Effects of long-term immobilisation on endomysium of the soleus muscle in humans

New Findings: What is the central question of this study? While muscle fibre atrophy in response to immobilisation has been extensively examined, intramuscular connective tissue, particularly endomysium, has been largely neglected: does endomysium content of the soleus muscle increase during bed rest? What is the main finding and its importance? Absolute endomysium content did not change, and previous studies reporting an increase are explicable by muscle fibre atrophy. It must be expected that even a relative connective tissue accumulation will lead to an increase in muscle stiffness. Abstract: Muscle fibres atrophy during conditions of disuse. Whilst animal data suggest an increase in endomysium content with disuse, that information is not available for humans. We hypothesised that endomysium content increases during immobilisation. To test this hypothesis, biopsy samples of the soleus muscle obtained from 21 volunteers who underwent 60 days of bed rest were analysed using immunofluorescence-labelled laminin γ-1 to delineate individual muscle fibres as well as the endomysium space. The endomysium-to-fibre-area ratio (EFAr, as a percentage) was assessed as a measure related to stiffness, and the endomysium-to-fibre-number ratio (EFNr) was calculated to determine whether any increase in EFAr was absolute, or could be attributed to muscle fibre shrinkage. As expected, we found muscle fibre atrophy (P = 0.0031) that amounted to shrinkage by 16.6% (SD 28.2%) on day 55 of bed rest. ENAr increased on day 55 of bed rest (P < 0.001). However, when analysing EFNr, no effect of bed rest was found (P = 0.62). These results demonstrate that an increase in EFAr is likely to be a direct effect of muscle fibre atrophy. Based on the assumption that the total number of muscle fibres remains unchanged during 55 days of bed rest, this implies that the absolute amount of connective tissue in the soleus muscle remained unchanged. The increased relative endomysium content, however, could be functionally related to an increase in muscle stiffness

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 μm 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. © 2002 Harcourt Publishers Ltd.link_to_subscribed_fulltex

Heat shock protein 65 increases arterial contractility

Background An association has been shown between raised serum antibody levels to 65-kDa heat shock protein (HSP 65) and vascular disease suggesting that HSP 65 might play a role in its pathogenesis. The aim of this study was to investigate the direct effects of HSP 65 on the vasculature. Methods Male Dark Agouti rats were immunized with 200 mu g HSP 65 in Freund's incomplete adjuvant (HSP group). Five rats were used as control group. The animals were killed at 6 and 10 weeks following immunization. Rings of thoracic aorta were assessed for contraction and relaxation in response to phenylephrine and acetylcholine using an organ bath technique. The distal thoracic aorta was fixed and stained to determine intima/media thickness and to detect proliferating cellular nuclear antigen (PCNA). T-helper (Th) cell type 1 and 2 status was also assessed using flow cytometry. Results The HSP group showed increased contractility in response to phenylephrine compared with controls (mean(s.e.) maximum contraction 0.701(0.030) mu g (n = 9) versus 0.533(0.020) mu g (n = 5); P = 0.004, analysis of variance, Bonferoni/Dunn). The dose-response curve for acetylcholine was not significantly different. Controls reached 93 per cent maximal relaxation and rats in the HSP group 92 per cent suggesting intact endothelial nitric oxide synthesis. No significant difference could be found in intima/media thickness between the two groups (control group: intimal, 3.8 mu m; media, 80.5 mu m versus HSP group: intima, 3.8 mu m; media, 78.0 mu m). Staining for PCNA showed no difference between the groups. Cytokine expression was shifted from Th1 towards a Th2 status. Conclusion This study has shown a change in vasoactivity produced by immunization with HSP 65. This novel finding may have implications for understanding the aetiology of vasospastic disorders and may give new ideas for possible therapeutic strategies such es the use of Th1 adjuvants in patients with vascular disease

Role of heat shock protein 65 kDa on vascular smooth muscle cell proliferation

Stable URL: http://www.jstor.org/stable/3515900Introduction. Proliferation of vascular smooth muscle cells (vSMC) is one of the most important pathological processes in atherosclerosis. Recently, heat-shock protein (Hsp65) has been implicated in the pathogenesis and propagation of this disease. The aim of this study was to examine the effect of Hsp65 on the proliferation of vSMCs. Materials/Methods. Male Dark Agouti rats were immunized with 200 gg of Hsp65 (n = 6) or saline control (n = 5) and then sacrificed at 10 weeks. The thoracic aortae were removed and from sections, vSMCs were grown using standard explant technique. Cells were grown to confluence using DMEM+ 10% fetal calf serum (FCS), trypsinized, plated into 96-well plates, and made quiescent. These were subsequently stimulated with FCS (0-20% concentration) over 24 and 48 h. Proliferation was determined by the uptake of 5-bromo-2' deoxyuridine using colorimetric ELISA. Results. At 24 h, there was no difference in proliferation in any of the groups. However, at 48 h, vSMC proliferation was significantly reduced in the rats immunized with Hsp65 compared to controls. Data are expressed as means?SEM (see Figure 1). Conclusions. These findings suggest that prior in vivo inoculation with Hsp65 subsequently inhibited smooth muscle cell proliferation in vitro. Therefore Hsp65 per se could not account for the proliferation of vSMC seen in atherosclerosis

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.link_to_OA_fulltex

Heat shock protein 65 increases arterial contractility which can be reversed with Mycobacterium vaccae

Stable URL: http://www.jstor.org/stable/3515900Introduction. An association has been shown between raised serum antibody levels to 65 kDa heat-shock protein (Hsp65) and vascular diseases suggesting that it might play a role in its pathogenesis. It was the aim of this study to investigate the effects of Hsp65 on the vasculature. Methods. Male Dark Agouti rats were either immunised with 200 gg Hsp 65 kDa in Freunds Incomplete Adjuvant on day 14 only (Hsp group) or with 10' Mycobacterium vaccae on day 0 and Hsp65 on day 14 (My group). Six rats were used as controls. Four animals were sacrificed at 4 weeks following immunization, the others, including the control, after 10 weeks. Rings of thoracic aorta were assessed for contraction and relaxation in response to phenylephrine and acetylcholine using an organ bath technique. The distal thoracic aorta was used for histology to determine intima/media thickness. Thl/Th2 status (IFNy/IL-10) was also assessed using flow cytometry. Results.The Hsp group showed significantly increased contractility both at 4 and 10 weeks. In the MV group at 4 weeks, contractility was significantly decreased, but rose again after 10 weeks. The dose-response curve for relaxation in both groups was not significantly different from the control, suggesting intact endothelial nitric oxide synthesis. No significant difference could be found in intima/media thickness in both groups. The cytokine expression was shifted from Thl towards a Th2 status at 4 weeks in the Hsp group. The MV group, however, showed a shift from Th2 to Thl1 status at 4 weeks. Both were reversed again after 10 weeks. Conclusions. This study has shown a change in vasocontractility produced by immunization with Hsp65; short-term this could be reversed with MV. Similar short-term changes could be seen in the Thl1/Th2 status. This novel finding may have implications for understanding the aetiology of vasospastic disorders and may open new ideas for therapeutic strategies modifying the Th I /Th2 status in vascular patients