Molecular markers and targeted therapy of skin rejection in composite tissue allotransplantation.

Skin rejection remains a major hurdle in reconstructive transplantation. We investigated molecular markers of skin rejection with particular attention to lymphocyte trafficking. Skin biopsies (n = 174) from five human hand transplant recipients were analyzed for rejection, characteristics of the infiltrate and lymphocytic adhesion markers. The cellular infiltrate predominantly comprised CD3+ T cells. CD68, Foxp3 and indoleamine 2, 3-dioxygenase expression and the CD4/CD8 increased with severity of rejection. Lymphocyte adhesion markers were upregulated upon rejection, intercellular adhesion molecule-1 and E-selectin correlated best with severity of rejection. Guided by the findings, a specific E- and P-selectin inhibitor was investigated for its effect on skin rejection in a rat hind limb allotransplant model. While efomycine M (weekly s.c. injection into the graft) alone had no effect, long-term allograft survival was achieved when combined with antithymocyte globulin and tacrolimus (control group without efomycine M rejected at postoperative day [POD] 61 +/- 1). Upregulation of lymphocyte trafficking markers correlates with severity of skin rejection and time after transplantation in human hand transplantation. Blocking E- and P-selectin in the skin holds potential to significantly prolong limb allograft survival.

The effect of secretory leukocyte protease inhibitor (SLPI) on ischemia/reperfusion injury in cardiac transplantation.

We investigated the role of secretory leukocyte protease inhibitor (SLPI) in ischemia/reperfusion injury in cardiac transplantation. SLPI-/- mouse hearts and wild-type (WT) controls were transplanted immediately or after 10 h of cold ischemia (CI). Recombinant SLPI (rSLPI) was added to the preservation solution or given systemically. After evaluation of myocardial performance, grafts were investigated for histology, SLPI, TNF-alpha, TGF-beta, NF-kappaB and protease expression at indicated time points. Early myocardial contraction was profoundly impaired in SLPI-/- hearts exposed to CI and associated with high intra-graft protease expression. Systemic administration of rSLPI had no effect, however, when SLPI was added to the preservation solution, myocardial contraction was restored to normal. At 10 days, inflammation, myocyte vacuolization and necrosis were significantly more severe in SLPI-/- hearts. SLPI gene expression was detected in WT mice at 12 and 24 h and was significantly higher after CI. SLPI protein was observed at 24 h and 10 days. High intra-graft concentrations of SLPI after administration of rSLPI were inversely correlated with protease levels early and TGF-beta expression late after reperfusion. SLPI plays a crucial role in early myocardial performance and postischemic inflammation after cardiac transplantation. A dual inhibitory effect on protease and TGF-beta expression might be the underlying mechanism.

Ep-CAM expression in pancreatic and ampullary carcinomas: frequency and prognostic relevance.

AIMS: Pancreatic adenocarcinoma is an aggressive gastrointestinal malignancy with only a few long-term survivors even after radical surgery. Patients with ampullary cancer have a better prognosis but adjuvant therapy needs further improvement. Epithelial cell adhesion molecule (Ep-CAM) is strongly expressed in a variety of epithelial cancers and represents a promising target for immunological tumour therapy. Thus, the aim of this study was to investigate Ep-CAM expression and its potential prognostic impact in pancreatic and ampullary carcinomas. METHODS: Ep-CAM expression was investigated retrospectively by immunohistochemistry in paraffin-embedded primary tumour tissue samples from a series of consecutive patients with pancreatic (n = 153) and ampullary cancer (n = 34). RESULTS: Ep-CAM overexpression was observed in 85 of 153 pancreatic cancer specimens (56%) and in 29 of 34 ampullary cancer samples (85%). Overall, Ep-CAM failed to be an independent prognostic marker. However, subgroup analyses showed that Ep-CAM overexpression correlated with shorter overall survival among patients with ampullary cancer and advanced stage pancreatic cancer. In the latter subgroup, survival gradually worsened with increasing Ep-CAM scores. Furthermore, in ampullary cancer, Ep-CAM overexpression was found to correlate with tumour stage. CONCLUSIONS: Ep-CAM overexpression was detectable in the majority of cases with pancreatic and ampullary cancer. Therefore, Ep-CAM represents an attractive target for immune-based therapeutic interventions in these tumour entities. However, the prognostic value of Ep-CAM overexpression remains undetermined.

Lipocalin-2 regulates the inflammatory response during ischemia and reperfusion of the transplanted heart.

Ischemia and reperfusion (IR) are known to negatively affect early allograft function following solid organ transplantation. Lipocalin-2 (Lcn-2) has been described as a marker and potential positive modulator of acute inflammation during these processes. Using a heterotopic murine heart transplant model we previously found that IR resulted in a pronounced upregulation of Lcn-2 mRNA in the heart at 12 (22.7-fold increase) and 24 h (9.8-fold increase) of reperfusion. We now confirm this increase at the protein level and provide evidence for infiltrating polymorphonuclear cells as the primary source of Lcn-2 protein. Lcn-2 levels are increased 6.6-fold at 12 h, 11.4-fold at 24 h and 6.4 fold at 48 h after reperfusion. In Lcn-2(-/-) grafts the number of infiltrating granulocytes is reduced by 54% (p < 0.05) at 2 h, 79% (p < 0.01) at 12 h, 72% (p < 0.01) at 24 h and 52% (p < 0.01) at 48 h after reperfusion compared to Lcn-2(+/+) grafts, without any differences in cardiomyocyte apoptosis. These data suggest a function of Lcn-2 in the initiation of the inflammatory response. Moreover, an increase in Lcn-2 is not only restricted to the transplanted heart, but is also observed in the kidney, hinting at a possible involvement of Lcn-2 in the systemic response to IR.

Aberrant tetranectin expression in human breast carcinomas as a predictor of survival.

AIMS: Tetranectin (TN), a plasminogen kringle 4 binding protein, is thought to play a prominent role in the regulation of proteolytic processes via binding to plasminogen. The aim of this study was to evaluate the expression of TN in human breast cancer and adjacent normal breast tissue and to determine the impact of this expression on survival. METHODS: A retrospective analysis was performed on 189 patients with breast cancer, with a median follow up time of 10.6 years. The expression of TN was assessed in tumour tissue and adjacent normal breast tissue by immunohistochemistry, and the prognostic relevance of its expression in tumour cells was evaluated. RESULTS: TN was highly expressed in connective tissue fibres surrounding normal breast epithelium, but not in normal epithelial cells. High expression of TN in tumour cells was found in 131 (69%) of the tumour samples. By western blot analysis, no significant difference in the amount and molecular weight of TN was seen between tumour tissue and normal tissue. Strong TN immunoreactivity in tumour tissue was predictive of poor disease free and tumour specific overall survival. By multivariate analysis, high TN expression in cancer cells was an independent prognostic factor for disease free and tumour specific overall survival. CONCLUSIONS: Our results demonstrate differential TN expression in normal and malignant breast tissue and a prognostic impact of TN protein expression in breast carcinoma tissue. These data suggest a possible role of TN in invasiveness and the metastatic spread of human breast cancer.

Cytokine-regulated expression of collagenase-2 (MMP-8) is involved in the progression of ovarian cancer.

Matrix metalloproteinases (MMPs) have been implicated in ovarian cancer progression. Among them, MMP-8 that degrades type I collagen may play a crucial role. The aim of our study was to determine MMP-8 expression and regulation in ovarian cancer and its association with other MMPs and tissue inhibitors of metalloproteinases (TIMPs). Tissue microarrays (TMAs) containing tissue cylinders from 302 patients were used for immunohistochemical studies. In addition, MMP-8 expression in vitro was analysed by a specific immunoassay and PCR-analysis. MMP-7 (81%), MMP-8 (95%), MT3-MMP (100%), TIMP-2 (100%), and TIMP-3 (96%) were expressed in all the OVCAs, but the staining intensities varied. MMP-3 (6%), MMP-9 (57%) and TIMP-1 (43%) expressions were more rarely detected. Only MMP-8 expression levels correlated with tumour grade (P<0.01), tumour stage (P<0.01), and a poor prognosis (P<0.05). MMP-8 protein and gene expression in vitro was found to be significantly upregulated by interleukin-1beta (IL-1beta, P<0.01). The data indicate that MMP-8 overexpression in OVCAs is regulated by IL-1beta and that pro-inflammatory cytokines may promote the invasive potential of ovarian cancer.

Heparanase-1 gene expression in normal, hyperplastic and neoplastic prostatic tissue.

Heparanase-1 (Hpa-1) has been implicated in tumour invasion and metastasis. In the present study, we evaluated the clinicopathological significance of Hpa-1 mRNA expression in prostate cancer and non-cancerous prostatic tissue by one-step polymerase chain reaction (PCR) of laser microdissected prostatic gland cells. In addition, cell type-specific expression of Hpa-1 mRNA in prostatic tissue was analysed by in situ hybridisation. Hpa-1 mRNA expression was found in 50% of normal and 40% of hyperplastic prostatic tissue. In situ hybridisation showed that Hpa-1 mRNA was strongly expressed in prostate gland cells. Of the 26 prostate carcinomas tested, 42% were positive for Hpa-1 mRNA. However, in non-cancerous prostatic tissue, Hpa-1 mRNA was significantly more often expressed than in less differentiated or more invasive prostate cancers (P<0.05). In situ hybridisation revealed only focal Hpa-1 mRNA expression in the neoplastic gland cells. Hpa-1 mRNA expression in the tumours significantly correlated with tumour differentiation and tumour stage (P<0.05). Our data indicate that Hpa-1 gene expression may be lost during dedifferentiation of prostatic gland cells.

Apoptosis of CD34+ cells after incubation with sera of leukopenic patients with systemic lupus erythematosus.

Leukopenia and anaemia are observed in about a fifth of all patients with systemic lupus erythematosus (SLE) and may be due either to the destruction of blood cells or their decreased production. The former may be humoral or cell-mediated or result from apoptosis of peripheral blood cells. Several observations suggest the occurrence of the latter reduced in vitro proliferation of pluripotent bone marrow progenitors from the bone marrow aspirates of SLE patients,reduced counts of CD34+ cells in bone marrow aspirates in SLE patients, apoptosis of lymphopoietic progenitors and apoptosis of bone marrow cells. The aim of our study was to investigate whether humoral factors may induce suppression of haematopoiesis by increased apoptosis of CD34+ cells. For this purpose, we incubated allogeneic CD34+-enriched cells with sera of 18 leukopenic SLE patients. Apoptosis was induced by four of 18 sera. This effect was independent of complement-inhibition and FAS-blockade. Although reduced proliferation of autologous pluripotent bone marrow progenitors has been attributed to an IgG serum inhibitor, removal of IgG from these four proapoptotic sera had no effect on apoptosis of allogeneic CD34+ cells. The proapoptotic effect was associated with high titres of anti-dsDNA antibodies and low haemoglobin concentrations, but not with high titres of antinuclear antibodies, TNF-alpha and IFN-alpha of the sera tested.

Correlation of COX-2 and Ep-CAM overexpression in human invasive breast cancer and its impact on survival.

Recent studies have demonstrated cyclooxygenase 2 (COX-2) overexpression in various human malignancies, especially in breast cancer, where COX-2 turned out to be a predictor of poor survival. To evaluate the relation of COX-2 and Ep-CAM overexpression and its prognostic significance, we performed a retrospective study on 212 breast cancer patients with a median follow-up time of 10.5 years. Overexpression of COX-2 in tumour tissue samples was assessed by immunohistochemistry. COX-2 overexpression was found in 48.6% of the tumour samples and was predictive for poor disease-free and overall survival. Univariate analysis revealed a strong correlation between COX-2 and Ep-CAM overexpression (P=0.009). Concurrent COX-2 and Ep-CAM overexpression was present in 21.7% of tumour specimens and had an additive negative impact on disease-free and overall survival. Determination of both tumour markers should help in guiding new therapeutic strategies in patients with invasive breast cancer.

Protease Inhibitors as a Potential Target in Modulation of Postischemic Inflammation.

Prevention of ischemia-reperfusion injury represents a main objective in infarction, cardiac surgery and organ transplantation. In the context of cellular homeostasis, postischemic inflammation may be understood in part as an initial physiological response to ischemia and reperfusion, aiming at restoration of tissue integrity. Polymorphonuclear cell infiltration and subsequent protease production, however, are crucial mechanisms contributing to tissue damage, cell necrosis and subsequent functional deficits. Therefore, inhibition of protease activity appears a promising target for modulating destructive processes of postischemic inflammation, while preserving its restorative nature. Recently, effects of secretory leukocyte protease inhibitor elafin and other protease inhibitors have been investigated in vivo and in vitro, which may provide a basis for future therapeutic strategies in postischemic inflammation. (c) 2002 Prous Science. All rights reserved.

Increased lactate production follows loss of mitochondrial membrane potential during apoptosis of human leukaemia cells.

Acute tumour-lysis syndrome (ATLS) is a frequently fatal complication after cytoreductive leukaemia therapy. Lactic acidosis is associated with ATLS and its extent is correlated with the severity of ATLS. In the course of cytoreductive therapy, apoptosis is induced in tumour cells, which results in loss of mitochondrial function. We hypothesize that loss of mitochondrial function leads to compensatory glycolysis, which is the main cause of lactate accumulation and acidosis. We tested this hypothesis using the model of glucocorticoid-induced apoptosis in the human acute lymphoblastic leukaemia cell line CCRF-CEM. After induction of glucocorticoid-induced apoptosis, a biphasic course of lactate production was observed. Prior to the onset of apoptosis, i.e. prior to the loss of membrane potential, lactate production was reduced. However, subsequent to loss of mitochondrial membrane potential a massive increase in lactate production was observed (15.5 +/- 0.5 versus 10.17 +/- 0.09 mmol/10(6) cells, P = 0.001). We also demonstrated that inhibition of respiratory chain activity by antimycin A resulted in excess lactate production. In the model cell line used, conditional bcl-2 expression delayed glucocorticoid-induced apoptosis by protecting against loss of mitochondrial membrane potential; bcl-2 expression delayed the increase in lactate production and had no effect on the pre-apoptotic drop in lactate production. Apoptosis-induced lactate production was also observed in other cell lines (HL60, THP1 and OPM2) with various cytotoxic agents [doxorubicin, gemcitabine and vumon (VM26)]. Thus, the data suggest that lactate acidosis can be caused by apoptotic loss of mitochondrial function and massive apoptosis of a tumour mass via lactic acidosis may be the essential pathological event in ATLS.

The influence of atracurium, cisatracurium, and mivacurium on the proliferation of two human cell lines in vitro.

We tested the influence of atracurium and cisatracurium (final concentrations: 0, 0.96, 3.2, 9.6, 32, and 96 microM) on proliferation of human cells (hepatoma HepG2 cells and human umbilical vein endothelial cells) in vitro. In additional experiments, glutathione, N-acetylcysteine, or carboxyl esterase was added before the addition of either relaxant. The number of cells counted after 72 h of incubation was expressed as a percentage of the mean cell number in wells incubated without additives. Atracurium and cisatracurium progressively decreased cell proliferation in a concentration-dependent pattern. With human umbilical vein endothelial cells, atracurium or cisatracurium (3.2 microM) decreased the cell count to 67.7 % (SD, 14.8%) and 50% (SD, 8.6%), respectively. Cell proliferation was not inhibited by mivacurium. The results were similar to those with HepG2 cells. Glutathione, N-acetylcysteine, and carboxyl esterase partially reversed the effects of atracurium and cisatracurium. When incubated in a buffer with glutathione, atracurium decreased the number of glutathione-sulfhydryl groups. The findings that atracurium and cisatracurium inhibit proliferation of human cell lines in vitro, but that mivacurium does not, and that this effect is alleviated by glutathione and N-acetylcysteine, as well as by the carboxyl esterase, indicate that the inhibition may be caused by the reactive acrylate metabolites.

A subpopulation of mitochondria prevents cytosolic calcium overload in endothelial cells after cold ischemia/reperfusion.

BACKGROUND: Calcium represents a key mediator of cold ischemia/reperfusion (CIR) injury presumably by affecting mitochondrial function. In this study, we investigated cellular and mitochondrial changes of calcium homeostasis in sublethally damaged human endothelial cells. METHODS: Changes in cellular and mitochondrial calcium concentrations were studied after cold ischemia in University of Wisconsin solution for 12 hr and reperfusion in ringer solution. Cytosolic-free calcium concentration ([Ca2+]c) and mitochondrial-free calcium content ([Ca2+]m) were analyzed by fura-2 and rhod-2 fluorescence, respectively. Pretreatment of cells with ruthenium red (RR) or a H+-ionophore was used to inhibit mitochondrial calcium uptake. Mitochondrial membrane potential (DeltaPsim) was measured by 5,5',6,6'-tetrachloro- 1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide and 3,3'-dihexyloxacarbocyanine iodide fluorescence. RESULTS: Twelve-hr cold ischemia did not induce apoptosis in endothelial cells. In such sublethally damaged cells, [Ca2+]c rose from approximately 20 nmol/L after cold ischemia to approximately 120 nmol/L during reperfusion. Pretreatment with RR leads to an approximately 5-fold rise in [Ca2+]c. Image analysis revealed a significant increase of [Ca2+]m in a subpopulation of mitochondria during reperfusion. This was not the case in RR-pretreated cells. DeltaPsim decreased significantly during cold ischemia and was sustained during reperfusion. The loss of DeltaPsim can be related to a reduced portion of mitochondria exhibiting high DeltaPsim. CONCLUSIONS: Our results suggest that cytosolic calcium influx during CIR is buffered by a selective portion of mitochondria in human umbilical vein endothelial cells. These mitochondria protect cells against cytosolic calcium overload and probably against subsequent cell injury.

Inhibition of store-operated calcium entry contributes to the anti-proliferative effect of non-steroidal anti-inflammatory drugs in human colon cancer cells.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit proliferation and angiogenesis in colorectal cancer. We examined a possible involvement of store-operated calcium (SOC) entry in human colon carcinoma cells (HRT-18), which require calcium for proliferation. Acetyl-salicylic-acid (ASA), mefenamic acid (MEF) and sulindac sulfide (SUS) inhibited cell proliferation with the following order of potency: SUS > MEF >> ASA. SUS but not MEF and ASA induced apoptosis following low-dose treatment. Furthermore, SUS and MEF significantly altered the cell cycle distribution. The ability of NSAIDs to inhibit SOC entry was assessed by measuring the intracellular calcium concentration ([Ca2+]i) in response to calcium store depletion using the endoplasmic calcium ATPase inhibitor thapsigargin. SUS and MEF, but not ASA significantly inhibited SOC entry. A causal link between SOC entry inhibition and anti-proliferative activity was tested using the inorganic SOC entry inhibitor La3+ and the specific organic inhibitor N-1-n-octyl-3,5-bis-(4-pyridyl)triazole (DPT). Both La3+ and DPT inhibited cell proliferation and SOC entry. Analogous to MEF, the anti-proliferative effect of DPT was mediated by cell cycle arrest and not by induction of apoptosis. These data indicate a role of SOC entry for cell proliferation in cancer cells and suggest a novel anti-proliferative NSAID mechanism in addition to its known influence on lipid metabolism.

Mitochondrial defects by intracellular calcium overload versus endothelial cold ischemia/reperfusion injury.

Questions as to the critical stress factor and primary targets of cold ischemia/reperfusion (CIR) injury were addressed by comparing mitochondrial defects caused by (1) CIR injury and (2) intracellular Ca2+ overload. CIR was simulated in transformed human umbilical vein endothelial cell cultures (tEC) by 8 h cold anoxia in University of Wisconsin solution and reoxygenation at 37 degrees C. Intracellular Ca2+ concentrations were changed by permeabilization of suspended cells with digitonin in culture medium (RPMI, 0.4 mM Ca2+). Binding of free Ca2+ by ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid in RPMI or mitochondrial incubation medium served as controls. Extracellular Ca2+ protected the cell membrane against permeabilization. Mitochondrial functions were determined before and after permeabilization of the cell membrane. After CIR, mitochondrial respiratory capacity declined, but oxygen consumption remained coupled to adenosine triphosphate (ATP) production. In contrast, Ca2+ overload caused uncoupling of mitochondrial respiration. High intracellular Ca2+ overload, therefore, does not reproduce cold ischemia/reperfusion injury in endothelial cells.

Mitochondrial calcium overload is restricted to a few mitochondria in endothelial cells after cold ischemia/reperfusion.

Changes in cytosolic and mitochondrial calcium content were studied in an endothelial cell model after simulating cold ischemia reperfusion injury. Image analysis demonstrated that only a subpopulation of mitochondria in endothelial cells accumulate calcium. Observations led to the hypothesis that mitochondria which are in close contact with the plasma membrane are mainly affected by the Ca2+ efflux across that membrane, while those in other parts of the cell remain unaffected.

Growth factor-induced morphological, physiological and molecular characteristics in cerebral endothelial cells.

The capacity of vascular endothelial cells to modulate their phenotype in response to changes in environmental conditions is one of the most important characteristics of this cell type. Since different growth factors may play an important signalling role in this adaptive process we have investigated the effect of endothelial cell growth factor (ECGF) on morphological, physiological and molecular characteristics of cerebral endothelial cells (CECs). CECs grown in the presence of ECGF and its cofactor heparin exhibit an epithelial-like morphology (type I CECs). Upon removal of growth factors, CECs develop an elongated spindle-like shape (type II CECs) which is accompanied by the reorganization of actin filaments and the induction of alpha-actin expression. Since one of the most important functions of CECs is the creation of a selective diffusion barrier between the blood and the central nervous system (CNS), we have studied the expression of junction-related proteins in both cell types. We have found that removal of growth factors from endothelial cultures leads to the downregulation of cadherin and occludin protein levels. The loss of junctional proteins was accompanied by a significant increase in the migratory activity and an altered protease activity profile of the cells. TGF-beta1 suppressed endothelial migration in all experiments. Our data provide evidence to suggest that particular endothelial functions are largely controlled by the presence of growth factors. The differences in adhesiveness and migration may play a role in important physiological and pathological processes of endothelial cells such as vasculogenesis or tumor progression.

Methylation and silencing of the retinoic acid receptor-beta2 gene in breast cancer.

BACKGROUND: A growing body of evidence supports the hypotheses that the retinoic acid receptor beta2 (RAR-beta2) gene is a tumor suppressor gene and that the chemopreventive effects of retinoids are due to induction of RAR-beta2. RAR-beta2 expression is reduced in many malignant tumors, and we examined whether methylation of RAR-beta2 could be responsible for this silencing. METHODS: RAR-beta2 expression was studied by reverse transcription-polymerase chain reaction (RT-PCR) analysis in eight breast cancer cell lines that were either treated with the demethylating agent 5-aza-2'-deoxycytidine and subsequently with all-trans-retinoic acid (ATRA) or left untreated. Sodium bisulfite genomic sequencing was used to determine the locations of 5-methylcytosines in the RAR-beta2 genes of three of these cell lines. In 16 breast cancer biopsy specimens and non-neoplastic breast tissue, methylation-specific PCR was used to determine the methylation status of RAR-beta2, and, in 13 of the specimens, RT-PCR analysis was used to detect RAR-beta2 expression. RESULTS: Cell lines SK-BR-3, T-47D, ZR-75-1, and MCF7 exhibited expression of RAR-beta2 only after demethylation and treatment with ATRA. The first exon expressed in the RAR-beta2 transcript was methylated in cell lines ZR-75-1 and SK-BR-3. Six breast cancer specimens showed methylation in the same region of the gene. No expression of RAR-beta2 was found in any grade III lesion. An inverse association between methylation and gene expression was found in all grade II lesions. The RAR-beta2 gene from non-neoplastic breast tissue was unmethylated and expressed. CONCLUSIONS: Methylation of the RAR-beta2 gene may be an initial step in breast carcinogenesis; treatment of cancer patients with demethylating agents followed by retinoic acid may offer a new therapeutic modality.

Fluid shear stress induces heat shock protein 60 expression in endothelial cells in vitro and in vivo.

Recent investigations indicate that the initial event in the pathogenesis of atherosclerosis involves an (auto)immunologic injury to the vessel wall. Heat shock proteins (hsps), which are expressed on the endothelial cell surface, constitute possible autoantigens. After being exposed to shear stress of 30 dyne/cm(2) in vitro by means of a rotational viscometer, human umbilical vein endothelial cells were immunohistochemically stained for hsp 60 by the monoclonal antibody ML-30; static control cells were negative. Maximal hsp 60 induction was observed after 12 hours of hemodynamic stress. In Northern blots, the level of hsp 60 mRNA was markedly increased after only 1 hour of shear stress in human umbilical vein endothelial cells compared with static control cells. In vivo investigations in Lewis rats confirmed these in vitro findings: the intima and media of frozen sections of the right common carotid artery exposed to increased wall shear stress (after ligation of the left common carotid artery) were stained for hsp 60. The vessel wall of the left low-shear-stress-exposed side was negative. These findings demonstrate that shear stress results in hsp 60 induction in endothelial cells in vivo and in vitro, providing the prerequisite for humoral and cellular reactions to endothelial hsp in the earliest stages of atherosclerosis.

Myocardial injury leads to a release of heat shock protein (hsp) 60 and a suppression of the anti-hsp65 immune response.

OBJECTIVE: While atherosclerosis is associated with high titers of autoantibodies to bacterial hsp65 crossreacting with human hsp60 (anti-hsp60 autoantibodies), myocardial infarction entails decreased humoral immune response to hsp65. We previously hypothesized that myocardial ischemia and subsequent infarction not only induce myocardial hsp60 expression, but also trigger release of myocardial hsp60 into the circulation, influencing the systemic hsp immune response via immune complex formation. METHODS: In the present study, organ culture of rat hearts under circulatory arrest provided a model of myocardiocyte injury due to ischemia. RESULTS: Reperfusion of ischemic hearts confirmed the occurrence of myocardial injury by a rise of heart enzymes. Myocardial hsp60 expression was induced up to threefold in response to ischemia, and most of hsp60 expression was localized to the muscle fibers. Analysis of coronary eluate revealed release of hsp60 from myocardium. In addition, hsp60-containing, but not hsp60-free, coronary eluate was recognized by anti-hsp65 serum antibodies and induced proliferation of hsp65-specific T cells. When hsp60-containing coronary eluate was reinjected into an hsp65-primed rat, both humoral and cellular hsp65-immune responses were strongly downregulated. CONCLUSION: Our findings demonstrate the release of highly immunogenic and crossreactive hsp60 into the circulation in response to myocardial ischemia and myocardiocyte injury.