Bucillamine prevents myocardial reperfusion injury.

Injury during reperfusion can partially offset the benefit of relief of ischemia in myocardial infarctions rapidly treated with thrombolytic drugs or angioplasty. We assessed whether bucillamine (N-[2-mercapto-2-methylpropionyl]-L-cysteine) is potentially useful to treat myocardial reperfusion injury. Bucillamine is a potent sulfhydryl donor not previously tested as a treatment of reperfusion injury. Cardiac myocytes were exposed to hydrogen peroxide or a xanthine/xanthine oxidase system resulting in injury-induced release of lactate dehydrogenase. Bucillamine (125-500 microM) prevented lactate dehydrogenase release in a concentration-dependent manner. Bucillamine, which has two donatable thiol groups, was twice as protective as N-2-mercaptopropionyl glycine, which contains a single donatable thiol group. Dogs were then exposed to 90 min of coronary artery occlusion and 48 h of reperfusion before sacrifice. Beginning at the onset of reperfusion, bucillamine, 11 or 22 mg/kg per hour, or vehicle (saline) was administered intravenously for 3 h. There was a dose-related response to bucillamine for infarct size, normalized for size of the region at risk and adjusted for collateral blood flow to the ischemic region. Infarct size was reduced by 41% in the group treated with bucillamine 22 mg/kg per hour, compared with the vehicle group. Bucillamine, probably through an antioxidant mechanism, reduced infarct size when administered during reperfusion.

An iron-binding exochelin prevents restenosis due to coronary artery balloon injury in a porcine model.

Background- Vascular smooth muscle cell (VSMC) proliferation is a critical factor in the neointima formation that causes restenosis after coronary angioplasty (PTCA). Desferri-exochelin 772SM (D-EXO), a highly diffusible, lipophilic iron chelator secreted by Mycobacterium tuberculosis, inhibits proliferation of VSMCs in culture. We hypothesized that treatment with D-EXO would inhibit neointima formation in balloon-injured vessels in vivo. Methods and Results- We subjected 24 pigs to overstretch coronary artery injury with standard PTCA balloons and then administered intramural injections of either D-EXO (n=14) or vehicle (n=10) through an Infiltrator catheter. Treatments were randomized, and the investigators were blinded with regard to treatment group until data analysis was completed. One month later, we euthanized the pigs, excised the injured coronary segments, made multiple sections of each segment, and identified the site of maximal neointima formation. An injury score based on the degree of disruption of the internal or external elastic lamina or media was assigned. D-EXO reduced stenosis index (neointima area divided by the area within the internal elastic lamina), adjusted for injury score, by 47%. Neointima thickness was also reduced. Conclusions- D-EXO, injected intramurally, substantially inhibited formation of neointima in a porcine vascular injury model.

A novel iron chelator in combination with a P-selectin antagonist prevents ischemia/reperfusion injury in a rat liver model.

BACKGROUND: Hepatic ischemia/reperfusion (I/R) injury is associated with early and late graft failure after liver transplantation. A major mechanism is leukocyte adhesion to endothelium followed by release of reactive oxygen intermediates. We examined whether desferriexochelin 772SM (D-Exo), a lipid soluble iron chelator that prevents hydroxyl radical formation, can enhance the capacity of recombinant P-selectin glycoprotein ligand immunoglobulin (rPSGL-Ig), a glycoprotein that binds to P-selectin and inhibits neutrophil adhesion, to protect against I/R injury in an ex vivo rat liver model. METHODS: Rat livers were harvested and stored for 6 hr at 4 degrees C in University of Wisconsin solution and then perfused with oxygenated whole blood for 2 hr. Three groups were studied (n=6 rats/group): an untreated control group; a group that received 0.4 mg/kg rPSGL-Ig intraportally at the time of harvest; and a group that received 0.4 mg/kg rPSGL-Ig plus 1 micromol D-Exo intraportally both at the time of harvest and at the onset of reperfusion. Liver portal venous blood flow was assessed during perfusion, and at the end of each experiment, liver samples were collected for blinded histological evaluation and biochemical analyses. RESULTS: Livers treated with D-Exo + rPSGL-Ig had significantly higher blood flow than livers treated with rPSGL-1Ig alone (P<0.05), and both treatment groups had higher blood flow than controls (P<0.001). Production of carbonyl proteins, a protein oxidation product, was significantly reduced in the D-Exo + rPSGL-1Ig group (P<0.02 vs. controls), but not in the rPSGL-Ig alone group. Total reduced glutathione was significantly higher than controls in the D-Exo + rPSGL-Ig group (P<0.001 vs. controls), but not in the rPSGL-Ig alone group, indicating less oxidative stress in the D-Exo-treated group. Production of malondialdehyde, an index of lipid peroxidation, was significantly less than controls in both treatment groups (P<0.03). Histopathological findings paralleled these results with Banffs scores of 3.3+/-0.5, 1.8+/-0.4, and 1.3+/-0.5 in the control, rPSGL-Ig alone, and D-Exo plus rPSGL-Ig groups, resp. CONCLUSION: rPSGL-Ig provides partial protection against I/R injury to ex vivo rat livers; however, the addition of D-Exo substantially increases protection by reducing oxidative injury. These findings may have clinical relevance in preventing the consequences of I/R injury after liver transplantation.

Desferri-exochelin induces death by apoptosis in human breast cancer cells but does not kill normal breast cells.

A major goal of cancer chemotherapy is the identification of cytotoxic compounds that are highly selective for cancer cells. We describe here one such compound - a novel iron chelator, desferri-exochelin 772SM. This desferri-exochelin has unique chemical and pharmacological properties, including extremely high iron binding affinity, the capacity to block iron-mediated redox reactions, and lipid solubility which enables it to enter cells rapidly. At low concentrations, this desferri-exochelin kills T47D-YB and MCF-7 human breast cancer cells by inducing apoptosis, but only reversibly arrests the growth of normal human mammary epithelial cells without cytotoxicity. Since iron-loaded exochelin is ineffective, iron chelation accounts for the efficacy of desferri-exochelin. For both the killing of breast cancer cells and the growth arrest of normal breast epithelial cells, desferri-exochelin was effective at much lower concentrations than the lipid-insoluble iron chelator deferoxamine, which has shown only limited potential as an anti-cancer agent. Growth arrest of progesterone receptor positive T47D-YB cells with the progestin R5020 transiently protects them from the cytotoxic effects of desferri-exochelin, but the cells are killed after cell growth resumes. Similarly, MCF-7 cells arrested with the estrogen antagonist ICI182780 are transiently resistant to killing by desferri-exochelin. Thus the desferri-exochelin is cytotoxic only to actively growing tumor cells. Since desferri-exochelin 772SM can selectively and efficiently destroy proliferating cancer cells without damaging normal cells, it may prove useful for the treatment of cancer.

Narcissistic leadership in psychotherapy groups.

Narcissistic leaders in groups are capable of impeding progress of their patients and, at worst, can produce iatrogenic effects. Significant interferences may occur when the therapist is unable to tolerate the expression of negative transferences and when they need to be idealized by their patients. The rare therapist who is a malignant narcissist is capable of inflicting severe damage by sadistically exploiting the group to satisfy his or her own pathological needs. Less severe interferences consist of inhibition in making transference interpretations, reluctance to seek out training or supervision, and a difficulty in protecting patients against being scapegoated as a result of the displacement of negative feelings toward the therapist onto a member. The universality of these issues among therapists is discussed and possible remediation is proposed.

Estrogen receptors alpha and beta: prevalence of estrogen receptor beta mRNA in human vascular smooth muscle and transcriptional effects.

BACKGROUND: Estrogens have vascular effects through the activation of estrogen receptors (ERs). In addition to ERalpha, the first ER to be cloned, a second subtype called ERbeta has recently been discovered. METHODS AND RESULTS: Using a reverse-transcriptase polymerase chain reaction assay that employs the same primer pair to simultaneously amplify ERalpha and ERbeta transcripts, we found that ERbeta is the ER form that is predominantly expressed in human vascular smooth muscle, particularly in women. The transcriptional effects of the 2 ERs in transfected HeLa cells differed. In response to 17beta-estradiol, ERalpha is a stronger transactivator than ERbeta at low receptor concentrations. However, at higher receptor concentrations, ERalpha activity self-squelches, and ERbeta is a stronger transactivator. Tamoxifen has partial agonist effects with ERalpha but not with ERbeta. CONCLUSIONS: The protective effects of estrogens in the cardiovascular system of women may be due to the genomic effects of ERbeta in vascular tissue.

An exochelin of Mycobacterium tuberculosis reversibly arrests growth of human vascular smooth muscle cells in vitro.

Proliferation of vascular smooth muscle cells (VSMC) is characteristic of restenosis following balloon angioplasty. We show here that a low concentration of a novel iron chelator, desferri-exochelin 772SM, reversibly arrests the growth of human VSMC in vitro, specifically in G(0)/G(1) and S phases. The lipophilic desferri-exochelin is effective more rapidly and at a 10-fold lower concentration than the nonlipophilic iron chelator deferoxamine. Treatment of growth-synchronized VSMC with the desferri-exochelin results in down-regulation of cyclin E/ Cdk2 and cyclin A/Cdk2 activity but does not affect the cyclin D/Cdk4/retinoblastoma phosphorylation pathway. Both DNA replication and RNA transcription are inhibited in exochelin-treated cells, but protein synthesis is not. The ability of desferri-exochelin 772SM to reversibly block the growth of VSMC in vitro with no apparent cytotoxicity suggests that the exochelin may be useful as a therapeutic agent to limit restenosis in injured vessels.

Complexity of effector mechanisms in cyclosporine-induced syngeneic graft-versus-host disease.

Administration of the immunosuppressive drug cyclosporine after syngeneic or autologous bone marrow transplantation elicits a T-lymphocyte-dependent autoimmune syndrome similar to graft-versus-host disease (GVHD). The onset of this autoaggression syndrome, termed syngeneic GVHD, is associated with the development of a highly restricted repertoire of CD8+ autoreactive T cells that recognize a peptide from the invariant chain, termed CLIP, presented by major histocompatibility complex (MHC) class II molecules. Clonal analysis reveals 2 distinct subsets of autoreactive T cells defined by their activation requirement for either the N-terminal or the C-terminal flanking regions of CLIP and by their cytokine profile. The studies here reveal that the autoreactive T-cell clones requiring the N-terminal flanking region of CLIP produce type 1 cytokines (interferon [IFN]-gamma, interleukin [IL]-2, and tumor necrosis factor-alpha). In contrast, the autoreactive T-cell clones that require the C-terminal flanking region of CLIP produce type 2 cytokines (IL-4, IL-10, transforming growth factor-beta). As assessed in a local graft-versus-host reaction assay, the N-terminal flanking-restricted clones mediate changes consistent with acute GVHD, whereas the clones responsive to the C-terminal flanking region do not. Moreover, the autoreactive T-cell clones restricted by the C-terminal flanking region of CLIP ameliorate the pathogenic potential of the cells responsive to the N-terminal flanking region of CLIP. The mechanism accounting for this regulatory affect appears to be the downregulation of mRNA message for type 1 cytokines (IFN-gamma and IL-2). The C-terminal-restricted autoreactive T-cell clones, however, could manifest disease with dermal changes similar to those seen in chronic syngeneic GVHD, provided that IFN-gamma was present. Consistent with these observations was the demonstration that type 1 cytokines are preferentially detected during the acute phase of syngeneic GVHD, whereas type 2 cytokines dominate during the chronic phase. The results suggest that acute and chronic syngeneic GVHD is mediated by distinct autoreactive T cells, which are separated by their fine specificity for the CLIP-MHC class II complex and by their cytokine profiles.

C6 produced by macrophages contributes to cardiac allograft rejection.

The terminal components of complement C5b-C9 can cause significant injury to cardiac allografts. Using C6-deficient rats, we have found that the rejection of major histocompatibility (MHC) class I-incompatible PVG.R8 (RT1.A(a)B(u)) cardiac allografts by PVG.1U (RT1.A(u)B(u)) recipients is particularly dependent on C6. This model was selected to determine whether tissue injury results from C6 produced by macrophages, which are a conspicuous component of infiltrates in rejecting transplants. We demonstrated that high levels of C6 mRNA are expressed in isolated populations of macrophages. The relevance of macrophage-produced C6 to cardiac allograft injury was investigated by transplanting hearts from PVG. R8 (C6-) donors to PVG.1U (C6-) rats which had been reconstituted with bone marrow from PVG.1U (C6+) rats as the sole source of C6. Hearts grafted to hosts after C6 reconstitution by bone marrow transplantation underwent rejection characterized by deposition of IgG and complement on the vascular endothelium together with extensive intravascular aggregates of P-selectin-positive platelets. At the time of acute rejection, the cardiac allografts contained extensive perivascular and interstitial macrophage infiltrates. RT-PCR and in situ hybridization demonstrated high levels of C6 mRNA in the macrophage-laden transplants. C6 protein levels were also increased in the circulation during rejection. To determine the relative contribution to cardiac allograft rejection of the low levels of circulating C6 produced systemically by macrophages, C6 containing serum was passively transferred to PVG.1U (C6-) recipients of PVG.R8 (C6-) hearts. This reconstituted the C6 levels to about 3 to 6% of normal values, but failed to induce allograft rejection. In control PVG.1U (C6-) recipients that were reconstituted with bone marrow from PVG.1U (C6-) donors, C6 levels remained undetectable and PVG.R8 cardiac allografts were not rejected. These results indicate that C6 produced by macrophages can cause significant tissue damage.

Tamoxifen resistant breast cancer: coregulators determine the direction of transcription by antagonist-occupied steroid receptors.

Pharmacological antagonists of steroid receptor action had been thought to exert their effects by a passive mechanism driven principally by the ability of the antagonist to compete with agonist for the ligand binding site. However, recent analyses of antagonist-occupied receptor function suggest a more complex picture. Antagonists can be subdivided into two groups, type I, or pure antagonists, and type II, or mixed antagonists that can have variable transcriptional activity based upon differential dimerization and DNA binding properties. This led us to propose that receptor antagonism may not simply be a passive competition for the ligand binding site, but may, in some cases, involve active recruitment of corepressor or coactivator proteins to produce a mixed transcriptional phenotype. We used a yeast two-hybrid screen to identify proteins that interact specifically with antagonist-occupied receptors. Two proteins have been characterized: L7/SPA, a ribosome-associated protein that is localized in both the cytoplasm and nucleus, but with no known extranucleolar nuclear function; and hN-CoR, the human homolog of the mouse thyroid receptor corepressor mN-CoR. In in vivo transcription assays we show that L7/SPA enhances the partial agonist activity of type II mixed antagonists, and that N-CoR and the related corepressor, SMRT, suppresses it. The coregulators do not affect agonists or pure antagonists. Moreover, the net agonist activity seen with mixed antagonists is a function of the ratio of coactivator to corepressor. Based upon these results, we proposed that in breast tumors the inappropriate agonist activity seen with therapeutic antagonists such as tamoxifen is responsible for the hormone-resistant state. To confirm this, we are quantitating coactivator/corepressor ratios in breast tumor cells lines and clinical breast cancers. Results should provide new insights into the mechanisms underlying the progression of breast cancer to hormone resistance, and may suggest strategies for delaying or reversing this process.

Iron-mediated cardiovascular injury.

Iron is an essential element for normal cellular function and general health. However, iron may play a pathologic role in certain cardiac conditions including reperfusion injury, hemochromatosis, beta-thalassemia and coronary atherosclerosis. It also may play a role in injury due to anthracycline cardiotoxicity. Removal of iron via phlebotomy for hemochromatosis and chelation therapy for beta-thalassemia are proven treatments. Cell culture, and isolated organ and animal studies suggest that depleting iron stores may prevent reperfusion injury, restenosis and even atherogenesis. This article will review mechanisms by which iron overload states and normal iron stores contribute to cardiovascular pathophysiology and the accumulating evidence that iron chelation may prevent restenosis and atherogenesis.

Variant estrogen and progesterone receptor messages in human vascular smooth muscle.

BACKGROUND: Estrogens stimulate growth of breast or uterine cells but have the opposite effect on vascular smooth muscle cells, in which they protect against coronary artery disease with or without concomitant administration of progesterone. A possible cause of differences in hormone action is variable tissue-specific expression of hormone receptor. Therefore, we analyzed the structure of estrogen receptors (ERs) and progesterone receptors (PRs) in human vascular smooth muscle. METHODS AND RESULTS: RNA was isolated from human vascular smooth muscle, and the functional domains of ER-alpha and PR were characterized by reverse transcriptase and polymerase chain reaction. Interestingly, in addition to wild-type ER-alpha and PR, 5 variant ER-alpha and 2 variant PR transcripts were found. These variants contained precise deletions of exons encoding regions of the hormone-binding domain. The PR transcripts lacked exon 4 (PRDelta4) and exon 6 (PRDelta6). The ER-alpha transcripts were missing exon 4 (ERDelta4), exon 5 (ERDelta5), exon 6 (ERDelta6), exon 7 (ERDelta7), and exons 6 and 7, (ERDelta6,7). ER-beta variants were also detected. The PR variants were functionally characterized, and PRDelta6 was found to be a dominant-negative transcription inhibitor of wild-type receptors. Variant PR was present in premenopausal women but absent in postmenopausal women. CONCLUSIONS: Variant PR and ER transcripts are extensively expressed in human vascular smooth muscle. The complex tissue-specific effects of sex hormones may be mediated by the expression of heterogeneous forms of their cognate receptors. The presence of variant ERs and PRs may be of importance in altering the physiological effects of estrogens or progestins in vascular smooth muscle.

Ovarian ablation alone promotes aortic intimal hyperplasia and accumulation of fibroblast growth factor.

BACKGROUND: Estrogen-mediated cardiovascular protection is incompletely explained by its beneficial lipid-modifying effects. Previous studies interrogating direct vascular effects of estrogens have used models of either diet- or injury-induced atherosclerosis. The purpose of this study was to determine the influence of ovarian ablation alone on vascular remodeling. We hypothesized that estrogens are atheroprotective, independent of their influence on lipid metabolism, by directly influencing the production and effects of a prototypical atherogenic mediator, basic fibroblast growth factor (bFGF). METHODS AND RESULTS: Twenty-five female sheep were randomized to sham operation, ovariectomy, or ovariectomy plus 17beta-estradiol replacement. Serum cholesterol and triglyceride levels were serially measured for 1 year and were similar among groups and in the normal range (30 to 60 mg/dL). At 6, 9, and 12 months, ovariectomy resulted in aortoiliac intimal hyperplasia compared with sham (P<0.01) and hormone replacement (P<0.01) groups. The neointima of ovariectomized animals was characterized immunohistochemically by increased vascular smooth muscle cells (VSMCs). Levels of bFGF protein were determined in adjacent aortic segments. Ovariectomized sheep had 2-fold more FGF than sham or ovariectomized sheep that received hormone replacement. In vitro, estradiol inhibited the mitogenic effect of bFGF on human aortic VSMCs. CONCLUSIONS: Without dietary manipulation, ovarian ablation alone induces aortic intimal hyperplasia in the ewe. Estradiol abrogates this response independently of its effects on serum lipids. Hormone replacement decreases the accumulation of the atherogenic peptide bFGF in vivo and inhibits the mitogenic response of VSMCs to bFGF in vitro. These results suggest that estrogens may provide atheroprotection both by modulating local production and by attenuating the influence of bFGF on VSMC growth.

Promiscuous recognition of major histocompatibility complex class II determinants in cyclosporine-induced syngeneic graft-versus-host disease: specificity of cytolytic effector T cells.

BACKGROUND: Administration of the immunosuppressive drug cyclosporine after syngeneic/autologous bone marrow transplantation paradoxically elicits a systemic autoimmune syndrome resembling graft-versus-host disease (GVHD). This syndrome, termed autologous or syngeneic GVHD, is associated with the development of a highly restricted repertoire of cytolytic T lymphocytes that promiscuously recognizes major histocompatibility complex class II determinants, including self. METHODS: Vbeta8.5+CD8+ effector lymphocytes and T-cell clones were isolated from Lewis rats with cylosporine-induced syngeneic GVHD. The specificity of the effector T cells and T-cell clones was examined in vitro. The pathogenicity of the T-cell clones was confirmed in vivo using a local graft-versus-host reaction assay. RESULTS: Clonal analysis reveals that the pathogenic effector T cells recognize a peptide from the invariant chain termed CLIP in association with major histocompatibility complex class II determinants. Moreover, there appears to be an additional interaction between the N-terminal flanking region of CLIP and the Vbeta segment of the T cell receptor. CONCLUSION: The results suggest that recognition of this highly conserved peptide along with the additional interaction between the flanking region and the T cell receptor may account for the promiscuous activity of the autologous/syngeneic GVHD autoreactive T cells.

Estrogen replacement inhibits intimal hyperplasia and the accumulation and effects of transforming growth factor beta1.

BACKGROUND: The role of estrogens in providing atheroprotection has been well documented in both epidemiologic and experimental studies. This phenomenon has traditionally been attributed to the beneficial lipid-modifying effects of estrogens. Previous studies have used models of either diet- or injury-induced atherosclerosis. As such, the interrelationship between estrogens, lipids, and atherosclerosis remains unclear. We hypothesized that estrogens are atheroprotective independent of changes in serum lipids by directly influencing the accumulation and effects of the peptide growth factor transforming growth factor beta1 (TGF-beta1). MATERIAL AND METHODS: Thirteen female sheep (8 years old) were randomized to sham, ovariectomy, or ovariectomy with 17beta-estradiol replacement. Serum lipid levels were serially measured. At 9 months, necropsy was performed with histologic morphometric analysis of the aortoiliac bifurcation. Levels of TGF-beta1 were determined in serum and aortic tissue. Human aortic smooth muscle cells were isolated and cultured. RESULTS: Serum triglyceride, lipoprotein a, and total, low-density lipoprotein, and high-density lipoprotein cholesterol levels were similar and normal between groups. Ovariectomy resulted in aortoiliac intimal hyperplasia compared with sham (P < 0.001) and hormone replacement (P < 0.001) groups. Compared with ovariectomy, estrogen replacement attenuated aortic accumulation of TGF-beta1 (P < 0.02). In vitro, estradiol potentiated TGF-beta1 inhibition of human vascular smooth muscle cell (VSMC) proliferation and increased TGF-beta1 release in stimulated VSMCs (P < 0.001). CONCLUSIONS: Without dietary manipulation, ovarian ablation induces aortic intimal hyperplasia in the ewe. Estradiol abrogates this response independently of its effects on serum lipids. Hormone replacement decreases the accumulation of TGF-beta1, suggesting that estrogens may provide atheroprotection both by modifying local production and by modulating the influence of TGF-beta1 on VSMC growth.

Prevention of coronary vascular abnormalities early in reperfusion with TGF-beta may not prevent late coronary vascular injury.

Endothelial injury, manifest by increased protein leak and decreased endothelium-dependent relaxation, occurs during reperfusion after ischemia. Transforming growth factor-beta (TGF-beta) has been shown to improve endothelium-dependent relaxation and reduce infarct size after short periods (from 20 min to 4.5 h) of reperfusion even when administered 24 h before the ischemic period. However, whether this represents a transient delay in the process leading to endothelial injury or prevention of injury has not been clear. To examine this issue, we measured protein leak, an index of coronary microvascular permeability, and endothelium-dependent relaxation, a measure of coronary endothelial function, after brief (1-h) and lengthy (48-h) reperfusion periods in dogs treated 30 min before ischemia with TGF-beta (30 microg/kg, i.v.) and control dogs. The left anterior descending coronary artery (LAD) was ligated for 1 h followed by 1 h of reperfusion (n = 10) or 48 h of reperfusion (n = 12). Protein leak was assessed by a dual-isotope technique by using radiolabeled transferrin and erythrocytes, and endothelium-dependent relaxation was assessed in epicardial coronary rings by using adenosine diphosphate (ADP), an endothelium-dependent vasodilator, and sodium nitroprusside (SNP), an endothelium-independent dilator. In control animals, there was a marked increase in the protein leak index (PLI) in the infarct zone (8.3 +/- 1.4 in 1-h dogs, and 8.7 +/- 0.9 in 48-h dogs) compared with the nonischemic myocardium (3.1 +/- 0.8 at 1 h, and 3.8 +/- 0.9 at 48 h). In TGF-beta treated dogs, there was a marked improvement in PLI in the infarct zone in 1-h dogs (PLI, 4.1 +/- 1.1; p < 0.05; or a 50% reduction compared with untreated dogs). However, the 48-h dogs treated with TGF-beta failed to demonstrate an improvement in PLI (PLI, 8.5 +/- 0.9; p = NS). Endothelium-dependent relaxation was impaired in the LAD in control dogs, and treatment with TGF-beta failed to improve relaxation after 1 or 48 h of reperfusion. Microvascular permeability was increased and endothelium-dependent relaxation was decreased after ischemia at both 1 and 48 h of reperfusion. Pretreatment with TGF-beta reduced the increase in permeability at 1 h of reperfusion but not at 48 h.