. In this paper the relative value of neurology, compared with orthopedics and surgery, in the diagnosis and treatment of whiplash injury to the cervical spine is discussed. The newer options available--particularly in neurophysiology--are highlighted, as are the differential diagnosis and problems concerned with official expert assessments, which also involve other specialties.

Homocysteine and vascular disease.

There is increasing evidence that a raised blood level of homocysteine (HC) is a risk factor for premature atherosclerosis. With a gene frequency between one in 70 and one in 200 this condition may be more common than previously thought. It should be suspected especially in young patients in whom other risk factors are absent. The diagnosis may be made by demonstrating raised plasma HC levels, either basally or after methionine loading. Studies have shown significantly increased levels of HC in patients with premature coronary artery, peripheral vascular and cerebrovascular disease. The mechanisms by which HC produces vascular damage are, as yet, not completely understood but endothelial injury is probably a central factor. The principle of treatment is to lower HC levels in the blood by administration of vitamin B6, vitamin B12, folate or betaine. How effective this strategy will be in preventing complications is not yet known.

Does vascular stapling improve compliance of vascular anastomoses?

Elastic properties of vessel walls are altered by vascular anastomoses. Such alterations may lead to neointimal hyperplasia, which is a common cause of reocclusion following vascular surgery. The severity of paraanastomotic hypercompliant zones and anastomotic compliance drop depend on suturing material and on elastic properties of the anastomotic vessel segments. This study compares paraanastomotic hypercompliance and anastomotic compliance drop when using a new vascular closure system (VCS) and a conventional, continuous suture line in the preparation of end-to-end anastomoses. Compliance of artery-artery, vein-artery, and polytetrafluoroethylene-artery anastomoses was measured in an artificial circulation system at mean pressures of 60, 90, and 120 mm Hg, comparing conventional suturing and the VCS. When using the VCS for vein-artery anastomoses, significantly less postanastomotic hypercompliance was achieved at mean pressures of 60 mm Hg (14.2 +/-3.8% above remote postanastomotic area), compared to suture (55.1 +/-14.8%, p<0.05). At 90 mm Hg, respective values were 11.0 +/-2.3% for VCS and 54.7 +/-10.1% for suture, p<0.01. At 120 mm Hg, in polytetrafluoroethylene-artery anastomoses, the anastomotic compliance drop was significantly less when using the continuous suture line (93.9 +/-1.1% below remote postanastomotic compliance), compared to VCS (97.2 +/-0.2%, p<0.05). Compared to conventional suturing, use of the VCS reduced postanastomotic hypercompliance in vein-artery anastomoses.

Endothelial seeding of compliant polyurethane vascular graft material.

Compliance was measured in a new compliant polyurethane vascular graft material and in polytetrafluoroethylene (PTFE) graft material using an ultrasonic device; attachment of indium-111 oxine-labelled human endothelial cells to both surfaces with a range of surface coatings was assessed. Compliant polyurethane was six to eight times more compliant than PTFE (P < 0.01) at all pressures in the range 50-120 mmHg, and endothelial cell attachment to uncoated polyurethane was three times better than to uncoated PTFE at times up to 90 min (P < 0.01). Attachment to polyurethane was also better after blood clot, collagen and fibronectin treatment at times up to 30 min (P < 0.05). Endothelial seeding of compliant graft material may provide a prosthetic vascular substitute with characteristics similar to those of autologous vein.

Prevention of experimental myointimal hyperplasia by immunomodulation.

INTRODUCTION: we have tested the hypothesis that treatment with a mycobacterial preparation that modulates the antibody response, would diminish restenosis in a rat angioplasty model. MATERIALS/METHODS: male Sprague-Dawley rats were used. All immunisations were given subcutaneously. Group A (control) received normal saline on days 0, 21, and 42. Group B received SRL172 on days 0, 21, and 42. Group C received SRL172 on days 0, 21, and 42, and hsp65/Incomplete Freund's on days 21 and 42. Group D received hsp65/Freund's on days 21 and 42. Right common carotid arteries were balloon-injured on day 63 using a standard technique known to produce MIH and animals were sacrificed on day 77. For each carotid 6 microm cross sections were cut from paraffin blocks. Cross-sectional areas were measured by computerised planimetry. RESULTS: balloon injury resulted in MIH in all animals. Data represents mean+/-SEM for the percentage of area enclosed within the internal elastic lamina occupied by MIH (% MIH); which for groups A, B, C, and D was 85+/-11, 24+/-3, 27+/-7, and 17+/-3 respectively. All the treatment groups had significantly less MIH when compared to the control group but no statistically significant difference was found between any of the treatment groups. CONCLUSIONS: this is the first report that immunomodulation with mycobacterial material suitable for use in man, can reduce MIH. Since such modulation has low risk, this raises the prospect of an important new therapeutic modality to combat restenosis.

Endotoxemia during supraceliac aortic crossclamping is associated with suppression of the monocyte CD14 mechanism: possible role of transforming growth factor-beta1.

PURPOSE: Monocyte CD14 and its soluble form (sCD14) mediate the proinflammatory response to endotoxemia. The aim of this study was to measure the changes to these factors after major aortic surgery and the possible inhibitory role of transforming growth factor-beta(1) (TGF-beta(1)) during these procedures. METHODS: Twenty-four patients with supraceliac aortic crossclamping during thoracoabdominal aortic aneurysm (TAAA) repair and 12 patients with infrarenal aortic crossclamping as part of infrarenal aneurysm repair (AAA) were studied. Blood was collected at incision, aortic clamping, and reperfusion and at 1, 8, and 24 hours after reperfusion. Samples were assayed for endotoxin, peripheral blood monocyte CD14 expression, sCD14, tumor necrosis factor-alpha, and TGF-beta(1). RESULTS: Although there was significant endotoxemia on reperfusion in both groups of patients, peak plasma endotoxin levels were significantly higher in patients with TAAA (P =.001). Monocyte CD14 and plasma sCD14 were significantly decreased in patients with TAAA at reperfusion and 1 hour after reperfusion (P <.01, both points). In patients with AAA, a significant upregulation of CD14 was observed at 24 hours after reperfusion (P <.01), but no significant changes in sCD14 were observed. TNF-alpha showed no significant changes during the study period in both groups. In patients with TAAA, TGF-beta(1) showed significant elevation at all time points (P <.01); whereas in patients with AAA, TGF-beta(1) showed no significant changes. CONCLUSION: Splanchnic ischemia reperfusion in patients who undergo supraceliac aortic clamping is associated with peripheral blood monocyte CD14 suppression and significant elevation of TGF-beta(1). TGF-beta(1) may play an important role in modulating the immune response to endotoxemia during major aortic aneurysm surgery.

Myocardial injury in major aortic surgery.

PURPOSE: The purpose of this study was to examine the effects of major aortic surgery and its associated oxidative stress and injury on the myocardium. METHODS: Plasma from 27 patients who underwent thoracoabdominal aortic aneurysm (TAAA) repair and 17 patients who underwent infrarenal aortic aneurysm (AAA) repair was collected at incision, aortic crossclamping, and reperfusion and 1, 8, and 24 hours thereafter. Samples were assayed for the myocardial specific protein troponin-T, total antioxidant status, and lipid hydroperoxides. RESULTS: Ten patients experienced cardiac dysfunction in the first 24 hours after surgery (eight patients in the TAAA group and two patients in the AAA group). Immediately after reperfusion, total antioxidant status levels dropped in all patients with TAAA and with AAA; this was more marked in patients with TAAA, leading to a significant difference between the two groups at this time point and for up to 1 hour thereafter (P <.01). Patients with TAAA showed a sharp rise in lipid hydroperoxide levels immediately after reperfusion, and levels were significantly higher than in patients with AAA (P =.0007). In patients with AAA, no significant change in troponin-T was observed throughout the study period; whereas in patients with TAAA, levels were significantly elevated at 8 and 24 hours after reperfusion (P <.01). Troponin-T levels significantly correlated with total antioxidant status (r = -0.5) and lipid hydroperoxides (r = 0.78) but not with systolic blood pressure. CONCLUSION: Supracoeliac aortic crossclamping is associated with a significant release of the myocardial injury marker troponin-T. This seems to correlate with the severity of oxidative rather than hemodynamic stresses. Ameliorating oxidative injury during TAAA surgery may therefore have a cardioprotective effect.

Anti-heat-shock protein 70 kDa antibodies in vascular patients.

INTRODUCTION AND AIM OF STUDY: there is recent evidence that the immune system plays an essential role in the pathogenesis of atherosclerosis, with both cellular and humoral mechanisms being involved. Heat-shock proteins (HSPs) have been detected in atherosclerotic lesions, and antibodies to HSPs have also been found to be raised in patients with carotid stenoses. The aim of our study was to examine the level of anti-HSP70 antibodies in patients with other vascular diseases. MATERIALS AND METHODS: a questionnaire was designed for the subjects in the study, with documentation of clinical details and ankle-brachial pressure index. Patients with concomitant infection, malignancy, hepatorenal failure, or recent surgery were excluded. Enzyme-linked immunosorbent assay (ELISA) was used to identify anti-HSP70 antibodies in the sera in different dilutions. Graphs of optical density (OD) vs. negative log dilution were plotted, the gradient of which was taken to be the estimated optical density for each subject (proportional to antibody level). Our groups consisted of controls (n =21, mean age 59.0+/-19.2), lower limb claudicants ( n =19, mean age 60.0+/-12.6), patients with lower-limb critical ischaemia ( n =22, mean age 68.5+/-10.07), and patients with abdominal aortic aneurysms ( n =20, mean age 69.9+/-6.2). RESULTS: we found no correlation between age and the estimated OD in our subjects (Spearman's correlation coefficient ( r )=0.123, one-tailed p value was 0.135). Patients with intermittent claudication, critical lower limb ischaemia, and aneurysms had higher estimated OD, and therefore higher anti-HSP70 antibody levels, than controls (Mann-Whitney test p =0.0127, 0.0037, 0.0008, respectively). CONCLUSIONS: our data provide the first evidence of a correlation between anti-HSP70 antibodies and different types of vascular diseases, suggesting that HSP70 might be involved in the pathogenesis and propagation of atherosclerosis. Since the immune response to HSPs can be modulated, this opens up the possibility of new therapeutic approaches.